The Parathyroids, Second Edition: Basic and Clinical Concepts

Parathyroids Basic and Clinical Concepts SECOND E D I T I O N

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The Parathyroids Basic and Clinical Concepts SECOND E D I T I O N

Editor-in-Chief

John P. Bilezikian, M.D. Professor of Medicine and Pharmacology Chief, Division of Endocrinology Director, Metabolic Bone Diseases Program Departments of Medicine and Pharmacology College of Physicians and Surgeons Columbia University New York, New York Associate Editors R o b e r t Marcus, M.D. Professor of Medicine Department of Medicine Stanford University School of Medicine Stanford, California and Director, Aging Study Unit VeteransAffairs Medical Center Palo Alto, California

M i c h a e l A. L e v i n e , M.D. Professor of Pediatrics, Medicine, and Pathology Direct~ PediatricEndocrinology TheJohns Hopkins University School of Medicine Baltimore, Maryland

ACADEMIC PRESS A Harcourt Science and Technology Company

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This book is printed on acid-free paper. 0 Copyright © 2001, 1994 by John E Bilezikian, Robert Marcus, and Michael Levine M1 Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without the permission in writing from the publisher. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Inc., 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 A c a d e m i c Press A Harcourt Science and Technology Company 525 B Street, Suite 1900, San Diego, California 92101-4495, US http://www, academicpress, com A c a d e m i c Press Harcourt Place, 32 Jamestown Road, London NW1 7BY, UK http://www.academicpress.com Library of Congress Catalog Card Number: 00-111700 International Standard Book Number: 0-12-098651-5 PRINTED IN THE UNITED STATES OF AMERICA 01 02 03 04 05 06 MM 9 8 7 6 5 4

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2

1

Contents Contributors

.................................................

Preface to the Second Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preface to the First Edition

......................................

ix xv

xvii

Section I: Basic Elements of the Parathyroid System 1.

Parathyroids: Morphology and Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . .

Virginia A. LiVolsi 2.

Parathyroid H o r m o n e Biosynthesis a n d Metabolism . . . . . . . . . . . . . . . . . . .

17

Henry M. Kronenberg, E Richard Bringhurst, Gino V. Segre, and John T. Potts, Jr. 3.

Parathyroid H o r m o n e - R e l a t e d Protein: Gene Structure, Biosynthesis, Metabolism, and Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

31

William M. Philbrick 4.

Interactions of Parathyroid H o r m o n e a n d Parathyroid H o r m o n e - R e l a t e d Protein with Their Receptors . . . . . . . . . . . . . . . . . . . . . .

53

Michael Chorev, Joseph M. Alexander, and Michael Rosenblatt 5.

Receptors for Parathyroid H o r m o n e a n d Parathyroid H o r m o n e - R e l a t e d Protein: Signaling a n d Regulation . . . . . . . . . . . . . . . . . .

93

Robert A. Nissenson 6.

Nuclear Actions of P T H r P

.......................................

105

Andrew C. Karaplis and M. T. Audrey Nguyen 7.

............................

117

Receptors a n d Signaling for Calcium Ions . . . . . . . . . . . . . . . . . . . . . . . . . . .

127

Signal Transduction of P T H a n d P T H r P

Lee S. Weinstein and Michael A. Levine 8.

Edward M. Brown, Arthur Conigrave, and Naibedya Chattopadhyay 9.

Immunoassays for P T H a n d PTHrP: Clinical Applications . . . . . . . . . . . . . .

143

L. J. Deftos

Section II: Physiological Aspects of the Parathyroid 10. 11.

Physiology of Calcium Homeostasis

................................

167

Edward M. Brown Parathyroid H o r m o n e : Anabolic a n d Catabolic Effects on Bone a n d Interactions with Growth Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Janet M. Hock, Lawrence G. Raisz, and Ernesto Canalis

183

vi

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12.

Cellular Actions of Parathyroid H o r m o n e on Osteoblast and Osteoclast Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

199

Physiologic Actions of PTH and PTHrP: I. Skeletal Actions . . . . . . . . . . . . . .

213

Physiologic Actions of PTH and PTHrP: II. Renal Actions

227

Jane E. A ubin and Johan N. M. Heersche

13. 14.

15.

GordonJ. Strewler

E Richard Bringhurst

..............

E n d o c h o n d r a l Bone Formation: Regulation by Parathyroid Hormone-Related Peptide, Indian Hedgehog, and Parathyroid H o r m o n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

245

Physiologic Actions of PTH and PTHrP: IV. Vascular, Cardiovascular, and Neurologic Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

261

Physiologic Actions of PTH and PTHrP: V. Epidermal, Mammary, Reproductive, and Pancreatic Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

275

Gino V. Segre and Kaechoong Lee

16.

Thomas L. Clemens and Arthur E. Broadus

17.

JohnJ. Wysolmerski, Andrew E Stewart, and John T. Martin

Section III: Clinical Aspects of Primary Hyperparathyroidism 18.

Parathyroid Growth: Normal and Abnormal . . . . . . . . . . . . . . . . . . . . . . . . .

19.

Molecular Basis of Primary Hyperparathyroidism

2O.

293

A. Michael Parfitt Andrew Arnold

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331

Clinical Presentation of Primary Hyperparathyroidism in the United States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

349

Clinical Presentation of Primary Hyperparathyroidism: Europe . . . . . . . . . .

361

Clinical Presentation of Primary Hyperparathyroidism: India, Brazil, and China . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

375

23.

Clinical Course of Primary Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . .

387

24.

Molecular Markers of Bone Metabolism in Parathyroid Disease . . . . . . . . . .

399

ShonniJ. Silverberg and John P. Bilezikian

21. 22.

Jonas Rastad, Ewa Lundgren, and Sverker LjunghaU

Ambrish Mithal, Francisco Bandeira, Xunwu Meng, ShonniJ. Silverberg, Yifan Shi, Saroj K. Mishra, Luiz Griz, Geisa Macedo, Gustav Celdas, Cristina Bandeira, John P. Bilezikian, and D. Sudhaker Rao

25.

ShonniJ. Silverberg and John P. Bilezikian

MarkusJ. Seibel Cytokines in Primary Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . .

411

26.

H i s t o m o r p h o m e t r i c Analysis of Bone in Primary Hyperparathyroidism . . . .

423

27.

Nephrolithiasis in Primary Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . .

437

Inaam A. Nakchbandi, Andrew Grey, Urszula Masiukiewicz, Maryann Mitnick, and Karl Insogna May Parisien, David W. Dempsteg, Elizabeth Shane, and John P. Bilezikian Vanessa A. Klugman, Murray J. Favus, and Charles Y. C. Pak

Contents

28.

Guidelines for the Medical and Surgical M a n a g e m e n t of Primary Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michael Kleerekopeg, Robert Udelsman, and Michael A. Levine

29.

Medical M a n a g e m e n t of Primary Hyperparathyroidism John L. Stock and Robert Marcus

30.

Preoperative Localization of Parathyroid Tissue in Primary Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . John L. Doppman

................

459

475 487

31.

The Surgical M a n a g e m e n t of Hyperparathyroidism Samuel A. Wells,Jr. and Gerard M. Doherty

32.

Ectopic Locations of Parathyroid Glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . Norman W. Thompson and Paul G. Gauger

499

33.

Parathyroid Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Elizabeth Shane

515

34.

Acute Primary Hyperparathyroidism Lorraine A. Fitzpatrick

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527

Multiple Endocrine Neoplasia Type 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . StephenJ. Marx

535

36.

Multiple E n d o c r i n e Neoplasia Type 2 Robert E Gagel

585

37.

Familial Forms of Primary Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . . Lawrence Mallette and Robert Marcus

38.

Familial Benign Hypocalciuric Hypercalcemia and Neonatal Severe Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ghada E1-Hajj Fuleihan and Hunter Heath III

35.

...................

451

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601

607

Section IV: Secondary Hyperparathyroidism 39.

The Parathyroids in Renal Disease: Pathophysiology . . . . . . . . . . . . . . . . . . . Kevin J. Martin, Esther A. Gonzdlez, and Eduardo Slatopolsky

625

40.

Renal Bone Diseases: Clinical Features, Diagnosis, and M a n a g e m e n t . . . . . . Jack W. Coburn and Isidro B. Salusky

635

Section V: Special Considerations 41.

Evaluation of the Hypercalcemic Patient: Differential Diagnosis . . . . . . . . . David Heath

663

42.

Hypercalcemia Due to P T H r P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Richard Kremer and David Goltzman

671

43.

O t h e r Local and Ectopic H o r m o n e Syndromes Associated with Hypercalcemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gregory R. Mundy and Babatunde Oyajobi

691

Genetic Disorders Caused by Mutations in the P T H / P T H r P Receptor: Jansen's Metaphyseal Chondrodysplasia and Blomstrand Lethal Chondrodysplasia . . . . Caroline Silve and HaraldJi2ppner

707

44.

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vii

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45. 46.

Acute M a n a g e m e n t of Hypercalcemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

729

Jean E. Mulder and John P. Bilezikian Primary H y p e r p a r a t h y r o i d i s m a n d O t h e r Causes of Hypercalcemia in Children a n d Adolescents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

743

Emily L. Germain-Lee and Michael A. Levine

Section VI: The Hypoparathyroid States 47.

H y p o p a r a t h y r o i d i s m in the Differential Diagnosis of Hypocalcemia . . . . . . .

48.

Magnesium Deficiency in Parathyroid Function . . . . . . . . . . . . . . . . . . . . . . .

755

Robert W. Downs 763

Robert K. Rude 49.

T h e Molecular Genetics of H y p o p a r a t h y r o i d i s m . . . . . . . . . . . . . . . . . . . . . .

779

R. V. Thakker

50.

A u t o i m m u n e Hypoparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

51.

Pseudohypoparathyroidism: Clinical, Biochemical, and Molecular Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

791

Michael P. Whyte 807

Suzanne M. Jan de Beur and Michael A. Levine

52.

T r e a t m e n t of Hypoparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

827

Marc K. Drezner

Section VII: The Parathyroids in Osteoporosis 53.

Parathyroid Function in the N o r m a l Aging Process . . . . . . . . . . . . . . . . . . . .

54.

Parathyroid Function and Responsiveness in Osteoporosis . . . . . . . . . . . . . .

55.

Parathyroid H o r m o n e and Growth H o r m o n e in the T r e a t m e n t of Osteoporosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

835

Sundeep Khosla, L.J. Melton III, and B. L. Riggs 843

ShonniJ. Silverberg and John P. Bilezikian 853

Robert Marcus Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

865

Contributors Harvard Medical School Boston, Massachusetts 02114

Joseph M. Alexander (53)* Division of Bone and Mineral Metabolism Charles A. Dana and Thorndike Laboratories Department of Medicine Beth Israel Deaconess Medical Center, and Harvard Medical School Boston, Massachusetts 02215

Arthur E. Broadus (261) Section of Endocrinology Department of Internal Medicine Yale University School of Medicine New Haven, Connecticut 06510

Andrew Arnold (331) Center for Molecular Medicine and Division of Endocrinology and Metabolism University of Connecticut School of Medicine Farmington, Connecticut 06030

Edward M. Brown (127, 167) Endocrine-Hypertension Division Department of Medicine Brigham and Women's Hospital, and Harvard Medical School Boston, Massachusetts 02115

Jane E. Aubin (199) Department of Anatomy and Cell Biology and Department of Medical Biophysics Faculty of Medicine University of Toronto Toronto, Ontario, Canada M5S 1A8

Ernesto Canalis (183) Department of Medicine The University of Connecticut School of Medicine Farmington, Connecticut 06030; and Departments of Research and Medicine Saint Francis Hospital and Medical Center Hartford, Connecticut 06105

Cristina Bandeira (375) Endocrine Unit Hospital dos Servidores do Estado, and Hospital Agamenon MagalhSes Secretaria da Saude de Pernambuco University of Pernambuco Pernambuco, Brazil

Gustav Celdas (375) University of Brazil 0020-020 Recife-PE, Brazil

Francisco Bandeira (375) Endocrine Unit Hospital Agamenon MagalhSes Secretaria da Saude de Pernambuco University of Pernambuco Pernambuco, Brazil

Naibedya Chattopadhyay (127) Endocrine-Hypertension Division Department of Medicine Brigham and Women's Hospital, and Harvard Medical School Boston, Massachusetts 02115

John P. Bilezikian (349, 375, 387, 423, 729, 843) Departments of Medicine and Pharmacology College of Physicians and Surgeons Columbia University New York, New York 10032

Michael Chorev (53) Division of Bone and Mineral Metabolism Charles A. Dana and Thorndike Laboratories Department of Medicine Beth Israel Deaconess Medical Center, and Harvard Medical School Boston, Massachusetts 02215

E Richard Bringhurst (17, 227) Endocrine Unit Massachusetts General Hospital, and Department of Medicine *Numbers in parentheses indicate the pages on which authors'contributions begin. ix

x

/ Contributors

Thomas L. Clemens (261) Division of Endocrinology and Metabolism University of Cincinnati College of Medicine Cincinnati, Ohio 45267 Jack W. Coburn (635) Departments of Medicine and Pediatrics UCLA School of Medicine, and Nephrology Section West Los Angeles Veterans Affairs Medical Center Los Angeles, California 90095 Arthur Conigrave (127) Endocrine-Hypertension Division Department of Medicine Brigham and Women's Hospital, and Harvard Medical School Boston, Massachusetts 02115 L.J. Deftos (143) Department of Medicine University of California, San Diego, and San Diego VA Medical Center LaJoUa, California 92161 David W. Dempster (423) Department of Pathology College of Physicians and Surgeons Columbia University New York, New York 10032; and Regional Bone Center Helen Hayes Hospital West Haverstraw New York, New York 10993 Gerard M. Doherty (487) Department of Surgery Washington University School of Medicine St. Louis, Missouri 63110 John L. Doppman (475)* Diagnostic Radiology Department National Institutes of Health Bethesda, Maryland 20892

Murray J. Favus (437) Department of Medicine University of Chicago Pritzker School of Medicine Chicago, Illinois 60637 Lorraine A. Fitzpatrick (527) Division of Endocrinology, Metabolism, Nutrition, and Internal Medicine Mayo Clinic and Foundation Rochesteg, Minnesota 55905 Ghada EI-Hajj Fuleihan (607) Calcium Metabolism and OsteoporosisProgram American University of Beirut Medical Center Beirut 113-6044, Lebanon Robert F. Gagel (585)' Division of Internal Medicine University of Texas M.D. Anderson Cancer Center Houston, Texas 77030 Paul G. Gauger (499) Division of Endocrine Surgery Department of Surgery University of Michigan Ann Arbor, Michigan 48105 Emily L. Germain-Lee (743) Division of Pediatric Endocrinology Department of Pediatrics The Johns Hopkins University School of Medicine Baltimore, Maryland 21287 David Goltzman (671) Departments of Medicine and Physiology McGiU University, and Calcium Research Laboratory Royal Victoria Hospital Montreal, Quebec Canada H3A 1A1 Esther A. GonzAlez (625) Division of Nephrology St. Louis University, and Renal Division Washington University St. Louis, Missouri 63110

Robert W. Downs (755) Division of Endocrinology and Metabolism Department of Internal Medicine Virginia Commonwealth University School of Medicine Richmond, Virginia 23298

Andrew Grey (411) Department of Medicine University of Auckland 92019 Auckland, New Zealand

Marc K. Drezner (827) University of Wisconsin-Madison Madison, Wisconsin 53792

Luiz Griz (375) University of Brazil 0020-020 Recife-PE, Brazil

*Deceased

Contributors David Heath (663) Department of Medicine SeUy Oak Hospital Birmingham B29 6JD United Kingdom Hunter Heath III (607) United States Medical Division Eli Lilly and Company Indianapolis, Indiana 46285 Johan N. M. Heersche (199) Faculty of Dentistry University of Toronto Toronto, Ontario, Canada M5G 1G6 Janet M. Hock (183) Department of Periodontics Indiana University School of Dentistry Indianapolis, Indiana 46202

Karl Insogna (411) Department of Medicine Yale University School of Medicine New Haven, Connecticut 06520 Suzanne M. Jan de Beur (807) Division of Endocrinology Department of Medicine and Metabolism The Johns Hopkins University School of Medicine Baltimore, Maryland 2128 7 Harald Jiippner (707) Endocrine Unit Department of Medicine and Children's Service Massachusetts General Hospital, and Harvard Medical School Boston, Massachusetts 02114 Andrew C. Karaplis (105) Division of Endocrinology Department of Medicine Sir Mortimer B. Davis-Jewish General Hospital, and Lady Davis Institute for Medical Research McGiU University Montreal, Quebec Canada H3T 1E2 Sundeep Khosla (835) Mayo Clinic and Foundation Rochest~ Minnesota 55905 Michael Kleerekoper (451) Department of Medicine Wayne State University Detroit, Michigan 48201

Vanessa A. Klugman (437) West Suburban Hospital Oak Park, Illinois 60302 Richard Kremer (671) Department of Medicine McGiU University, and Calcium Research Laboratory Royal Victoria Hospital Montreal, Quebec Canada H3A 1A1 Henry M. Kronenberg (17) Endocrine Unit Massachusetts General Hospital, and Department of Medicine Harvard Medical School Boston, Massachusetts 02114 Kaechoong Lee (245) Endocrine Unit Massachusetts General Hospital, and Department of Medicine Harvard Medical School Boston, Massachusetts 02114 Michael A. Levine (117, 451,743, 807) Departments of Pediatrics, Medicine, and Pathology The Johns Hopkins University School of Medicine Baltimore, Maryland 21287

Virginia A. LiVolsi (1) Department of Pathology and Laboratory Medicine University of Pennsylvania Medical Center Philadelphia, Pennsylvania 19104 Sverker Ljunghall (361) Global Clinical Sciences AstraZeneca Research and Development S-431 83 M61ndal, Sweden Ewa Lundgren (361) Department of Surgery Endocrine Unit University Hospital S-751 85 Uppsala, Sweden Geisa Macedo (375) University of Brazil 0020-020 Recife-PE, Brazil

/

xi

xii

/ Contributors

Lawrence Mallette (601) Department of Medicine Stanford University School of Medicine, and Aging Study Unit VA Medical Center Palo Alto, California 94304 Robert Marcus (459, 601,853) Department of Medicine Stanford University School of Medicine, and Aging Study Unit VA Medical Center Palo Alto, California 94304 John T. Martin St. Vincent's Institute of Medical Research Fitzroy, VIC3065 Australia Kevin J. Martin (625) Division of Nephrology St. Louis University, and Renal Division Washington University St. Louis, Missouri 63110 Stephen J. Marx (535) Metabolic Diseases Branch National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892 Urszula Masiuldewicz (411 ) Department of Medicine Yale University School of Medicine New Haven, Connecticut 06520 L.J. Melton III (835) Mayo Clinic and Foundation Rochesteg, Minnesota 55905 Xunwu Meng (375) Peking University Medical College Hospital 100730 Beijing, China Saroj K. Mishra (375) Department of Surgery Sanjay Gandhi Post Graduate Institute of Medical Sciences 226 O14 Lucknow, India Ambrish Mithal (375) Indraprastha Apollo Hospital 110044 New Delhi, India Maryann Mitnick (411) Mineral Metabolism Laboratory

Yale University School of Medicine New Haven, Connecticut 06520 Jean E. Mulder (729) Department of Medicine College of Physicians and Surgeons Columbia University New York, New York 10032 Gregory R. Mundy (691) Medicine~Endocrinology University of Texas Health Science Center San Antonio, Texas 78284 Inaam A. Nakchbandi (411) Mannheim Faculty of Medicine University of Heidelberg 68135 Mannheim, Germany M. T. Audrey Nguyen (105) Division of Endocrinology Department of Medicine Sir Mortimer B. Davis-Jewish General Hospital, and Lady Davis Institute for Medical Research McGiU University Montrgal, Quebec Canada H 3 T 1E2 Robert A. Nissenson (93) Endocrine Unit San Francisco VA Medical Centeg, and Departments of Medicine and Physiology University of California, San Francisco San Francisco, California 94121 Babatunde Oyajobi (691) Medicine~Endocrinology University of Texas Health Science Center San Antonio, Texas 78284 Charles Y. C. Pak (437) Department of Internal Medicine University of Texas Southwestern Medical Center at Dallas Dallas, Texas 75390 A. Michael Parfitt (293) Division of Endocrinology and Centerfor Osteoporosis and Metabolic Bone Disease University of Arkansas for Medical Sciences Little Rock, Arkansas 72205 May Parisien (423) Department of Pathology College of Physicians and Surgeons Columbia University New York, New York 10032

Contributors William M. Philbrick (31) Section of Endocrinology Department of Internal Medicine Yale University School of Medicine New Haven, Connecticut 06520 John T. Potts, Jr. (17) Endocrine Unit Massachusetts General Hospital, and Department of Medicine Harvard Medical School Boston, Massachusetts 02114 Lawrence G. Raisz (183) Department of Medicine The University of Connecticut School of Medicine Farmington, Connecticut 06030 D. Sudhaker Rao (375) Bone and Mineral Metabolism Department of Medicine Henry Ford Health System Detroit, Michigan 48202 Jonas Rastad (361) Department of Surgery Endocrine Unit • University Hospital S-751 85 Uppsala, Sweden B. L. Riggs (835) Mayo Clinic and Foundation Rochester, Minnesota 55905 Michael Rosenblatt (53) Division of Bone and Mineral Metabolism Charles A. Dana and Thorndike Laboratories Department of Medicine Beth Israel Deaconess Medical Center, and Harvard Medical School Boston, Massachusetts 02115 Robert K. Rude (763) University of Southern California School of Medicine Los Angeles, California 90089 Isidro B. Salusky (635) Departments of Medicine and Pediatrics UCLA School of Medicine, and Nephrology Section West Los Angeles Veterans Affairs Medical Center Los Angeles, California 90095 Markus J. Seibel (399) Division of Endocrinology and Metabolism Department of Internal Medicine I

University of Heidelberg 69115 Heidelberg, Germany

Gino V. Segre (17, 245) Endocrine Unit Massachusetts General Hospital, and Department of Medicine Harvard Medical School Boston, Massachusetts 02114 Elizabeth Shane (423, 515) Department of Medicine College of Physicians and Surgeons Columbia University New York, New York 10032 Yifan Shi (375) Peking University Medical College Hospital 100730 Beijing, China Caroline Silve (707) INSERM U. 42 6 Facult~ de M~decine Xavier Bichat 75018 Paris, France Shormi J. Silverberg (349, 375, 387, 843) Department of Medicine College of Physicians and Surgeons Columbia University New York, New York 10032 Eduardo Slatopolsky (625) Division of Nephrology St. Louis University, and Renal Division Washington University St. Louis, Missouri 63110 Andrew E Stewart University of Pittsburgh Medical Center Pittsburgh, Pennsylvania 15213 John L. Stock (459) Department of Medicine University of Massachusetts Medical School Worcester, Massachusetts O1605; and Eli Lilly and Company Indianapolis, Indiana 46285 Gordon J. Strewler (213) VA Boston Healthcare System West Roxbury, Massachusetts 02132; and Department of Medicine Harvard Medical School Boston, Massachusetts 02114

/

xiii

xiv

/ Contributors

R. V. Thakker (779) Nuffield Department of Clinical Medicine University of Oxford Headington, Oxford OX3 9DU United Kingdom Norman W. Thompson (499) Division of Endocrine Surgery Department of Surgery University of Michigan Ann Arbor, Michigan 48105 Robert Udelsman (451) ~ Department of Surgery The Johns Hopkins University School of Medicine Baltimore, Maryland 21287 Lee S. Weinstein (117) Metabolic Diseases Branch National Institute of Diabetes and Digestive and Kidney Diseases

National Institutes of Health Bethesda, Maryland 20892 Samuel A. Wells, Jr. (487) Department of Surgery Washington University School of Medicine St. Louis, Missouri 63110 Michael P. Whyte (791) Centerfor Metabolic Bone Disease and Molecular Research Shriners Hospitals for Children St. Louis, Missouri 63131; and Division of Bone and Mineral Diseases Washington University School of Medicine at Barnes-Jewish Hospital St. Louis, Missouri 63110 John J. Wysolmerski Yale University School of Medicine New Haven, Connecticut 06520

Current affiliation: Department of Surgery, Yale University, New Haven, Connecticut 06520.

Preface to the S e c o n d Edition The first edition of The Parathyroids was published in 1994. It marked a milestone in the field, carrying on the tradition of Albright and Reifenstein whose 1948 classic The Parathyroid Glands and Metabolic Disease established a key role for the parathyroids in calcium homeostasis and metabolic bone disease. In The Parathyroids, we assembled a body of knowledge that had been accumulating over a 30-year period. The spectacular pace of discovery placed the tiny parathyroid glands at an epicenter of an enormous research effort in metabolic bone disease. The first edition was used widely and filled an essential gap in reference literature. Over the past seven years, as this field has continued to grow, with newer and greater appreciation of the role of the parathyroids in the overall governance of calcium homeostasis, a second edition appears to be particularly apt. The second edition of The Parathyroids contains chapters that have been extensively revised and expanded and many new chapters as well. The chapters d o c u m e n t our new knowledge about virtually every facet of this field and reexamine classic precepts that have stood the test of time. We understand better than ever before the structure and function of the parathyroid h o r m o n e gene and protein as well as the regulatory control of parathyroid h o r m o n e synthesis and secretion, the physiological and pathophysiological aspects of parathyroid hormone-related protein (PTHrP), the mechanisms of parathyroid h o r m o n e and PTHrP action, and the cell biology of PTH and PTHrE With regard to primary hyperparathyroidism, we now appreciate a spectrum of clinical presentations according to where in the world it is detected. Information about the course of primary hyperparathyroidism with and without parathyroid surgery is also new, as are the molecular genetics, biochemical, and histomorphometric dynamics of primary hyperparathyroidism. Advances in preoperative localization of parathyroid tissue and newer operative approaches to parathyroid gland surgery are noteworthy. The hypoparathyroid disorders are understood better with regard to their molecular genetics, pathophysiology, and mechanism. Finally, newer information is available about how parathyroid h o r m o n e can be both a catabolic and anabolic h o r m o n e for bone. This newer knowledge has fueled provocative ideas about the pathophysiology of osteoporosis and is heralding a new era in the therapeutics of osteoporosis. The second edition, thus, is still a comprehensive examination of basic and clinical concepts of the parathyroids. It is intended for students, teachers, practitioners, and investigators. In light of these newer developments in the field, the second edition has been reorganized to provide the reader with information that follows best the changing scientific logic. Fifty-five chapters are divided into seven sections. In Section I, nine chapters are devoted to basic concepts of parathyroid h o r m o n e and PTHrP, covering embryology, anatomy, and pathology of parathyroid tissue; gene structure, biosynthesis, and metabolism of PTH and PTHrP; receptors, nuclear targeting, and signal transduction for PTH, PTHrP, and calcium ion; and a comprehensive review of the immunoassays for PTH and PTHrE In Section II, eight chapters are devoted to the physiological aspects of calcium metabolism and the anabolic and catabolic effects of PTH at the level of bone and bone cells. Five chapters cover in detail all aspects of PTH and PTHrP with regard to traditional and nontraditional target organs. In Section III, 21 chapters are devoted to clinical aspects of primary hyperparathyroidism. Chapters on the growth of normal and abnormal parathyroid cells and the molecular genetics of primary hyperparathyroidism are followed by three chapters that describe different clinical presentations of primary hyperparathyroidism t h r o u g h o u t the world. Detailed coverage of bone dynamics and stone disease is followed by information relevant to the medical and surgical m a n a g e m e n t of primary hyperparathyroidism. Also covered are other presentations of primary

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/ Preface to the Second Edition hyperparathyroidism: as a malignancy, as an acutely hypercalcemic disorder, and in association with the multiple endocrine syndromes I and II. A chapter on familial hypocalciuric hypercalcemia completes this section. Two chapters in Section IV cover the parathyroids in renal disease. These are followed by six chapters in Section V that focus on special considerations. The first three chapters review the differential diagnosis of hypercalcemia, including syndromes caused by the local and systemic production of hypercalcemic factors such as PTHrR Jansen's disease, the acute m a n a g e m e n t of hypercalcemia, and hypercalcemia in children are considered in separate chapters. In Section VI, the hypoparathyroid states are reviewed in six chapters, which cover molecular, ionic, and immunological defects in the hypoparathyroid states and the role of hypoparathyroidism in the differential diagnosis of hypocalcemia. In Section VII, the role of parathyroid function in osteoporosis is covered in three chapters describing changes in parathyroid function with aging, parathyroid function and responsiveness in osteoporosis, and the potential of parathyroid h o r m o n e as a therapy for osteoporosis. As was true for the first edition, we recognize that this book is not likely to be read from cover to cover. Thus, each chapter has been written to provide a body of knowledge that can stand alone. The chapters, however, are also liberally cross-referenced to help the reader continue reading more directly related material if desired. The first edition of this book was dedicated to the m e m o r y of Gerald D. Aurbach, whose untimely and tragic death was its catalyst and inspiration. Virtually all the principal authors of the first edition had known and worked with Jerry. We r e m e m b e r e d him then for his "wisdom, scientific acumen, investigative skills, and daring insights." We r e m e m b e r him now in m u c h the same way. We were and still are mindful of the role Jerry had not only for us but also for the entire field, which he helped to create. We were his scientific progeny. It is 10 years since Jerry's death, virtually a generation in the world of science. As a result, some of the leading figures in this field have e m e r g e d without having had the special privilege of working with or knowing Jerry. The authorship of the second edition has been broadened, therefore, to include the very best in our field, recognizing that although Jerry's legacy is still alive, it now extends to an even broader cross section of the field. We wish to thank Jasna Markovac of Academic Press, who was instrumental in both the first and current editions of The Parathyroids. Mica Haley of Academic Press was also most helpful in attending to the many details required to ensure a rapid t u r n a r o u n d time to final publication. Enjoy the book.

John P. Bilezikian Robert Marcus Michael A. Levine

Preface to the First E d i t i o n One of us (JPB), d r e a m e d of this book about five years ago. It seemed then that advances in our knowledge of the parathyroids represented nothing less than a 30-year revolution of spectacular progress. We gained knowledge over this period at an explosive pace with a concomitant new appreciation of the basic and clinical ramifications of these four tiny endocrine glands. The major secretory product, parathyroid h o r m o n e (PTH), was isolated, sequenced, assayed, and cloned. PTH became one of the first h o r m o n e s to be shown to utilize cAMP as a second messenger. Regulation of PTH synthesis and secretion by calcium and 1,25-dihydroxyvitamin D was appreciated, as well as the cellular effects of PTH on its two major target organs, bone and kidney. The discovery of parathyroid hormone-related protein (PTHrP) as a cause of hypercalcemia of malignancy and a more general appreciation of PTHrP and PTH as polypurpose factors with many diverse biological effects represent exciting new advances in our field. The recent cloning of a bona fide receptor for both PTH and PTHrP is a tremendous achievement, as is the thinking that both PTH and PTHrP may utilize more than one second messenger pathway, and perhaps interact with more than one receptor. At the clinical level, we have seen a remarkable evolution in the presentation of primary hyperparathyroidism and are beginning to understand molecular features of this disease. Pseudohypoparathyroidism is now appreciated, in its classical form, to be a G protein deficiency disease. A u t o i m m u n e and molecular features of hypoparathyroidism have been identified and studied. New knowledge of the pathophysiology of secondary hyperparathyroidism associated with renal failure has had direct impact on m a n a g e m e n t and clinical outcome. PTH is now appreciated to have important anabolic properties in bone that may have implications for its use as a therapeutic agent in osteoporosis. This incomplete summary argues persuasively for how fast and how far this field has advanced. This is not to say that we were in the dark ages before Aurbach isolated parathyroid h o r m o n e . Certainly, it was Fuller Albright who in 1948 correctly pointed out that "back in the dark ages of endocrinology, in the early 1920s, hyperparathyroidism was an u n k n o w n fact." It was also Albright who r e m i n d e d us of the work of Sandstrom, who in 1880, 40 years before the first known cases of hyperparathyroidism wrote, "The existence of a hitherto u n k n o w n gland in animals that have so often been a subject of anatomical examination called for a t h o r o u g h approach to the region a r o u n d the thyroid gland even in man. Although the probability of finding something hitherto unrecognized seemed so small that it was exclusively with the purpose of completing the investigations rather than with the hope of finding something new that I began a careful examination of this region, so m u c h the greater was my astonishment therefore when in the first individual I examined, I found on both sides at the inferior b o r d e r of the thyroid gland an organ of the size of a small pea, which j u d g i n g from its exterior, did not appear to be a lymph gland, or an accessory thyroid gland, and u p o n histological examination showed a rather peculiar structure." The first chapters on the parathyroids were indeed written by Albright and a band of spectacular clinical investigators of the 1920s, 1930s, and 1940s. These chapters are recorded in the Albright and Reifenstein classic The Parathyroid Glands and Metabolic Disease. We r e c o m m e n d this insightful 45-year-old book as important and provocative reading. The Parathyroids is designed to follow the Albright and Reifenstein text. Certainly all endocrinology reference texts routinely include a section on the subject matter of this book. O t h e r texts that are more focused on calcium metabolism provide more information than the standard endocrinology texts on the parathyroids. However, there is no book that is exclusively devoted to a comprehensive examination of basic and clinical concepts

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/ Preface to the First Edition of the parathyroids. As indicated by the size and scope of The Parathyroids, it is clear that a book devoted to this subject is worthy and long overdue. It is time for such a book to stand on the endocrine shelf near its anatomical partner, the thyroid gland, which in Werner and Ingbar's The Thyroid has had its own literary repository since 1955. This book is intended for students, teachers, practitioners, and investigators of this field. It covers in a current and concise yet complete m a n n e r virtually all that we know about the parathyroids. Thus, it is both a basic and a clinical text. The 51 chapters are divided into a presentation of basic knowledge of the parathyroids and the clinical disorders associated with dysfunction of these glands. Section I, Basic Concepts of the Parathyroids, consists of 22 chapters. Chapters 1-7 cover the embryology, anatomy, and pathology of the parathyroid glands; calcium homeostasis; regulation of parathyroid hormone by dietary calcium and vitamin D; anabolic and catabolic effects of parathyroid hormone; cellular actions of parathyroid hormone on osteoblast and osteoclast function; autocrine and paracrine functions of parathyroid tissue; and the chemistry and biology of parathyroid h o r m o n e secretory protein. In Chapters 8-16, parathyroid hormone is considered with respect to the discovery by Aurbach of one of its second messengers, cAMP; regulation of it biosynthesis and metabolism; the parathyroid hormone gene; structure-function analysis of parathyroid hormone and parathyroid hormone-related protein; measurement of parathyroid hormone in the circulation; parathyroid hormone and parathyroid hormone-related protein as polyhormones; receptors for parathyroid hormone and parathyroid hormone-related protein; G proteins as transducers of parathyroid hormone action; biochemical mechanisms of parathyroid hormone action. The book proceeds in Chapters 17-20 to a consideration of PTHrP: its structure, physiological processing, and actions; its causative role in hypercalcemia of malignancy; it skeletal and renal actions; and its measu r e m e n t in the circulation. Other causes of hypercalcemia, besides PTHrP, and the management of PTH and PTHrP-dependent hypercalcemia complete this section (Chapters 21-22). Section II, Clinical Concepts of the Parathyroids, begins with an 18-chapter section on primary hyperparathyroidism (Chapters 23-40). This segment is a full exploration of the hyperparathyroid state from theoretical aspects of parathyroid cell growth to the molecular basis of primary hyperparathyroidism. A discussion of the spectrum of parathyroid tumors leads to a consideration of its modern clinical presentations and the course of primary hyperparathyroidism. The change in clinical presentation of primary hyperparthyroidism from a disease of bones and stones and groans to a relatively asymptomatic disorder does not lose sight of a major clinical complication, nephrolithiasis, which is still seen in patients on a regular basis. A chapter devoted to newer markers of bone turnover in primary hyperparathyroidism is followed by a discussion of the histomorphometric features of the disease. Medical and surgical management of primary hyperparathyroidism and the role of preoperative localization techniques are covered completely. Unusual manisfestations of primary hyperparathyroidism include separate discussions of parathyroid carcinoma and acute primary hyperparathyroidism. The MEN syndromes I and II focus on the parathyroids, as does the chapter on familial hypocalciuric hypercalcemia. In Chapters 41 and 42, the parathyroids in renal disease are reviewed with respect to pathophysiology, clinical profile, and management. Chapters 43-47 cover the hypoparathyroid states with respect to differential diagnosis, autoimmune etiologies, molecular genetics, and a special consideration of the clinical, biochemical, and molecular features of pseudohypoparathyroidism. A separate chapter is devoted to the therapy of hypoparathyroidism. The last four chapters of the book, Chapters 48-51, cover unusual aspects of the parathyroids: parathyroid function in the pathophysiology of osteoporosis and parathyroid hormone as a potential therapy of osteoporosis. Parathyroid functions in Paget's disease of bone and in magnesium deficiency complete the treatise. We recognize that few readers will read this book from cover to cover, although many of the chapters are closely interrelated. In order to permit virtually all chapters to "stand alone" but also to be connected to the rest of the book, we have liberally included cross-references to other chapters where appropriate. The reader can thus easily refer to other chapters for more information on a given subject. This design also necessarily calls for some interdigitation between chapters so that the reader in not always required to refer to another chapter but, rather, can get a brief summary in the chapter being read of an area that is covered more completely elsewhere.

Preface to the First Edition If it was true that we n e e d e d a b o o k on this subject five years ago w h e n the idea was first germinating, why did it take so long to get it d o n e a n d what was the impetus for finally accomplishing the task? T h e first of these two questions has a simple answer. Ideas for books are r a t h e r easy to develop but it is quite a n o t h e r m a t t e r to mobilize an army of over 90 experts to bring that idea to reality. As is true for so m a n y things, this idea was p u t on the shelf to be a d m i r e d for its own sake a n d to be c o m p l e t e d later. T h e mobilizing impetus a n d the inspiration for this effort eventually did come. Regrettably, it came in the f o r m of a tragic event in o u r lives, the death of Gerald D. Aurbach. T h e death of Jerry on a street in Charlottesville, Virginia, on N o v e m b e r 4, 1991, was r a n d o m , senseless, a n d violent. At 64 years of age, J e r r y was still alive with love for his work, his family, a n d his friends. In a m o m e n t , we suddenly lost a m a n who g u i d e d the very definition of o u r field for over 30 years. We lost a m a n who was o u r t e a c h e r a n d o u r friend. We lost a brilliant scientist who was involved in most of the major advances in this field over the past three decades. We lost a m a n who trained an e x t r a o r d i n a r y n u m b e r of us for successful careers in basic a n d clinical investigation of the parathyroids. We lost a gentle m a n who consistently b r o u g h t out the best of us. A s u m m a r y of the m a n y a c c o m p l i s h m e n t s that came f r o m Jerry's laboratory a n d the trainees, collaborators, a n d associates who worked with h i m is depicted in the time-line on pages xxvi-xxvii of this book. It is an e x t r a o r d i n a r y legacy. T h e two IN MEMORIA, by Bilezikian (Journal of Bone and Mineral Research 7:ix-x, 1992) a n d by Potts a n d Spiegel (Journal of Clinical Endocrinology and Metabolism 75:1386-1388, 1992), speak volumes to his career, to his accomplishments, a n d to his persona. In a flash, the d r e a m shelved in the recesses of consciousness a n d relegated to "when I get to it" b e c a m e an u r g e n t need. The Parathyroids h a d to be written in the m e m o r y a n d h o n o r of Gerald D. Aurbach, a n d it s e e m e d altogether fitting that it be written by those who were close to Jerry. We who knew h i m so well a n d respected h i m so m u c h would write a volume for the field. Virtually all of the principal authors of this text fit into that category. Maurice Attie, who also belongs in this book, was tragically killed in a bicycle accident in Philadelphia only a few m o n t h s after Jerry's death. We r e m e m b e r Maurice a n d wish that he too were still with us. It is e x t r a o r d i n a r y that a b o o k designed to be as c o m p r e h e n s i v e as this could be assembled by a collective a u t h o r s h i p whose scientific roots were established by Jerry. His contributions to this field are r e p r e s e n t e d not only by his science b u t also by his scientific p r o g e n y who are the n e x t g e n e r a t i o n of investigators to study a n d write a b o u t it. We took up this task with time in mind. The Parathyroids h a d to be published with a short lag time because the b o o k is a timely dedication to Jerry's memory. It h a d to be published soon because this field is in "fast forward" a n d if one used the n o r m a l publication time for a b o o k of this m a g n i t u d e , it would r u n the risk of rapidly b e c o m i n g outdated. To the credit a n d thanks to all the authors, virtually all 51 chapters were submitted within a six-month p e r i o d of time. T h e dedication of the authors to this task is gratefully acknowledged by us. We also are grateful to J a s n a Markovac of Raven Press, who h e l p e d to ensure that the process ran as efficiently as possible a n d whose efforts also were i n s t r u m e n t a l in ensuring a rapid t u r n a r o u n d time to final publication.

John P. Bilezikian Robert Marcus Michael A. Levine

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Th e Parathyroids Basic and Clinical Concepts

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Para

thyr o l"ds

Morphology and Pathology

VIRGINIA A. LIVOLSI Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Centeg, Philadelphia, Pennsylvania 19104

INTRODUCTION

The morphologic abnormalities seen in the parathyroid glands are predominantly those related to hyperfunction, i.e., primary hyperparathyroidism. Thus the focus here is on this aspect of parathyroid pathology, because almost all surgical specimens of parathyroid lesions are derived from patients with hyperparathyroidism. Because morphologic abnormalities are an important factor in the surgical treatment of this disease, a review of parathyroid embryologic development, anatomy, and normal histology is included. A brief discussion of parathyroid pathology in h y p o p a r a t h y roidism is also included, as is discussion of the pathology of the glands in humoral hypercalcemia of malignancy.

D E V E L O P M E N T O F P A R A T H Y R O I D GLANDS In the 8- to 10-mm embryo, the parathyroids begin to develop from the third and fourth branchial pouches. The third branchial pouch gives rise to the thymus and the parathyroid complex. The parathyroids migrate to and remain at the lower poles of the thyroid. Thus, in the usual case, the inferior parathyroids migrating with the thymus come to rest below the parathyroid derived from branchial pouch four (1). Embryologic studies in animals have demonstrated that ablation of the ventral half of the third branchial arch leads to nonformation of the upper parathyroid gland (2). Hoxa3 mutant homozygotes show defects in development and migraThe Parathyroids, Second Edition

tion pathways of thymus, thyroid, and parathyroid glands; the molecular events underlying the actions of the Hoxa3 genes remain to be determined (3). The fourth branchial pouch, or the fourth-fifth pharyngeal complex, gives rise to the superior parathyroid glands and via the ultimobranchial body to the parafollicular or C cells in the lateral thyroid. The superior parathyroids lie adjacent to the upper poles of the thyroid.

A N A T O M Y O F P A R A T H Y R O I D GLANDS Both the number and the location of the parathyroid glands vary in normal individuals. Variation in location of the glands can lead to problems during surgical exploration of the neck. For example, there may be difficulty in locating the diseased, abnormal parathyroid tissue in patients with hypercalcemia; conversely, surgery on the neck for other reasons, such as thyroid or laryngeal disease, may inadvertently cause trauma or removal of parathyroid glands because of the normal variability in their anatomic position (1,4-7). A report by Lee et al. indicates that almost 12% of patients undergoing thyroid resection have one parathyroid gland removed inadvertently (8). Although from one to twelve parathyroid glands can be found, (1), 84% of normal adults have four parathyroids (4). From 1 to 7% of adults have three glands and 3 to 13% have five glands (1,4-7). The variability of the location of the parathyroid glands is usually greater in the lower parathyroids. The superior parathyroids may be found close to the thyroid capsule or actually within Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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the thyroid capsule, but they may also be located behind the pharynx or the esophagus, lateral to the larynx, or behind any part of the thyroid. The lower glands, which usually lie near the lower pole of the thyroid, may be found behind the thyroid, in the paratracheal area, or close to or within the thymus in the superior mediastinum. The glands tend to be bilaterally symmetrical in location, with approximately 75% of cases showing such symmetry (4,5). The parathyroid glands measure between 2 and 7 m m in length, 2 and 4 m m in width, and 0.5 and 2 m m in thickness. They are reniform, soft, and brown to rust in color. However, color varies with fat content, the degree of vascular congestion, and the n u m b e r of oxyphil cells present (5,9,10). Parathyroid tissue weight varies with sex, race, and overall nutritional status of the individual (11). The combined weight of all parathyroid tissues in a normal adult male is a r o u n d 120 mg; in females combined tissue weight is around 145 mg. Weights of individual glands range from 3 to 75 mg, with averages of a r o u n d 35 to 55 mg (5,9-11).

HISTOLOGY OF PARATHYROID GLANDS Microscopic examination shows that each parathyroid gland is invested by a thin connective tissue capsule that extends into the parenchyma as fibrous septae, dividing the gland into lobules. A rich capillary vascular network is s u r r o u n d e d by nests and cords of

parenchymal cells. Small clusters of cells are interspersed with foci of adipose tissue (Fig. 1). However, there is variability in the location and interrelationships between the fat and the parenchymal cells in the parathyroid gland, so that biopsies from specific areas of the parathyroid may be predominantly fat, predominantly parenchyma, or a mixture of these two. In the adult, the parathyroid is composed of chief and oxyphil cells, fibrous stroma that is usually thin and delicate, and variable amounts of fat. Historically, the ratio of 50:50 cells:fat has been accepted as normal for adults. However, numerous studies have indicated that individuals dying without h o r m o n a l dysfunction of any type have parathyroids in which the stromal fat content is significantly less than 50% in most cases. It may be as little as 10%. In fact, n u m e r o u s studies (11-14) have shown that an approximately 17% fat content is normal in an adult parathyroid gland. Indeed, cell:fat ratios in terms of stromal fat serve little purpose in microscopic interpretation of functional status. Densitometry measurements concur, indicating that parenchymal cell mass accounts for 74% of parathyroid weight (4,14). The cells that make up the parathyroid glands include chief cells, oxyphils, and clear cells (Fig. 2). These variable cell groups probably represent different morphologic expressions of the same parenchymal cell. The chief cell is polyhedral in shape, poorly outlined, and measures 6 to 8 nm in diameter (15). It has an amphophilic to slightly eosinophilic cytoplasm, a

FIG. 1 Normal parathyroid gland adjacent to thyroid (lower left). Note the cellularity of the gland and the relative paucity of fat (f clear spaces) in this section. Hematoxylin and eosin, x50.

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FIG. 2 Parathyroid chief cells (dark cells), oxyphils (larger cells), and fat in normal adult gland. Hematoxylin and eosin, × 150.

sharp nuclear membrane, and well-defined, abundant nuclear chromatin. Clear cells represent chief cells in which there is an excessive amount of glycogen in the cytoplasm. Oxyphils, which tend to be found initially around the time of puberty and rarely in childhood, apparently increase in n u m b e r with age and may form small micro-

scopic nodules. The oxyphil cell in the parathyroid, as in other organs, is large, measuring approximately 10 nm in diameter, has a well-demarcated cell membrane, and has eosinophilic granular cytoplasm (Fig. 3). This reflects a marked mitochondrial content (9,10,15). In contrast to stromal fat content, intracellular fat content may be helpful in defining functional status. Thus, in

FI6.3 Cluster of oxyphils in normal parathyroid gland. Hematoxylin and eosin, x250.

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chief cells, which are the predominant cells in the parathyroid, intracellular fat, i.e., intracytoplasmic fat, is found in the overwhelming majority of cells in the euparathyroid state (approximately 80% of cells) (11-14). Ultrastructurally, the chief cells undergo a cyclic process during synthesis and secretion of parathyroid hormones, with the hormone being synthesized on Golgi apparatus-associated membrane-bound secretory granules. These cells eventually secrete these particles of h o r m o n e into the surrounding milieu. Little lipid is present in the active parathyroid cell, which in the euparathyroid state is approximately 20% of the parenchymal cell population (15).

DISEASES OF THE PARATHYROIDmPATHOPHYSIOLOGY Surgical pathologists dealing with the parathyroids almost always evaluate parathyroid tissue in patients who have hypercalcemia. The predominant effect of parathyroid hormone, as noted, is to increase serum calcium. The usual clinical problem is not to distinguish normal from hypercalcemic patients, but rather to distinguish those who have hypercalcemia caused by hyperparathyroidism from those who have hypercalcemia arising from other causes. Primary hyperparathyroidism is defined as the disease in which, in the absence of a known stimulus, one or more parathyroid glands secrete excess parathyroid hormone, producing hypercalcemia. Serum calcium ranges from 11 to 18 mg/dl, with most asymptomatic patients found in the lower end of the spectrum (16). The prevalence of primary hyperparathyroidism in the United States is estimated to be 1-5 cases per 1000 adults (16). The etiology of the disease is unknown. In a certain number of individuals a history of irradiation to the head and neck may be found, although the magnitude and significance of this association are not clear (17,18). Prinz et al. (18) found that 67% of patients in their series with combined thyroid and parathyroid tumors gave a history of irradiation. In some patients, genetics plays a role [multiple endocrine neoplasia (MEN) syndromes; see also Chapter 19] (19-25). Mutations of the M E N - 1 gene (menin) have been identified in some irradiated patients with hyperparathyroidism (22-24).

Pathology of the Parathyroid Glands in Primary Hyperparathyroidism Three subgroups of pathologic lesions are found in patients with primary hyperparathyroidism: adenoma, multigland hyperplasia, and, rarely, carcinoma.

Parathyroid Adenoma The parathyroid adenoma is responsible for hyperparathyroidism in 30-90% of cases. The wide range of variation indicates both pathologic interpretation and surgical interpretation of the disease (9,10,26-29). Most researchers believe that 75-80% of primary hyperparathyroidism is caused by a solitary adenoma (9,10,26-29). Evidence supports a clonal origin for parathyroid adenomas. Although older studies using protein polymorphisms indicated that parathyroid adenomas were polyclonal (30,31), many studies (25,32-36) using the techniques of molecular biology show that sporadic parathyroid lesions are monoclonal neoplasms. Grossly, parathyroid adenomas tend to be located more commonly in the lower glands than in the upper glands. Typically, the adenoma is an oval red-brown nodule that is smooth, circumscribed, or encapsulated. The lesion, which often replaces one parathyroid gland, may show areas of hemorrhage and, if large, cystic degeneration. Occasionally in small adenomas, a grossly visible rim of normal yellow-brown parathyroid tissue may be seen. Weights of adenomas vary from 300 mg to several grams. The size ranges from 1 to over 3 cm (9,10,27,29). Microscopically, adenomas are usually encapsulated lesions composed of parathyroid chief cells arranged with a delicate capillary network, recapitulating endocrine tumors in general (Figs. 4 and 5). Rarely, lobules are seen, and sometimes nodules may be formed. Stromal fat is usually absent. Unless they are very large, about 50% of adenomas will appear to have a normal rim or even atrophic parathyroid tissue outside the adenoma capsule. The cells in the rim tend to be smaller and more uniform, with stromal and cytoplasmic fat abundant in the rim but absent in the adenoma (9,10,27,29,37). However, the absence of a rim does not preclude the diagnosis of adenoma, because large tumors may have overgrown the preexisting normal gland or the rim may have been lost during sectioning. In large tumors, zones of fibrosis may be found in addition to hemorrhage, cholesterol clefts, and hemosiderin, as well as occasional areas of calcification. Rarely, lymphocytes will be noted within an adenoma (38). Thymic tissue may be found in association with an adenoma or an adenoma may be found within the thymus. There may be atypical cells in an adenoma. Most cells comprising the lesion have relatively small, uniform, dark nuclei. Usually focally, bizarre multinucleated cells with dark, crinkled nuclei can be seen. These nuclei probably represent degenerative changes rather than malignant or premalignant potential. It has been stated that mitotic activity is never found in a parathyroid adenoma and that such activity should suggest the

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FIG. 4 Parathyroid adenoma. Almost all chief cells; no fat present. Hematoxylin and eosin, x200.

possibility of a malignant neoplasm. This particular diagnostic area, however, is fraught with difficulty and is u n d e r debate at the present time. The n o n a d e n o m a tous glands in a patient with a parathyroid a d e n o m a may show normal to increased cytoplasmic fat content and normal weight (9,10,13,14). In about 10% of cases microscopic examination of biopsies from "normal" glands will show areas of hypercellularity, so-called microscopic hyperplasia. Although

this may represent a true parenchymal cell increase, the difficulty in defining "normal," or more likely sampling errors, probably accounts for this (39-41). Oxyphilic or oncocytic adenomas do occur and can function. These tumors tend to be larger than chief cell adenomas and the serum calcium levels tend to be minimally elevated (42-47). Because of the embryologic migration patterns, parathyroid adenomas can occur in ectopic locations.

FIG. 5 Parathyroid adenoma with follicle formation; rarely, this is mistaken for thyroid tissue, especially on frozen section. Hematoxylin and eosin, x300.

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Thus, when hyperparathyroidism occurs in such an individual and no a d e n o m a or abnormal glands are identified in the neck, ectopic locations that should be considered include the mediastinum, with or without associated thymic tissue, b e h i n d the esophagus, or even intrathyroidal (48-54). Double adenomas, if they occur, are very rare (55-57). Most patients who have socalled double adenomas will, over a period of time, have recurrent hyperparathyroidism and in fact have four-gland hyperplasia. The diagnosis of double aden o m a can be made only if two glands are enlarged and histologically abnormal; the remaining glands are normal, there is no family history of parathyroid disease, and p e r m a n e n t cure of hypercalcemia follows excision of only two enlarged glands (55-65). Indeed, heterogeneous size of four glands in primary hyperplasia may account for some cases interpreted as "double adenomas" (65).

Primary Parathyroid Hyperplasia Primary parathyroid hyperplasia is divided into two main groups: chief cell hyperplasia, which is common, and water clear cell hyperplasia, which occurs less commonly (9,10,29). Chief cell hyperplasia accounts for 15% of hyperparathyroidism in most series, although some reports indicate that about half of primary hyperparathyroidism is produced by hyperplasia. The reasons for this probably lie in discrepancies in pathologic interpretation. About 30% of patients with chief cell hyperplasia have familial hyperparathyroidism or one of the syndromes of multiple endocrine neoplasia (9,10,29,66-73). Grossly, all four glands are enlarged equally or nonequally. If unequal in size, the lower glands are usually larger. Occasionally one gland will be much larger than the others and will convey the surgical impression of an adenoma. The weight of all four glands ranges from 150 mg to over 20 g, but usually is in the range of I to 3 g (9,10). Microscopically, diffuse chief cell hyperplasia may be characterized by solid masses of cells with minimal to no fat. Usually almost all cells are chief cells, with rare oxyphils. Nodular or pseudoadenomatous hyperplasia consists of circumscribed nodules of chief, transitional, or oxyphil cells, each nodule devoid of fat, and with there being little fat in the intervening stroma. Usually in hyperplasia there is no rim of normal tissue. Bizarre nuclei are rarely found in primary hyperplasia. Mitoses may occasionally, however, be identified (9). Therapy in this disease is directed to the removal of all parathyroid tissue, with or without autotransplantation. Clear cell (water clear cell) hyperplasia is very rare and is the only condition of the parathyroid in which the superior glands are larger than the lower. Total weights of such parathyroids always exceed 1 g and usu-

ally range from 5 to 10 g. The glands are irregular and show pseudopods and cysts; a distinct mahogany color is seen grossly. Histologically, the glands are composed of diffuse sheets of clear cells without any mixture of other cell types. No rim is present (9,10,74-76). An interesting association of clear cell hyperplasia with the blood group O allele has been reported (77).

Parathyroid Carcinoma Parathyroid carcinoma accounts for approximately 1% of primary hyperparathyroidism (78-94). There is clinically an unusual scenario with an almost equal sex ratio, which is u n c o m m o n in parathyroid adenomas and usual hyperplasias, in which women predominate. The incidence of benign hyperparathyroidism appears to increase with age; however, patients with parathyroid carcinoma tend to be somewhat younger and are almost always symptomatic with very high levels of serum calcium. Very rarely, parathyroid carcinoma can occur in the setting of familial endocrine disease (95-99) or as a complication of secondary parathyroid hyperplasia (100-104). Most of the latter cases occur in patients with renal failure (12 cases were d o c u m e n t e d in 1999) (104). Clinically, patients with parathyroid carcinoma show high calcium levels (up to 15 m g / d l ) . Many have polyuria, polydypsia, nausea, vomiting, weight loss, and constipation. They may also have bone pain, renal stones, and other symptoms related to hypercalcemia. An important clinical clue is the presence of a palpable mass in the neck on physical examination. The mass may be clinically thought to be an a d e n o m a of the thyroid (9,10,78-94). Parathyroid carcinomas tend to be large tumors (average weight 12 g) and characteristically show a histology with trabecular a r r a n g e m e n t of tumor cells divided by thick fibrous bands, with capsular and blood vessel invasion in the presence of mitotic figures (Fig. 6) (9,10,105). The cytology may be clear or rarely oxyphilic; nuclear atypia may be seen or may be absent (9,10,102,106). Because mitotic figures are almost never found in a benign parathyroid adenoma, their presence in tumor cells should raise the suspicion of malignancy. However, this has been called into question and parathyroid tumors with mitotic activity may in fact be benign. As a note of caution, long-term follow-up in the reported series is quite limited, and there is a long natural history to parathyroid carcinoma, so the answers are not all in yet (107,108). Mitotic activity in secondary hyperparathyroidism is not to be equated with malignancy, and mitotic activity may occasionally be found in primary hyperparathyroidism as well (9). The presence of capsular invasion is not equated with malignancy because large parathyroid adenomas

PARATHYROIDS: MORX'HOLOGYAND PATHOLOGY /

7

FI6. 6 Parathyroid carcinoma; note mitosis (+). This tumor recurred three times locally and eventually metastasized to the lungs. Hematoxylin and eosin, x300.

may have u n d e r g o n e prior hemorrhage, with consequent fibrosis and trapping of tumor cells within the capsule. Vascular invasion is difficult to define except if seen outside the vicinity of the neoplasm. An important clue to the diagnosis of parathyroid carcinoma is the surgical finding of adherence a n d / o r invasion into local structures, which should raise the suspicion of a carcinoma (9,10,78-94,102). Metastases at the time of presentation are unusual, but may be found in the regional lymph nodes. There may also be local invasion into nerves, soft tissue, and the esophagus. Rarely, nonfunctioning parathyroid carcinomas have been described. These lesions tend to be large and composed of clear or oxyphil cells (83,109,110). The prognosis of parathyroid carcinoma is usually one of an indolent malignancy. Metastases may occur in up to one-third of cases and are found in regional lymph nodes, bone, lung, and liver. Many patients survive long periods of time, however. Multiple recurrences are known to occur over a 15- to 20-year period (9,10,78-94,102). The severity of the symptoms due to metastatic disease is directly related to tumor burden, because this is related to parathyroid h o r m o n e p r o d u c e d (111). Some solitary parathyroid tumors show features that suggest carcinoma, such as large size, fibrous bands, etc., but not all of the characteristics of malignancy are present. We use the term "atypical adenoma" for these lesions; short-term follow-up suggests they are benign, but long-term studies are needed in this subset of lesions.

Multiple Endocrine Neoplasia Syndromes The syndromes of MEN-1 (Wermer's syndrome) and MEN-2 (Sipple syndrome) are associated with pathologic changes in the parathyroids. In MEN-l, pathologic changes similar to adenomatous or p s e u d o a d e n o m a t o u s chief cell hyperplasia as described above are found (9,10). I n MEN-2, the parathyroids tend to show a diffuse hyperplasia, but occasionally one gland is involved, suggesting an "adenoma." In this syndrome the hyperparathyroidism is considered to represent a genetically determined event and not a response to hypercalcitoninemia (9,10). Parathyroid abnormalities are m u c h less c o m m o n in other variants of MEN-2 syndromes. Familial hyperparathyroidism shows the pathologic alterations of chief cell hyperplasia similar to Wermer's syndrome; in familial hypercalciuric hypercalcemia, mild parathyroid hyperplasia has been described (9,10).

Unusual Lesions of the Parathyroid

Parathyroid Cysts Cysts of the parathyroid glands are unusual and may present and be misinterpreted clinically as thyroid nodules (112-124). They occur more frequently in women than in men, usually are large, ranging from 1 to 6 cm, and may be located in any parathyroid gland, although most are found in the lower glands. Occasionally they may be found in the mediastinum, mimicking super i o r / a n t e r i o r mediastinal masses (114,121,123).

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1

may be functional or nonfunctional and usually is circumscribed but rarely encapsulated. In unusual examples a rim of normal parathyroid tissue is present at the periphery. In some instances at least one other histologically normal parathyroid has been recognized. Also, in some, there is an unusual myxomatous stroma, and other mesenchymal elements including metaplastic bone may be found. Wolff and Goodman (125) suggest the term "parathyroid adenomas with stromal component." More than three-quarters of the reported cases functioned, although with relatively low levels of hypercalcemia. We have studied a woman presenting with an orbital brown tumor due to a parathyroid lipoadenoma that weighed over 10 g (128).

Grossly, these cysts are almost always unilocular and smooth walled and contain water fluid with a high parathyroid hormone content. Histologically, they are lined by one layer of clear epithelium containing glycogen. The cyst wall is fibrous, with fragments of smooth muscle and nests of normal parathyroid tissue. It is unclear how these cysts arise. Microcysts are found in about half of normal parathyroids and might possibly enlarge by accumulation of secretions, or may fuse and produce grossly visible cysts. The cysts may arise from embryologic remnants of pharyngeal pouches in the neck undergoing cystic degeneration and entrapping portions of parathyroid tissue. Many investigators believe, however, that parathyroid cysts represent degenerated parathyroid adenomas, and in some cases, in fact, that the cysts are associated with hyperparathyroidism (Fig. 7) (117-119). However, this is u n c o m m o n and only a few functional cysts have been reported. It may be that different parathyroid cysts have different origins, although pathologically they resemble one another. Cytologists may encounter parathyroid cysts during attempts to aspirate thyroid nodules (112,124). The cyst fluid can be assayed biochemically for parathyroid hormone to confirm the diagnosis.

Parathyromatosis In rare instances of hyperparathyroidism due to primary hyperplasia, nests of hyperplastic parathyroid cells are found in the neck, outside of hyperplastic glands (129-131). In the individuals for which this has been reported, these nests were discovered at the first neck exploration, so that spillage during prior surgery could be excluded. In each of these patients there was no evidence of malignancy. It has been postulated that during embryologic development nests of pharyngeal tissue containing parathyroid cells might be scattered throughout the adipose tissue of the neck and mediastinum. Normally these nests are inconspicuous. However, in the process of diffuse hyperplasia of the parathyroids, all functioning tissue may become hyperplastic and appear as separate fragments on histologic evaluation.

Lipoadenoma-Hamartoma of the Parathyroid These tumors present as masses that histologically are composed of parathyroid cells arranged in nests, similar to normal parathyroid but intimately associated with large areas of adipose tissue (125-128). The lesion

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FIG. 7 Parathyroid cyst. This tumor presented with hypercalcemia and extended from the lower neck to the upper mediastinum. It was 6 cm in size, but most of the lesion was cyst. However, about 10-15% was solid parathyroid tissue, making this a cystic adenoma. Hematoxylin and eosin, x 150.

PARATHYROIDS: MORPHOLOGYAND PATHOLOGY /

Infarction of Parathyroid Adenomas Twelve d o c u m e n t e d cases (132) of adenomas that spontaneously infarcted have been reported. This phen o m e n o n is associated with remission of hypercalcemia. The etiology of the infarction is unclear in most cases, although some have been associated with the intake of certain drugs that may predispose to vascular damage, thrombosis, or hemorrhage. Therapeutic infarction can also result in cure of the metabolic abnormalities (85).

INTRAOPERATIVE ASSESSMENT OF PARATHYROIDS--THE BANE OF THE SURGICAL PATHOLOGIST In normal parathyroid glands, 80% of the cells are in the nonsecretory phase and contain intracytoplasmic fat (12,13,15). Therefore, is the fat stain useful in distinguishing hyperplasia from adenoma, because all hyperfunctioning glands should be fat depleted? The advocacy of fat stains (Sudan IV or Oil Red O) on parathyroid tissue removed at surgery has come into vogue. The scenario is as follows. A sample of an enlarged parathyroid gland is sent for frozen section and by hematoxylin and eosin stain it is hypercellular with little or no stromal fat. Thus it either represents an a d e n o m a or a hyperplastic gland and is not normal. A biopsy of a second parathyroid is frozen and is normocellular or minimally hypercellular. Fat stain shows a b u n d a n t cytoplasmic fat in the latter biopsy; hence this is a normal gland. The enlarged gland, which shows minimal to no fat, represents an adenoma. Many authors have cautioned, however, that the fat stain cannot be the sole procedure on which to base a diagnosis, because although the fat stain is helpful, it is helpful in only about 80% of cases and must be considered as an adjunctive technique in light of gross findings, gland weight, and size, and cannot be relied on by itself (133-141). We have found it useful to perform a r a p i d (30-second) toluidine blue stain on frozen sections of parathyroid tissue. The intracellular fat is well defined by this stain and it is faster to perform and interpret compared to Oil Red O (Lyle S, et al., unpublished observations, 2000). Another rapid technique that may prove useful for intraoperative assessment is density gradient measurements (142). There is an almost linear relationship between density and parenchymal content of parathyroid tissue and thus such a technique can assess parenchymal cell mass. The technique is to take a sample of the gland and weigh it, and take a small piece from the center and a piece from the rim, determining their densities in a 25% mannitol solution. Abnormal parathyroid tissue sinks because of decreased fat and

9

high parenchymal mass. Wang and Ryder (142) have found that this is a simple test to be used by the surgeon in the operating room for distinguishing normal from abnormal glands. In the intraoperative assessment of parathyroid pathology it cannot be stated strongly enough that there must be close communication between the surgeon and pathologist during the operation. The pathologist needs to be apprised of the gross findings and cannot work in a vacuum. What is r e c o m m e n d e d is as follows: the largest parathyroid gland found is resected in toto, then the pathologist weighs it, measures it, and examines it histologically. If the gland shows diffuse growth of chief cells and perhaps a normal-appearing rim, a lack of fat, and bizarre nuclei, a diagnosis of presumed a d e n o m a can be rendered. If the histology is that of hypercellularity but criteria for a d e n o m a are not seen, biopsy of at least one more gland is needed, and, in fact, in many centers pathologists prefer to have the largest abnormal gland and at least a biopsy of one more gland. Weight ratio of parenchymal cells to fat, and normal or a b u n d a n t intracytoplasmic fat content in the second gland, strongly support that the first gland is an a d e n o m a (133-141). The success rate of identifying parathyroid tissue by frozen section is over 99% (143); distinguishing one-gland from multigland disease is much more problematic.

OTHER TYPES OF HYPERPARATHYROIDISM

Secondary Hyperparathyroidism Secondary hyperparathyroidism is usually due to renal disease and is relatively c o m m o n in the age of hemodialysis and renal transplantation. The role of the surgical pathologist in the evaluation of secondary hyperparathyroidism is basically to identify parathyroid tissue at the time of frozen section to allow for the surgeon to remove portions of this tissue for autotransplantation. Secondary hyperparathyroidism is really no different histopathologically from primary hyperparathyroidism (144-146). Mitotic activity may occasionally be found in such glands. Usually all four glands are enlarged, although one or two glands may be of very great size. Transplantation of parathyroid tissue is successful in the majority of cases and occasionally part of this tissue may be removed if hyperfunction again becomes a problem (147,148). Such lesions will have small nests and islands of vascularized parathyroid tissue growing in muscle or fat, usually having been implanted in the arm (149,150).

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Tertiary Hyperparathyroidism Although the existence of tertiary hyperparathyroidism has been questioned, most authors believe it represents the autonomous function of one parathyroid gland that develops in the face of long-standing secondary hyperparathyroidism (151). The pathology resembles that of secondary hyperparathyroidism, although one of the four glands is usually disproportionately enlarged.

Familial Hyperparathyroidism In addition to the multiple endocrine neoplasia syndromes, in which hyperparathyroidism is often a prominent clinical problem, familial parathyroid hyperplasia without other endocrine lesions has been reported. The lesions in all of these patients resemble those of primary chief cell hyperplasia (9,10,152-154), although a ribbon pattern of cell growth may be prominent in MEN-1.

Familial Hypocalciuric Hypercalcemia Familial hypocalciuric hypercalcemia, inherited as an autosomal dominant gene, is manifested clinically by familial occurrence, moderate to minimally elevated serum calcium, and reduced urinary calcium excretion (see Chapter 41). The parathyroid glands appear normal to mildly hypercellular, and subtotal parathyroidectomy fails to reverse the hypercalcemia. The defect appears not to be in the parathyroid glands (155,156).

SPECIAL STUDIES AND THE PARATHYROID Cytology Because most parathyroid lesions are not palpable, direct biopsy of a parathyroid tumor by fine-needle aspiration (FNA) is unusual. However, on occasion, parathyroid lesions present clinically as thyroid nodules or are large enough to be clinically evident. The FNA features of parathyroid adenoma include cellular fragments of epithelial cells arranged around vascular cores, an organoid or trabecular architecture, and microacini. Parathyroid chief cells contain uniform round nuclei; groups of oxyphilic cells are helpful in defining the tissue as parathyroid. If available, immunostains for parathyroid h o r m o n e may help (157).

Proliferative Markers Attempts at using immunocytochemical markers (158-164) of proliferation index (MIB1 for cell cycle-

associated Ki-67 antigen) for distinguishing between parathyroid adenomas and hyperplasia have met with varied success (159). Whereas statistically significant differences are found between normal (suppressed "rim") parathyroid tissue and hyperfunctioning glands, similar proliferative indices are noted between adenomas and hyperplasias (159,160). Loda et al. (160) identified higher numbers of labeled nuclei in adenomas than in hyperplasias by proliferating cell nuclear antigen (PCNA) immunostaining. The labeling index of individual cases of parathyroid tumors shows so much overlap that it cannot be used to distinguish benign from malignant lesions (161-164).

Flow Cytometry and the Parathyroid Several studies of DNA content have shown that aneuploidy may be found in parathyroid adenomas, and even in hyperplasia, as well as in carcinomas. Approximately 70% of parathyroid carcinomas, 30% of adenomas, and 30-50% of chief cell hyperplasia glands have aneuploid DNA populations (165-171). As in proliferations of other endocrine organs, the finding of aneuploid cell populations does not ensure a diagnosis of malignancy (169-173).

Clonality Modern molecular biology techniques, primarily using restriction fragment-length polymorphisms, have shown that most (if not all) parathyroid adenomas are monoclonal proliferations (25). In addition, about 40% of primary hyperplasias and 60% of secondary hyperplasia (secondary to chronic renal disease) are clonal. Different laboratories utilizing different probes as markers confirm these findings (25,174-176). The biologic meaning of these results is unclear.

Genetics The P R A D 1 oncogene has been implicated in parathyroid tumorigenesis. PRAD1 (for parathyroid adenoma), which encodes cyclin D1, results from a chromosome inversion that occurs as a dominant clonal event in some parathyroid adenomas. The inversion is created by a break in the vicinity of the parathyroid gene on the short arm of chromosome 11 (band 1 lp15), another break in the long arm (band 1 lq13), rotation of the center piece around the axis of the centromere, and rejoining (177). Cyclin D overexpression can be detected immunohistochemically in 18-38% of parathyroid adenomas, and in 91% of carcinomas (178,179). The retinoblastoma (Rb) gene is a tumor suppression gene that has growth inhibitory effects in the cell cycle. Inactivation of the Rb gene has been associated

PARATHYROIDS: MORPHOLOGY AND PATHOLOGY

with loss of an Rb allele by molecular analysis, and immunostaining for Rb protein may assist in the distinction between parathyroid adenomas and carcinomas (179-183). However, caution must be used in interpretation of the results, because some parathyroid carcinomas do not show loss of Rb protein and a few adenomas do (181,182). Studies of parathyroid neoplasms (benign and malignant) have not shown p53 mutations in such lesions (184). In another study of parathyroid tissues, there were significant differences between p27 protein expression in parathyroid hyperplasia, adenomas, and carcinomas, suggesting that this cell cycle protein may be useful in distinguishing between these two conditions (185,186).

HUMORAL HYPERCALCEMIA O F MALIGNANCY, O R ECTOPIC PARATHYROIDISM Hypercalcemia without bone metastasis in nonparathyroid malignancies may be found in association with a malignant tumor. Hypercalcemia is relieved by excision of the tumor and returns with its recurrence. This paraneoplastic endocrine syndrome is due in many cases to a peptide that resembles parathyroid hormone but is distinctly different. The factor responsible for the syndrome of humoral hypercalcemia of malignancy, which is due to parathyroid hormone-related protein (PTHrP), is discussed in other chapters in this volume. PTHrP binds to parathyroid h o r m o n e receptors on bone and kidney and mimics the actions of parathyroid hormone. The tumors most commonly associated with this syndrome include squamous carcinomas arising in a number of primary sites, including lung, vulva, esophagus, and head and neck, and clear cell cancers, especially of renal and ovarian origin (187-191). The parathyroid glands appear normal or atrophic histologically.

HYPOPARATHYROIDISM The most common parathyroid pathology found in patients with hypoparathyroidism is four normal glands. Unfortunately, they often have been surgically removed from the patient! Accidental excision of normal parathyroid glands during the course of neck surgery, especially thyroid surgery, is an u n c o m m o n but unfortunately not a rare event (8). In addition to actual excision of the glands, injury to their vascular supply may cause their infarction, or they may be so damaged that they become functionally absent.

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Infiltration Impaired parathyroid function caused by infiltration of parathyroid glands has been described in hemochromatosis, amyloidosis, and metastatic carcinomas. These are all rare causes of hypoparathyroidism (192).

Radiation Rarely, patients are reported who have developed hypoparathyroidism after radioactive iodine treatment for hyperthyroidism. The presumed mechanism is radiation damage to and fibrosis of the parathyroids (193).

Autoimmune Parathyroid Destruction Lymphocytic infiltration of parathyroid tissue, with subsequent autoimmune destruction of the glands, is probably the most common cause of hypoparathyroidism (noniatrogenic cause). It may occur as an isolated event or in association with autoimmune diseases of other endocrine organs, i.e., thyroid, adrenal, or ovary (194-197).

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41. Harrison TS, Duarte B, Reitz RE, et al. Primary hyperparathyroidism: Four to eight year postoperative follow-up demonstrating persistent functional insignificance of microscopic parathyroid hyperplasia and decreased autonomy of parathyroid hormone release. Ann Surg 1981;194:429-437. 42. McGregor DH, Lotuaio LG, Chu LH. Functioning oxyphil adenoma of parathyroid gland. An ultrastructural and biochemical study. A m J Patho11978;92:691-703. 43. Ordonez NG, Ibanez ML, MacKay B, et al. Functional oxyphil cell adenomas of parathyroid gland: Evidence of hormonal activity in oxyphil cells. A m J Clin Patho11982;78:681-689. 44. Rodriquez FH, Sarma DE Lunseth JH, Guileyardo JM. Primary hyperparathyroidism due to an oxyphil adenoma. Am J Clin Pathol 1983;80:878-880. 45. Bedetti CD, Dekker A, Watson CG. Functioning oxyphil cell adenoma of the parathyroid gland: A clinicopathologic study of ten patients with hyperparathyroidism. Hum Pathol 1984;15:1121-1126. 46. Jones SH, Dietler E Oxyphil cell adenoma as a cause of hyperparathyroidism. Am J Surg 1981 ;141:744-745. 47. Baloch ZW, LiVolsi VA. Oncocytic lesions of the neuroendocrine system. Semin Diagn Patho11999;16:190-199. 48. Nathaniels EK, Nathaniels AM, Wang CA. Mediastinal parathyroid tumors: A clinical and pathological study of 84 cases. Ann Surg 1970;171:165-170. 49. Russell CE Edis AJ, Scholz DA, et al. Mediastinal parathyroid tumors: Experience with 38 tumors requiring mediastinotomy for removal. Ann Surg 1981;193:805-809. 50. Russell CF, Grant CS, vanHeerden JA. Hyperfunctioning supernumerary parathyroid glands: An occasional cause of hyperparathyroidism. Mayo Clin Proc 1982;57:121-124. 51. Edis AJ, Purnell DC, vanHeerden JA. The undescended "parathymus": An occasional cause of failed neck exploration for hyperparathyroidism. Ann Surg 1979;190:64-68. 52. Sloane JA, Moody HC. Parathyroid adenoma in submucosa of esophagus. Arch Pathol Lab Med 1978;102:242-243. 53. Spiegel AM, Marx SJ, Doppmann JL, et al. Intrathyroidal parathyroid adenoma or hyperplasia. JAMA 1975;234:1029-1033. 54. Kobayashi T, Man IM, Shin E, et al. Hyperfunctioning intrathyroidal parathyroid adenoma: Report of two cases. Surgery Today 1999;29:766-768. 55. Schwindt WD. Multiple parathyroid adenomas. JAMA 1967;199:945-946. 56. Verdon CA, Edis AJ. Parathyroid "double adenomas." Fact or fiction? Surgery 1981;90:523-526. 57. Harness JK, Ramsbury SR, Nishiyama RH, Thompson NW. Multiple adenomas of the parathyroids; do they exist? Arch Surg 1979;114:468-474. 58. Seyfar AE, Sigdestad JB, Hirata RM. Surgical considerations in hyperparathyroidism: Reappraisal of the need for multigland biopsy. A m J Surg 1976;132:338-340.

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109. Merlano M, Conte P, Scarsi P, et al. Nonfunctioning parathyroid carcinoma. A case report. Tumori 1985;71:193-196. 110. Yamashita H, Noguchi S, Nakayama I, et al. Light and electron microscopic study of nonfunctioning parathyroid carcinoma. Acta PatholJpn 1984;34:123-132. 111. Zisman E, Buckle RM, Deftos LJ, et al. Production of parathyroid hormone by metastatic parathyroid carcinoma. Am J Med 1971 ;45:1068:619-623. 112. Wang CA, Vickery AL, Maloof E Large parathyroid cysts mimicking thyroid nodules. Ann Surg 1972;175:448-453. 113. Ginsberg J, Young JEM, Walfish PG. Parathyroid cysts. JAMA 1978;240:1506-1507. 114. Thacker WC, Wells VH, Hall ER. Parathyroid cysts of the mediastinum. Ann Surg 1971;174:969-975. 115. Hoehn JG, Beahrs OH, Woolner LB. Unusual surgical lesions of the parathyroid gland. A m J Surg 1969;118:770-778. 116. Troster M, Chiu HE McLarty TD. Parathyroid cysts: Report of a case with ultrastructural; observations. Surgery 1978;83:238-242. 117. Earll JM, Cohen A, Lundberg GD. Functional cystic parathyroid adenoma. Am J Surg 1969; 118:100-103. 118. Albertson DA, Marshall RB, Jarman WT. Hypercalcemic crisis secondary to a functioning parathyroid cyst. Am J Surg 1981;141:175-177. 119. Clark OH. Hyperparathyroidism due to primary cystic parathyroid hyperplasia. Arch Surg 1978;113:748-750. 120. SilvermanJE Khazanie PG, Norris T, Fore WW. Parathyroid hormone (PTH) assay of parathyroid cysts examined by fine needle aspiration biopsy. A m J Clin Patho11986;86:708-776. 121. Marco V, Carrasco MA, Marco C, Bauza A. Cytomorphology of a mediastinal parathyroid cyst. Acta Cyto11983;27:688-692. 122. Gough IR. Parathyroid cysts. Aust N Z J Surg 1999;69:404-406. 123. Shields TW, Immerman SC. Mediastinal parathyroid cysts revisited. Ann Thorac Surg 1999;67:581-590. 124. Shi B, Guo H, Tang N. Treatment of parathyroid cysts with fine needle aspiration. Lancet 1999;2:797-798. 125. Wolff M, Goodman EN. Functioning lipoadenoma of supernumerary parathyroid gland in the mediastinum. Head Neck Surg 1980;2:302-307. 126. Grimelius L, Johansson H, Lindquist B. A case of unusual stromal development in a parathyroid adenoma. Acta Chir Scand 1972; 138:628-629. 127. Ober WB, Kaiser GA. Hamartoma of the parathyroid. Cancer 1958; 11:601-606. 128. Perosio P, Brooks JJ, LiVolsi VA. Orbital brown tumor as initial manifestation of parathyroid lipoadenoma. Surg Pathol 1988;1:77-82. 129. Reddick RL, Costa JC, Marx sJ. Parathyroid hyperplasia and parathyromatosis. Lancet 1977; 1:549. 130. Fitko R, Roth SI, Hines JR, et al. Parathyromatosis in hyperparathyroidism. Hum Patho11990;21:234-237. 131. Kollmorgen CF, Aust MR, FerreiroJA, et al. Parathyromatosis: A rare yet important cause of persistent or recurrent hyperparathyroidism. Surgery 1994;116:111-115. 132. Kovacs KA, Gay JDL. Remission of primary hyperparathyroidism due to spontaneous infarction of a parathyroid adenoma: Case report and review of the literature. Medicine 1998;77:398-402. 133. Roth SI, Wang CA, Potts JT. The team approach to primary hyperparathyroidism. Hum Pathol 1975;6:645-658. 134. LiVolsi VA, Hamilton R. Introperative assessment of parathyroid gland pathology. A common view from the surgeons and the pathologist. A m J Clin Pathol 1994;102:365-373. 135. Dufour DR, Durkowski C. Sudan IV staining: Its limitations in evaluating parathyroid functional status. Arch Pathol Lab Med 1987;106:224-227.

136. King DT, Hirose FM. Chief cell intracytoplasmic fat used to evaluate parathyroid disease by frozen section. Arch Pathol Lab Med 1979;103:609-612. 137. Kasden EJ, Cohen RB, Rosen S, Silen W. Surgical pathology of hyperparathyroidism: Usefulness of fat stains and problems in interpretation. Am J Surg Pathol 1981 ;5:381-384. 138. Ljungberg O, Tibblin S. Perioperative fat staining of frozen sections in primary hyperparathyroidism. AmJPatho11979;95:633-642. 139. Dekker A, Watson CG, Barnes EL. The pathologic assessment of primary hyperparathyroidism and its impact on therapy: A prospective evaluation of 50 cases with oil-red-O stain. Ann Surg 1979;190:671-675. 140. Monchik JM, Farrugia R, Teplitz C, Brown S. Parathyroid surgery: The role of chief cell intracellular fat staining with osmium carmine in the intraoperative management of patients with hyperparathyroidism. Surgery 1983;94:877-886. 141. Bondeson AG, Bondeson L, Ljundberg O, Tibblin S. Fat staining in parathyroid disease mdiagnostic value and impact on surgical strategy. Hum Pathol 1985;16:1255-1263. 142. Wang CA, Ryder SV. A density test for the intraoperative differentiation of parathyroid hyperplasia from neoplasia. Ann Surg 1978;187:63-67. 143. Westra WH, Pritchett DD, Udelsman R. Intraoperative confirmation of parathyroid tissue during parathyroid exploration. Am J Surg Pathol 1998;22:538-544. 144. Roth SI, Marshall RB. Pathology and ultrastructure of human parathyroid glands in chronic renal failure. Arch Intern Med 1969;124:397-407. 145. Malmaeus J, Grimelius L, Johansson H, et al. Parathyroid pathology in hyperparathyroidism secondary to chronic renal failure. Scan J Urol Nephrol 1984;18:75-84. 146. Akerstrom G, Malmaeus J, et al. Histological changes in parathyroid glands in subclinical and clinical renal disease. ScandJ Urol Nephro11984; 18: 75-84. 147. Rattner DW, Marrone GC, Kasdon E, Silen W. Recurrent hyperparathyroidism due to implantation of parathyroid tissue. Am J Surg 1985;149:745-748. 148. Akerstrom G, Rudberg C, Grimelius L, Rastad J. Recurrent hyperparathyroidism due to preoperative seeding of neoplastic or hyperplastic parathyroid tissue. Acta Chir Scand 1988;154-219. 149. Jansson S, Tisell LE. Autotransplantation of diseased parathyroid glands into subcutaneous abdominal adipose tissue. Surgery 1987;101:549-556. 150. Max MH, Flint LM, Richardson JD, et al. Total parathyroidectomy and parathyroid autotransplantation in patients with chronic renal failure. Surg Obstet Gyneco11981;153:177-180. 151. Krause MW, Hedinger CE. Pathologic study of parathyroid glands in tertiary hyperparathyroidism. Hum Pathol 1985;16:772-784. 152. Jackson CE, Norum RA, Boyd SB, et al. Hereditary hyperparathyroidism and multiple ossifying jaw fibromas: A clinically and genetically distinct syndrome. Surgery 1990;108:1006-1013. 153. Mallette LE, Malini S, Rappaport ME Kirkland JL. Familial cystic parathyroid adenomatosis. Ann Intern Med 1987;107:54-60. 154. Harach HR, Jasane B. Parathyroid hyperplasia in multiple endocrine neoplasia type 1. Histopathology 1992;20:305-313. 155. Law WM, Carney JA, Heath H. Parathyroid glands in familial benign hypercalcemia (familial hypocalciuric hypercalcemia). A m J M e d 1989;76:1021-1026. 156. Thorgeirsson U, Costa J, Marx SJ. The parathyroid glands in familial hypocalciuric hypercalcemia. Hum Pathol 1981; 12:229-237. 157. Abati A, Skarulis MC, Shawker T, Solomion D. Ultrasoundguided fine needle aspiration of parathyroid lesions. A mor-

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CHAPTER2 Parathyroid H o r m o n e Biosynthesis and Metabolism

HENRY M. KRONENBERG, E RICHARD BRINGHURST, GINO V. SEGRE, AND JOHN T. POTTS, JR. Endocrine Unit,

Massachusetts General Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114

BI O SYNTHESIS OF PARATHYROID H O R M O N E

INTRODUCTION The parathyroid h o r m o n e gene has many jobs. It must encode a peptide that can bind to and activate receptors on target tissues. Equally importantly, the a m o u n t of p a r a t h y r o i d h o r m o n e (PTH) p r o d u c e d must be carefully controlled to maintain the blood level of calcium within a narrow range. Nature's solution to these problems has involved the specific synthesis of PTH primarily in the parathyroid chief cell, a cell designed to sense the blood level of calcium. In the chief cell, synthesis and secretion of the h o r m o n e can be carefully regulated. Furthermore, the structure of the h o r m o n e is designed for rapid metabolic degradation, even in the absence of receptor binding. In this way, the rapid turnover of the h o r m o n e can assure that blood levels of h o r m o n e change quickly in response to changes in h o r m o n e secretory rate. This rapid metabolism of h o r m o n e is required of a system designed to respond quickly to sudden changes in the amounts of calcium entering and leaving the bloodstream. Studies over the past two decades have shown that the sequences of PTH and its precursors are designed to steer the h o r m o n e through the chief cell's secretory pathway, to direct the h o r m o n e ' s binding to receptors, and to assure rapid metabolism of the hormone. More recent studies have begun to unravel the mechanisms whereby synthesis of PTH is regulated in the chief cell. Descriptions of the structure of the PTH gene and a summary of the current understanding of how this structure allows the gene to accomplish its multiple functions are presented in this chapter. The Parathyroids, Second Edition

PTH is synthesized as part of the larger precursor molecule, preproparathyroid h o r m o n e (preproPTH). Only trace amounts of this full-length precursor are found in parathyroid chief cells, because the "pre," or signal, sequence is cleaved from the amino terminus while the protein is being synthesized (see Fig. 1). As the signal sequence emerges from the ribosome, it binds to a signal recognition particle, an RNA-protein complex that recognizes signal sequences on most secreted proteins. The signal recognition particle then binds to a receptor on the rough endoplasmic reticulum (docking protein) and directs the nascent p r e p r o P T H molecule to a protein-lined channel, through which the p r e p r o P T H molecule is transported. A signal peptidase located on the inner surface of the m e m b r a n e of the endoplasmic reticulum then cleaves off the signal sequence, leaving the intermediate precursor, proparathyroid h o r m o n e (proPTH) in the cisternae of the endoplasmic reticulum. P r o P T H then travels via a series of vesicles to and through the Golgi apparatus (see Fig. 2). In the Golgi, the short, aminoterminal "pro" sequence is removed, leaving the mature PTH molecule. PTH is then concentrated in dense core secretory vesicles; these vesicles fuse with the plasma m e m b r a n e and release PTH in response to a decrease in extracellular calcium. The h o r m o n e secreted is predominantly the intact 84-residue PTH molecule, though a variable fraction made up of carboxy-terminal PTH fragments is secreted, as well.

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CHAPTER2

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®

O P

1. SRP

BINDING

O

2. DOCKING PROTEIN BINDING

4. MEMBRANE TRANSPORT, PEPTIDE CLEAVAGE

3. MEMBRANE INSERTION

5. TRANSPORT COMPLETED

,

I

FIG. 1 The signal, or pre, sequence directs the nascent polypeptide to the apparatus for transport across the membrane of the endoplasmic reticulum. SRP, Signal recognition particle.

Function of the Pre (Signal) Sequence The specific sequences of each of the three portions of the preproPTH molecule are responsible for directing the h o r m o n e through the complicated pathway of transport and cleavage. The known preproPTH sequences from h u m a n (1), bovine (2), rat (3), pig (4), chicken (5,6), and dog (7) tissues share a 25-residue pre sequence and a 6-residue pro sequence (see Fig. 3). Each pre sequence contains a hydrophobic stretch of

amino acids preceded by a positively charged residue. The signal sequence ends with a small amino acid at the last and third-to-last positions. These characteristics are typical of most signal sequences. The preproPTH signal sequence was first discovered (8) when parathyroid gland mRNA was translated in a cell-free extract devoid of endoplasmic reticulum. Directed mutations have demonstrated the importance of each of the regions of

AA, AAA

PTH "-------

FIG. 2 Multiple cleavages occur during the intracellular transport of PTH.

PTH BIOSYNTHESISAND METABOLISM /

human bovine porcine rat canine chicken

PRE $ PRO $ -31 -6 MIPAKDMAKVMIVNLAICFLTKSDG KSVKKR MMSAKDMVKVMIVNLAICFLARSDG KSVKKR MMSAKDTVKVMVVNLAICFLARSDG KPIKKR MMSASTMAKVMILMLAVCLLTQADG KPVKKR MMSAKDMVKVMIVMFAICFLAKSDG KPVKKR MTSTKNLAKAIVILYAICFFTNSDG RPMNKR

PTH +i +i0 SVSEIQLMHN AVSEIQFMHN SVSEIQLMHN AVSEIQLPIHN SVSEIQFMHN SVSEMQLMHN

human bovine porcine rat canine chicken

+20 +30 +40 +50 LGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAG SQNPRK L G K H L S S M N R V E W L R K K L Q D V H N F V A L G A S IA Y R D G S S Q N P R K LGKHL S SLNNVEWLRKKLQDVHNFVALGAS IVHRDGG SQRPRK LGKHLASVERMQWLRKKLQDVH FVSLGVQMAAREGSYQNPTK L G K H L S S M N N V E W L R K K L Q D V H N F V A L G A P IA H R D G S S Q N P L K L G E H R H T V E N Q D W L Q M K L Q D V H . . S A L E ...... D A R T Q R P R N

human bovine porcine rat canine chicken

+60 +70 +80 DKADVNVLTKAKSQ KEDNVLVE...SHEKSLGEA K E D N V L V E . . . S H Q K S L G E A .......... D K A D V D V L I K A K P Q K E D N V L V E . . . S H Q K S L G E A .......... D K A A V D V L I K A K P Q K E E N V L V D . . . G N S K S L G E G .......... D K A D V D V L V K A K S Q K E D N V L V E . . . S Y Q K S L G E A .......... D K A D V D V L T K A K S Q KEDIVLGEIRNRRLLPEHLRAAVQKKSIDLDKAYMNVLFKTKP. .

.

.

.

.

.

.

.

.

.

FIG. 3 Amino acid sequences of preproPTH from mammalian and avian species. Residues -31 to - 7 constitute the pre sequences; residues - 6 to -1 constitute the pro sequences. Dots represent residues found in chicken PTH without corresponding residues in the mammalian sequences. Amino acids are indicated by the single-letter code: A, Ala; R, Arg; N, Asn; D, Asp; C, Cys; Q, Gin; E, Glu; G, Gly; H, His; I, lie, L, Leu; K, Lys; M, Met; F, Phe; P, Pro; S, Ser; T, Thr; W, Trp; Y, Tyr; V, Val.

the preproPTH signal sequence for normal signal function (9-11). Further, when a synthetic prepro peptide was added to a cell-free extract, it blocked the transport and cleavage of preproPTH by microsomal membranes (12). Most strikingly, a point mutation was found in the signal sequence of a preproPTH gene in a family with inherited hypoparathyroidism (13). A point mutation at residue 18 changed the cysteine to arginine and thereby inserted a charged residue into the hydrophobic core of the signal sequence. When this mutant preproPTH was expressed in cell-free extracts or in cultured cells, the precursor was inefficiently transported and cleaved (14).

Function of the Pro Sequence The signal sequence of preproPTH, thus, resembles the signal sequences of other secreted proteins and performs the important role of directing the protein across the membrane of the endoplasmic reticulum and into the secretory pathway. The function of the pro sequence is less well established. In all known preproPTH sequences, the pro sequence is six residues long. The first is always positively charged, the third is hydrophobic, and the last two residues are Lys-Arg. This pattern closely resembles that found in rat proalbumin

19

(Arg-Gly-Val-Phe-Arg-Arg) and that predicted to be present in the pro sequence of preproparathyroid hormone-related peptide (Arg-Arg-Leu-Lys-Arg). ProPTH was first discovered as a large PTH-related molecule that was the predominant form of the h o r m o n e found in parathyroid cells after pulse labeling with radioactive amino acids (15,16). Subsequent chase incubations demonstrated that the proPTH was converted to PTH in about 15 minutes; this correlated in time with transport to the Golgi (17). After this time, no trace of the pro peptide or possible fragments could be found in the cell or medium (18). These data strongly suggest that the pro sequence serves an exclusively intracellular function, probably involved in movement through the secretory pathway. Wiren et al. (19) tested this hypothesis by deleting the DNA sequences encoding the pro hexapeptide from cloned cDNA encoding h u m a n preproPTH and by subsequently expressing the cDNA in cell-free protein-synthesizing extracts and in intact rat pituitary GH4 cells. The mutant precursor functioned abnormally in both expression systems. The precursor crossed the membrane of the endoplasmic reticulum inefficiently, and, consequently, the subsequent cleavage of the signal sequence was inefficient. Cells secreted PTH but also secreted a molecule slightly bigger than PTH. Sequence analysis showed that the abnormal protein included the last two residues of the signal sequence. Thus, the removal of the pro sequence resulted in imprecise and inefficient function of the signal sequence. The pro sequence of preproPTH should be considered part of the functional unit responsible for transport and cleavage of the precursor on its entry into the secretory pathway. This result is not surprising. In other precursor proteins, the sequences immediately distal to the signal sequence can affect signal sequence function. One can speculate that the constraints on this region conflict with the constraints on the amino terminus of the mature PTH molecule. The PTH receptor, for example, requires very specific residues at the amino terminus of PTH for subsequent activation of adenylyl cyclase. The experiments of Wiren et al. show that these residues cannot be placed immediately distal to the signal sequence. The pro sequence can be considered a linker region that allows efficient signal sequence function and physically separates the signal sequence from the mature h o r m o n e sequence, which has its own and separate evolutionary constraints. The possibility that the pro sequence has additional functions, such as the promotion of proper folding of the PTH molecule in the endoplasmic reticulum, has not been rigorously examined. The enzyme responsible for cleavage of the pro sequence of proPTH has not yet been characterized, but a n u m b e r of arguments suggest that the protease, furin (or a close relative), is the cleavage enzyme (20).

20

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CI4AeTV.R2

Furin is a subtilisin-like enzyme that is located in the Golgi cisternae of probably all mammalian cells. The enzyme cleaves sequences like the pro sequence of rat proalbumin, which ends in dibasic residues and is preceded by other basic residues. Unlike the related PC2 and PC1 proteases, which are found in cells with secretory granules, furin cleaves precursors in cells like hepatocytes, which have no secretory granules. Cleavage by furin probably explains why proPTH, in contrast to proinsulin, for example, is cleaved normally when the hormone is synthesized in all sorts of cells, from parathyroid chief cells to fibroblasts and kidney cells (21,22). One can only speculate as to why proPTH, which is normally synthesized virtually exclusively in specialized parathyroid chief cells, uses an enzyme designed for cleavage of proteins secreted from nonendocrine cells. One plausible explanation is an evolutionary argument. The parathyroid hormone gene may well be derived from the gene encoding parathyroid hormone-related peptide (PTHrP). The PTHrP gene is widely expressed, both in cells with secretory granules, such as parathyroid chief cells and neurons, and in cells without secretory granules, such as smooth muscle cells. Therefore, it would be expected that the pro sequence of proPTHrP would be designed for cleavage by an enzyme expressed in most cells. The pro sequence of proPTH may well share this property because of its evolutionary heritage, even though proPTH is normally expressed only in cells with a secretory granule apparatus.

Intracellular Roles of the Mature PTH Sequence Like the prepro sequence, portions of the mature PTH molecule serve to facilitate intracellular handling of PTH (23). Shortened versions of preproPTH are not stable in transfected cells. When the h u m a n preproPTH cDNA was modified to encode preproPTH(1-40) (in which the numbers refer to the mature PTH sequence), the signal sequence functioned, and proPTH(1-40) was produced in transfected cells. The proPTH (1-40) was not further cleaved to PTH(1-40), however. Instead, it was degraded intracellularly; no PTH peptides were secreted from the cells. A similar, though less dramatic, defect in secretion was exhibited by preproPTH(1-52). These short precursors were long enough for the signal sequence to direct them into the secretory pathway, but they were unstable and were not transported through the entire pathway. These results may partly explain the role of the carboxy-terminal portion of the PTH molecule. One function of the full 84-residue protein may be to allow stable and efficient transport through the secretory apparatus. Because all secreted peptides are syn-

thesized as rather large precursors, this need for a minimal length of translation product may be a general one for secreted proteins. Of course, this "length" requirement for PTH does not preclude other functions for the carboxy-terminal portion of PTH, such as binding to a distinct PTH receptor (24). Even the 84-residue PTH molecule is not completely stable in the parathyroid chief cell. PTH(1-84) is concentrated in secretory vesicles and granules that contain the proteases, i.e., cathepsins B and H (25,26). This colocalization of proteases and PTH may explain the observation that the hormone secreted by calves in vivo under conditions of hypercalcemia consists largely of carboxy-terminal fragments of PTH (27). Secretion of fragments of PTH was studied in detail by Habener et al. (28) and Chu et al. (29). These workers noted that the degradation of newly synthesized PTH is influenced by the level of extracellular calcium. Few fragments were secreted when the gland was stimulated in vitro by medium containing low levels of calcium. In contrast, most of the hormone secreted under conditions of hypercalcemic suppression consisted of fragments. Thus, calcium regulated the amount of available intact PTH by causing the intracellular degradation of hormone. This effect could have been caused by the activation of a PTH-degrading pathway. Alternatively, the intracellular degradation rate might have been constant; the decrease in total degradation of PTH associated with low calcium levels might simply have resulted from rapid secretion of hormone and the concomitant shorter time of exposure to the intracellular degradation mechanism. Phorbol ester treatment of parathyroid cells in vitro has also been shown to result in the secretion of an increased fraction of PTH fragments, both in high and low calcium concentration conditions (30). Phorbols are either activating a proteolytic mechanism or may be selectively stimulating secretion from secretory granules containing a high proportion of PTH fragments. The physiologic correlate in vivo of this action of phorbol esters has not yet been established. In any case, the parathyroid gland has the capability of varying the fraction of PTH secreted as the biologically active, intact molecule. This seemingly wasteful capability makes it possible for the gland to vary quickly and dramatically the amount of biologically active hormone secreted. This regulatory capability provides a rationale for the intracellular instability of the hormone. To sum up, it can be seen that all portions of the preproPTH molecule have intracellular functions. The prepro region is required for efficient introduction of the hormone to the secretory pathway. The carboxyterminal region of the mature hormone is required for efficient and stable transport of PTH through the secre-

PTH BIOSYNTHESISAND METABOLISM / tory pathway. I n h e r e n t instability of even the full-length h o r m o n e provides a regulatory mechanism that allows extracellular calcium to alter rapidly the a m o u n t of active h o r m o n e available for secretion.

THE PARATHYROID HORMONE GENE The genomic DNAs encoding h u m a n (31), bovine (32), rat (33), and chicken (34) p r e p r o P T H have been cloned; the complete sequences of the h u m a n (35) and bovine (32) genes have been determined. Each gene contains three exons separated by two introns (see Fig. 4). The introns vary in size from species to species, though the first intron is invariably large, and the second intron in the human, bovine, and rat genes is about 100 base pairs in length. This length is close to the m i n i m u m length that can be recognized by the splicing machinery. The introns interrupt the sequences encoding mRNA at precisely the same locations in each species. The first exon contains most of the 5' n o n c o d i n g sequence. The second exon encodes most of the prepro sequence; the second intron comes in the middle of the triplet encoding the lysine residue that precedes the dibasic cleavage sequence Lys-Arg found at the end of the known pro sequences. The third exon encodes the Lys-Arg sequence, the mature PTH sequence, and the 3' noncoding region of the gene. The h u m a n and bovine genes are preceded by two functional TATA boxes that determine the two closely spaced start sites of the h u m a n and bovine transcripts. The rat and chicken genes are preceded only by one TATA box, found in a position equivalent to the second TATA box in the h u m a n and bovine genes. T h o u g h both start sites of transcription are used in the h u m a n and bovine genes, no conditions have been found that favor the use of one start site over the other. No data suggest that the two transcripts have importantly different stabilities or translatability, but such questions have not been exhaustively studied. The 5' n o n c o d i n g regions of each gene extend approximately 120 base pairs. The 3' n o n c o d i n g

iii !il!i iiiii!!i!iiiiii!iii! !iiiii!iiii!iiii!i~i!i~ii ii i liiii i!i iililiii ~~ii~!iiii

iiii!i i~i!iilililiiii i iiiiiii!illi!i!!ii!iiiiiii ii!i!iiiiiiiil iiliiiii ii!iil iiili!iiiiil!iiiiiiiiiii!i~iiiii!iiiiiil

ii

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!!!!!!!!!!!!! !!

!!!ii i!ii!iiiiiiii!iii !iili!i!iiiiiiiii!i!i!i!iiiiiii!iiiiiiiiiiiiiiilii i!iiiii!i!iiiii!!i!i!!!!!!ii i i i!i i~i~~ii~iii! !ill!if!!i~!~ ili i! i!ii! i! !i!i!iiii!ii!!iiiii!ii! i iiiiiii ii!iil FIG. 4 The parathyroid hormone gene. NC, Noncoding.

21

regions of each gene vary substantially in length, from the bovine at 227 base pairs to the chicken at more than 1600 base pairs. The 3' n o n c o d i n g region binds proteins that may regulate the stability of the p r e p r o P T H mRNA (36,37). The human, rat, and bovine PTH genes are represented only once in the haploid genomes of each species. The h u m a n PTH gene is located on the short arm of chromosome 11 at band 1 lp15 (38-40). A series of restriction fragment length polymorphisms (41,42) have made it possible to show that the h u m a n PTH gene is linked to the genes encoding catalase, calcitonin, H-ras, insulin, and [3-globin (43). Two other polymorphisms have been identified through the use of denaturing gel electrophoresis (44). All of these polymorphisms have proved useful in defining the inheritance of specific alleles of the PTH gene in families with calcium disorders (45). Several features of the PTH gene suggest that the gene is related to that encoding PTHrP (46-48). Most importantly, the major coding exon of both genes starts precisely at the same nucleotide, one base before the codons encoding the Lys-Arg residues of the pro sequences of each hormone. After the Lys-Arg sequences, the PTH and PTHrP amino acid sequences are identical in 8 of the next 13 residues. Further, the PTHrP gene is located on c h r o m o s o m e 12, a chromosome known to encode many genes that resemble genes on c h r o m o s o m e 11; for this reason, the chromosomes are t h o u g h t to have arisen by an ancient duplication event (49). One can speculate that the PTH gene may represent a variation of the PTHrP gene; the PTH h o r m o n e takes advantage of the PTHrP receptor in order to regulate calcium metabolism. If this hypothesis is correct, then the gene had to change in order to assure expression primarily in the parathyroid chief cell and to assure appropriate regulation by modulators such as extracellular calcium and 1,25-dihydroxyvitamin D [ 1,25 (OH) zOo]. A hypothalamic peptide called TIP39 (50) has been found to activate the PTH2 receptor and to be distantly related in sequence to PTH and PTHrE The structure of the TIP39 gene has not yet been reported. This gene may represent a third member of the PTH gene family.

REGULATION OF PTH BIOSYNTHESIS The minute-to-minute stability of the level of blood calcium depends on the regulation of PTH secretion by calcium. Longer term homeostasis depends on several other levels of control (see Fig. 5). The n u m b e r of parathyroid chief cells is carefully regulated; when appropriately stimulated, the parathyroid glands can

22

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C~a'TER2

TRANSCRIPTION/mRNA STABILITY

Ca, 1,25 D, PO4 Ca

Ca, 1,25 D

P H SECRETION PROLIFERATION

FIG. 5

Levels of parathyroid cell regulation.

increase in size dramatically. The parathyroid chief cell is uniquely designed to express the PTH gene; the state of differentiation of the chief cell can, therefore, influence the rate of PTH biosynthesis. Specific blood-borne signals, most notably calcium and 1,25(OH)zD3, regulate the activity of the PTH gene, as well. These several levels of regulation of PTH biosynthesis are examined in the following discussions (see also Chapter 18).

Regulation of Parathyroid Cell Number Little is known about the regulation of parathyroid cell number. The relatively uniform morphology of chief cells suggests that all chief cells have the potential to divide, if appropriately stimulated, but the alternative hypothesis that a subset of chief cells has the unique, stem-cell-like capability to proliferate has not been evaluated. Further, there is the general impression that parathyroid cells are long-lived, because mitoses are seldom seen in normal glands of mature animals, because the observed rate of apoptosis is low, and because hyperplastic glands only slowly decrease in size after stimulation. Nevertheless, specific studies to define potential modulators of chief cell longevity have not been performed. Despite this paucity of information, the dramatic hyperplasia of parathyroid cells in patients and animals with renal failure demonstrates the likely roles of calcium, phosphate, and 1,25(OH)2D 3 in regulating parathyroid cell proliferation. Dietary manipulation alone can similarly lead to chief cell hyperplasia. Naveh-Many and Silver (51), for example, used flow cytometry to count parathyroid cells and showed that 3 weeks of a calcium- and vitamin D-deficient diet fed to weanling rats led to a 1.7-fold increase in parathyroid cell number. These investigators subsequently studied the mechanism of the increase in parathyroid cell number caused by hypocalcemia, hyperphosphatemia, vitamin D deficiency, and uremia in vivo (52). They found that hypocalcemia and

uremia led to increases in parathyroid cell proliferation, whereas hypophosphatemia led to decreases in parathyroid cell proliferation. Administration of 1,25(OH)2D 3 for 3 days had no effect on parathyroid cell proliferation. None of these conditions led to changes in the rate of parathyroid cell apoptosis. Further studies of the effects of calcium in the uremic model suggest that calcium works by acting on the same calcium-sensing rector that mediates the actions of calcium on PTH secretion. The calcimimetic compound NPS R-568, like calcium, suppressed parathyroid cell proliferation in uremic rats (53). The possibly i n d e p e n d e n t roles of calcium and 1,25(OH)2D 3 in the regulation of parathyroid cell proliferation have not been studied extensively. In vivo, these variables are difficult to manipulate independently in the intact animal. One particularly instructive in vivo model, the vitamin D receptor knockout mouse, has been studied, however (54,55). These mice develop hypocalcemia, hypophosphatemia, and secondary hyperparathyroidism in the days and weeks after weaning. When the hypocalcemia and hypophosphatemia are prevented by a diet high in calcium, phosphate, and lactose, the hyperparathyroidism and parathyroid gland enlargement are prevented. Because these mice lack vitamin D receptors, they must be able to regulate parathyroid cell n u m b e r without using the genomic actions of 1,25 (OH) 2D~. Presumably, the direct effects of normal calcium and phosphate are sufficient to prevent parathyroid cell replication. In studies of cultured parathyroid chief cells, it has been possible to vary the levels of calcium and 1,25(OH)2D ~ separately. Several groups have shown that 1,25(OH)2D 3 can regulate parathyroid cell proliferation in vitro. Whether the cells were grown in the presence of serum (56,57) or serum-free growth factors (58), administration of 1,25(OH)2D 3 decreased their rate of proliferation. Studies of the effects of calcium on parathyroid cell proliferation in vitro have yielded differing results. Several studies (59-61) have shown that lowering of calcium leads to increased cellular proliferation. Other studies of dispersed, early-passage chief cells have demonstrated no effect of calcium on the rate of cell proliferation, however (57,58,62). Though extracellular levels of calcium and 1,25(OH)2D~ can be independently regulated in vitro, it is hard to be sure that parathyroid cells in culture respond to modulators of proliferation in this setting in the same way that they do in vivo. Thus, though the combined effects of low calcium and low levels of 1,25(OH)2D ~ to stimulate parathyroid cell proliferation are well established, the individual roles of calcium, phosphate, and 1,25 (OH)2D~ in vivo remain uncertain.

PTH BIOSYNTHESISAND METABOLISM /

Cell-Specific PTH Gene Expression Expression of the parathyroid h o r m o n e gene occurs almost exclusively in the parathyroid chief cell. [Expression has also been noted in the rat hypothalamus (63).] Thus, genes required for parathyroid chief cell differentiation are possible candidates for genes that might regulate the PTH gene as well. These genes, identified through the study of knockout mice, include hoxa3 (64,65), pax9 (66), and glial cells missing 2 (67). The hoxa3 and pax9 mutant mice lack a range of branchial arch derivatives, whereas the glial cell missing knockout mouse exhibits highly selective parathyroid cell deficit. When the chief cell is disrupted by neoplastic transformation, the regulation of PTH gene expression can be altered. For example, parathyroid cancers may stop synthesizing PTH completely (68). Presumably, specific DNA sequences associated with the PTH gene respond to the environment of the chief cell to activate gene expression. Because no well-differentiated cell line expressing the PTH gene has been established, it has been difficult to determine the sequences responsible for chief cell-specific PTH gene expression. Occasional "experiments of nature" have provided important clues, however. Very rarely, h u m a n nonparathyroid tumors have been found to produce PTH ectopically, for example. In one case that was studied carefully (69), the PTH regulatory region upstream from the gene was disrupted in tumor cells. Presumably, this gene r e a r r a n g e m e n t allowed the gene to be expressed in nonparathyroid cells by providing new regulatory signals or abolishing normal silencing mechanisms found upstream of the gene. Further, in a subset of parathyroid adenomas, the entire upstream portion of the PTH gene along with the first, noncoding exon are separated from the rest of the gene and rearranged adjacent to the PRAD1 gene (70). As a consequence of this rearrangement, the PRAD1 gene (encoding cyclin D1), a regulator of the cell cycle, is dramatically overexpressed. These observations suggest that the PTH gene upstream region contains sequences that stimulate gene transcription in parathyroid chief cells. Further analysis of the sequences that determine chief cell expression of the PTH gene m u s t await studies of transgenic animals or the establishment of welldifferentiated parathyroid chief cell lines.

Modulators of PTH Gene Expression The effects of calcium on PTH gene expression were first demonstrated in experiments using primary parathyroid cells in culture. Russell et al. (71) found that high levels of calcium resulted in a decrease in PTH mRNA levels over a several-day period. In those

23

studies, no difference was noted between the effects of low and normal levels of extracellular calcium. The decrease in PTH mRNA levels in response to high calcium levels could be reversed by lowering the calcium level; thus, the suppressive effect of calcium was not an irreversible, toxic effect. These in vitro observations have been confirmed by Brookman et al. (72), who noted a slight increase in PTH mRNA u n d e r low calcium level conditions at one time point. Subsequent studies by Russell et al. (73) showed that the rate of transcription of the PTH gene in nuclei of dispersed bovine parathyroid cells fell within 6 hours in response to high levels of extracellular calcium. The rate of transcription of the actin gene was unchanged; therefore, the effect of calcium was shown to be specific. The lack of parathyroid cell lines that produce PTH has h a m p e r e d the search for DNA sequences responsible for the transcriptional effects of calcium noted in cultured parathyroid cells. Okazaki et al. (74) have identified short sequences several thousand base pairs upstream from the start site of PTH gene transcription that may well be important for calcium regulation, however. These investigators identified the region by showing that several short sequences in the region could decrease gene transcription from many different promoters, including the PTH gene p r o m o t e r (75). Further, when the level of extracellular calcium was varied, after transfection of fusion genes containing a short oligonucleotide from this region, high calcium levels further suppressed transcription from genes containing the sequence but had no effect on control plasmids. Intriguingly, almost identical sequences were found in the gene encoding rat atrial natruiretic polypeptide, another gene negatively regulated by calcium. This DNA sequence could also confer calcium sensitivity to a fusion gene in fibroblast transfection experiments. T h o u g h these experiments are very suggestive, further studies will be required to show that the regulatory region can confer calcium sensitivity in its normal location far upstream from the PTH gene transcription start site. Ultimately, studies using welldifferentiated parathyroid cells will be required. Two groups have studied the acute effects of changes in blood calcium on PTH mRNA levels in the intact rat. Both showed that acute lowering of blood calcium (with phosphate, calcitonin, or EDTA) led to a p r o m p t increase in PTH mRNA levels (76,77). Elevations in blood calcium, in contrast, led to no change in PTH mRNA levels after 6 hours (76) and to a slight decrease in PTH mRNA levels after 48 hours (77). The parathyroid gland apparently, then, in the normal state, rests near the bottom of the calcium dose-response curve. The gland is well equipped to increase PTH production, but poorly prepared to decrease production in the

24

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CHAPTER2

face of hypercalcemia. Subsequent studies showed that changes in PTH mRNA caused by hypocalcemia in vivo are not caused by a transcriptional mechanism, but rather are caused by changes in mRNA stability (37). 1,25(OH)zD 3 has been shown to be an i m p o r t a n t regulator of PTH gene transcription in studies both in vitro and in vivo. Silver et al. (78) used primary parathyroid cells in culture to show that exposure to 1,25(OH)2D 3 led to a decrease in PTH mRNA levels. This work has been confirmed by studies of Karmali et al. (79) and Brown et al. (80). Russell et al. (81) then showed that 1,25(OH)zD 3 lowers the PTH gene transcription rate as early as 2 hours after exposure of cells to 1,25 (OH)zD 3. Similarly, in intact rats, intraperitoneal injections of 1,25(OH)zD 3 rapidly led to decreased transcription of the PTH gene and decreased PTH mRNA levels (82). The doses of 1,25(OH)zD 3 were so low that blood calcium did not change; the precise blood levels of 1,25(OH)zD 3 required to suppress PTH gene transcription acutely in vivo have not been established, however. The effects of low levels of 1,25(OH)zD 3 have not been studied extensively in intact animals. Such studies are difficult to interpret, because of confounding effects of vitamin D deficiency on blood calcium and parathyroid cell number. Weanling rats fed a vitamin Ddeficient diet for 3 weeks had a modest increase in their PTH mRNA levels (51). This increase occurred with no a p p a r e n t decrease in blood calcium levels. In the intact organism, calcium and 1,25(OH)zD 3 seld o m vary independently; consequently, the effects of changes in both parameters simultaneously have important physiologic relevance. When rats were made acutely hypocalcemic with phosphate and were at the same time given 1,25(OH)zD 3 intraperitoneally, the suppressive effect of 1,25(OH)2D 3 reversed the effect of hypocalcemia and led to a decrease in PTH mRNA (76). In contrast, when rats were fed a low-calcium diet for 3 weeks, blood calcium levels decreased and blood 1,25 (OH) 203 levels increased dramatically. In this setting, PTH mRNA levels rose severalfold; thus, the effects of low calcium levels were more influential than the effects of high 1,25(OH)zD 3 levels. The partial vitamin D resistance of the parathyroid gland in the setting of hypocalcemia makes sense physiologically: in that setting the action of vitamin D to increase intestinal calcium absorption is needed, but the action to inhibit PTH synthesis is not. Sela-Brown et al. (83) studied the mechanism of hypocalcemia-induced resistance to vitamin D action on the parathyroid gland. They showed that hypocalcemia in vivo induces nuclear accumulation of calreticulin, a calcium-binding protein, in parathyroid chief cells, and that calreticulin can interfere with the actions of the vitamin D receptor on a negative vitamin D response e l e m e n t in transfected cells in vitro.

In experimental uremia, the double stimulus of hypocalcemia and low levels of 1,25(OH)zD 3 has consistently led to increases in PTH mRNA (84,85). Administration of 1,25(OH)zD 3 could reverse this increase. This effect of 1,25(OH)zD 3 is likely to contribute importantly to the decrease in PTH blood levels seen in dogs with experimental uremia (86) and in dialysis patients (87). A series of transfection studies and DNA binding assays have been used to identify DNA sequences in the PTH gene responsible for modulating transcription of the PTH gene in response to 1,25(OH)2D 3. When a fusion gene containing 684 base pairs (bp) of DNA upstream of the h u m a n PTH gene was introduced stably into rat pituitary GH4 cells, expression of the gene was specifically suppressed by 1,25(OH)2D 3 (88). Three groups have identified DNA sequences upstream of the PTH gene that bind to 1,25(OH)zD 3 receptors in vitro. Filter binding assays showed that 1,25(OH)zD 3 receptors can bind to bovine PTH gene sequences between - 4 8 5 and - 1 0 0 bp upstream from the transcription start site (89). Subsequently, gel mobility-shift assays were used to identify a specific 26-bp sequence, located 125 bp upstream from the start site of transcription of the h u m a n PTH gene, that binds 1,25 (OH)203 receptors (90). When this short sequence was linked to a reporter gene and expressed in pituitary GH4 cells, 1,25(OH)zD 3 decreased expression of the reporter gene. This suppression of transcription was even greater when the n u m b e r of 1,25(OH)zD 3 receptors in the GH4 cells was increased by cotransfection of a 1,25(OH)zD 3 receptor expression vector. The h u m a n negative 1,25 (OH)203 (vitamin D) response e l e m e n t (VDRE) contains one copy of a motif found in two copies in the mouse osteopontin gene, a gene up-regulated by 1,25(OH)zD 3. Negative VDREs have also been identified in the chicken (91) and rat (92) PTH genes. These sequences closely resemble positive VDREs and have been shown to bind heterodimers of the vitamin D receptor and RXR, just as positive VDREs do. Subtle differences in binding interactions may explain why these particular VDREs in the PTH gene can act as negative VDREs with vitamin D receptor-RXR heterodimers (93). Until recently, the effects of phosphate on the parathyroid cell were t h o u g h t to be indirectly mediated by the hypocalcemia associated with increases in blood phosphate. The rapid actions of changes in phosphate levels in vivo on PTH secretion work through such a mechanism. However, studies using intact rat parathyroid glands in vitro demonstrate that changes in phosphate levels can, after several hours, lead to changes in PTH secretion (94,95). PTH mRNA did not change in these studies in vitro, but analogous studies p e r f o r m e d in intact rats d e m o n s t r a t e d that phosphate, in the setting of apparently constant levels of calcium and

PTH BIOSYNTHESISAND METABOLISM / 1,25(OH)2D ~, increases PTH mRNA by a posttranscriptional mechanism (96). Though calcium and 1,25(OH)zD 3 are certainly the most important physiologic regulators of PTH gene transcription, other circulating factors are likely to modulate PTH gene transcription as well. The PTH gene contains a consensus cyclic AMP response element that can function in the context of a fusion gene in transfection experiments (97). Thus, hormones that stimulate adenylyl cyclase may increase PTH gene transcription. Glucocorticoids have been shown to increase PTH mRNA in dispersed, hyperplastic h u m a n parathyroid cells (98) and to abolish the decrease in PTH mRNA in response to 1,25(OH)zD ~ in dispersed bovine parathyroid cells (79). These cell culture studies need to be confirmed by studies in vivo to determine their physiologic significance. In ovariectomized rats, estradiol administration led within 24 hours to a fourfold increase of PTH mRNA (99). Estrogen receptors were identified in rat parathyroids. These observations may have important implications for an understanding of postmenopausal osteoporosis and hyperparathyroidism. The possibility that the effect of estrogen on PTH mRNA levels is a direct effect on the parathyroid gland needs to be tested by studies using cultured parathyroid cells.

Peripheral Metabolism of PTH Intact PTH is rapidly cleared from the circulation with a disappearance half-time of approximately 2 minutes (100-103). Removal of PTH from the blood occurs mainly (60-70%) in the liver but also in the kidneys (20-30%) and, to a much lesser extent, in other organs (100,102,103). Clearance of PTH by the liver is mediated mainly by a high-capacity, nonsaturable uptake by Kupffer cells and is followed by rapid and extensive proteolysis (104). Renal clearance occurs almost entirely by glomerular filtration. The hormone is also reabsorbed by the renal tubules and then extensively degraded, so that little or no intact PTH appears in the final urine (102). A large membrane-bound protein, megalin, binds PTH (but not carboxy-terminal fragments of PTH) in the lumen of the proximal tubule to initiate this reabsorption (105). In both the liver and kidney, as in bone, some PTH is removed by high-affinity binding to cell surface receptors, but this constitutes only a small fraction (<1%) of overall PTH clearance (102,106,107). Thus, it appears that the main role of the liver and kidney in PTH metabolism is rapid removal and degradation of circulating biologically active hormone, assuring that the concentration of hormone available to receptors in target tissues is dictated exclusively by the rate at which PTH is secreted by the parathyroid glands.

25

This simple first-order model, in which secreted intact PTH that is not bound to specific high-affinity cellular receptors is rapidly cleared by peripheral organs, was found to be inadequate when it was recognized that multiple species of immunoreactive PTH molecules are present in the circulation (108,109). Early observations with region-specific immunoassays in vivo and in medium from perfused organs in vitro, followed later by direct analysis of the peripheral metabolism of intravenously administered radioactive PTH, confirmed that intact PTH is not only rapidly cleared from the blood but also is rapidly cleaved by endoproteases to a series of carboxyl-terminal (C) fragments, some of which reenter the circulation (100,102,103,108,110,111). The chemical identities of these fragments have not been established definitively. Microradiosequencing of radioactive fragments recovered from blood of rats following intravenous administration of [125I-Tyr-43]bPTH or [SH-Tyr-43]bPTH has shown that PTH peptides produced by cleavages between residues 33-34, 36-37, 40-41, and 42-43 are the predominant large "signature" C fragments (103,104,107,110). These fragments exhibit apparent molecular masses of 4000-7000 Da and it is not certain that they all extend entirely to the original C terminus (i.e., to residue 84) of the intact hormone. Also, because fragments shorter than PTH(43-84) would not have been detected by these radiosequencing analyses, it is possible that such shorter fragments also exist in blood. Other evidence, derived from detailed analysis by region-specific immunoassays of circulating forms of PTH, points to the presence in blood of shorter, "midmolecule" fragments, presumably derived from proteolysis within both the amino (N-) and C-terminal portions of the intact hormone (108,112,113). In vitro, isolated hepatic Kupffer cells, but not hepatocytes, generate C fragments that are chemically identical to the major circulating form (104,114). Also, liver ablation, but not nephrectomy, blocks the production of circulating C fragments that otherwise are produced by proteolysis of administered radiolabeled intact PTH (103). These findings suggest that hepatic Kupffer cells are responsible for both the rapid clearance and the extensive proteolysis of PTH that occur in the liver. Moreover, Kupffer cells appear also to be the source of the major circulating C fragments identified so far that result from postsecretory metabolism of PTH. In the kidney, extensive proteolysis of PTH occurs also, but the kidney does not contribute significantly to the circulating pool of PTH C fragments (100,103,110). Studies of the cellular enzymes responsible for these cleavages are most consistent with a role for lysosomal enzymes of the cathepsin B/D family (107,115). Quantitatively, less than 10-20% of secreted intact PTH is converted to circulating C fragments by

26

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CHAPTER2

peripheral metabolism (100,103,110). On the other hand, C fragments comprise between 50 and 90% of total circulating PTH immunoreactivity (102,107,108,112,113,116). This disparity results from at least two factors. First, the clearance of C fragments, which does not occur in the liver but rather proceeds mainly via glomerular filtration in the kidney, is significantly slower than that of intact PTH (102,117). Second, it is now clear that C fragments are secreted along with intact PTH by the parathyroid glands, which thus constitute an independent source of these fragments (115,116,118-120). Remarkably, the major C fragments secreted by the parathyroids are chemically identical (at their N termini) with the principal circulating products of peripheral metabolism of PTH (115,119). The impact of these factors is especially obvious in renal insufficiency, in which delayed renal clearance (up to 100-fold) of C fragments, combined with their accelerated generation, both within the hyperplastic parathyroid glands and during peripheral metabolism of overproduced intact hormone, leads to massive accumulation of C fragments vs. intact hormone in the circulation. Metabolism of PTH, both peripherally and within the parathyroid glands, involves cleavages within the region PTH(33-43), which, at least potentially, could generate biologically active N-terminal fragments. Accordingly, considerable interest has focused on the possibility that such circulating N fragments might result from PTH metabolism and, further, that the overall rate or pattern of PTH proteolysis might be regulated physiologically to modulate the production of such fragments. Circulating amino-terminal PTH fragments have been demonstrated occasionally, almost exclusively in the setting of renal failure or hyperparathyroidism, or both, but it has been difficult to exclude postcollection proteolysis as the explanation in these circumstances (107,108,121). Moreover, immunochemical analyses of normal plasma have not provided convincing evidence of circulating N-terminal PTH fragments. More recently, direct analysis of the fate of the N terminus of PTH has been possible using [~5S]methionine to radiolabel biologically active hormone to high specific activity within the N-terminal region of the molecule. These studies in normal rats have shown that N-terminal PTH fragments produced by isolated Kupffer cells in vitro or by the liver or kidney in vivo are rapidly degraded in situ and do not reenter the circulation, at least in concentrations above 50 fM (100). Similar investigations have provided evidence that peripheral metabolism of PTH is not regulated physiologically in response to alterations in serum or dietary calcium, vitamin D intoxication, or parathyroid status (101,122). In contrast, secretion of C fragments by the parathyroid glands is strikingly influenced by

extracellular calcium, in that hypercalcemia increases the ratio of secreted C fragments to intact hormone whereas the opposite occurs in hypocalcemia (108,116,118,119). These alterations in the intracellular proteolysis of PTH are reflected by corresponding changes in the predominant immunoreactive forms of PTH in the circulation observed during hypercalcemia and hypocalcemia (108,112). The extensive metabolism of PTH(1-84) to fragments of PTH that accumulate in the circulation but cannot activate the P T H / P T H r P receptor has created an obstacle to the measurement of biologically active PTH in the circulation by immunologic methods. A series of two-site immunoassays, in which antibodies to the amino-terminal region and carboxy-terminal region of PTH must both bind the ligand simultaneously to register as a PTH molecule, have revolutionized the clinical usefulness of PTH assays (123). However, later generation versions of these assays have demonstrated unanticipated complexity in the PTH fragments present in the circulation (124-126). Some two-site PTH assays, but not others, recognize a large PTH fragment, roughly the size of PTH(7-84). This fragment is normally only a small fraction of the circulating PTH, but it can represent as much as 50% of the PTH recognized by some two-site PTH assays in the presence of renal failure. The fraction of immunoreactive PTH represented by the large PTH fragments is also increased in hypercalcemic states. Though PTH fragments missing the amino terminus of PTH are not expected to activate the P T H / P T H r P receptor, the possibility that these fragments might antagonize the actions of PTH (1-84) or might have unique activities of their own is currently being explored. The precise structure, site of origin, and biologic activities of these fragments, thus, are important questions for the future. In summary, the extremely rapid peripheral clearance and proteolysis of intact PTH play an important role in limiting the duration of hormone action and in assuring that the secretory activity of the parathyroid glands is the overriding determinant of the circulating concentration of biologically active PTH. Whereas a small percentage of degraded PTH molecules reappears in the blood, these are exclusively composed of large carboxyl-region and midregion fragments that are devoid of classic PTH bioactivity. Additional fragments are released directly by the parathyroid glands, reflecting an intraglandular mechanism involved in calcium regulation Of hormone secretion, but these too are biologically inactive. It remains possible that such carboxylregion and midregion PTH fragments may exert novel biologic actions in some tissues via receptors other than those known to be activated by intact PTH or N-terminal PTH fragments, but there is little evidence at present in support of a critical physiologic role for such fragments.

P T H BIOSYNTHESIS AND METABOLISM

CONCLUSION The blood level of PTH is regulated at several levels, each designed to respond to different challenges to calcium homeostasis. Over short time frames, the regulation of PTH secretion by calcium, coupled to the rapid metabolism of the hormone, assures that the blood level of PTH can adjust to sudden changes in calcium flux. Turnover of PTH within the parathyroid gland decreases under hypocalcemic conditions; this adjustment provides rapid increases in available hormone. Over a longer time frame both calcium and 1,25(OH)zD 3 regulate PTH biosynthesis. It is, of course, reasonable that 1,25(OH)zD ~ should regulate PTH biosynthesis, because the need for PTH can be expected to be great in face of vitamin D deficiency, no matter what the instantaneous level of blood calcium. Though the physiologic studies are not extensive, the synthetic machinery seems designed particularly to respond dramatically to decreases in blood calcium and 1,25 (OH) 203 levels. Over a longer time period, calcium and, perhaps to a greater extent, 1,25(OH)zD ~ regulate the n u m b e r of parathyroid cells. This is a relatively crude and slow process. The slow turnover of parathyroid chief cells suggests that the parathyroid gland is not meant to rely on frequent changes in parathyroid cell number, but uses this method of amplifying its signal only when other alternatives prove insufficient. This perspective on parathyroid control, with its emphasis on multiple levels of regulation, provides a framework for understanding the alterations caused by disease and may suggest therapeutic strategies as well.

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25. Hashizume Y, Waguri S, Watanabe T, Kominami E, Uchiyama Y. Cysteine proteinases in rat parathyroid cells with special reference to their correlation with parathyroid hormone (PTH) in storage granules. J Histochem Cytochem 1993;41:273-282. 26. MacGregor RR, Hamilton JW, Shofstall RE, Cohn DV. Isolation and characterization of porcine parathyroid cathepsin B. J Biol Chem 1979;254: 4423-4427. 27. Mayer GP, Keaton JA, Hurst JG, Habener JE Effects of plasma calcium concentration on the relative proportion of hormone and carboxyl fragments in parathyroid venous blood. Endocrinology 1979;104:1778-1784. 28. Habener JE Kemper B, Potts JT, Jr. Calcium-dependent intracellular degradation of parathyroid hormone: A possible mechanism for the regulation of hormone stores. Endocrinology 1975;97:431-441. 29. Chu LLH, MacGregor RR, Anast CS, et al. Studies on the biosynthesis of rat parathyroid hormone and proparathyroid hormone: Adaptation of the parathyroid gland to dietary restriction of calcium. Endocrinology 1973;93:915-924. 30. Tanguay KE, Mortimer ST, Wood PH, Hanley DA. The effects of phorbol myristate acetate on the intracellular degradation of bovine parathyroid hormone. Endocrinology 1991;128:1863-1868. 31. Vasicek T, McDevitt BE, Freeman MW, et al. Nucleotide sequence of the human parathyroid hormone gene. Proc Natl Acad Sci USA 1983;80:2127. 32. Weaver CA, Gordon DE Kissil MS, et al. Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone. Gene 1984;28:319-329. 33. Heinrich G, Kronenberg HM, Potts JT, Jr, Habener JE Gene encoding parathyroid hormone: Nucleotide sequence of the rat gene and deduced amino acid sequence of rat preproparathyroid hormone. JBiol Chem 1984;259:3320. 34. Russell J, Olivera A, Liu S, Sherwood LM. Isolation and complete nucleotide sequence of the avian parathyroid hormone gene. Endocrine Society Program and Abstracts, 73rd Annual Meeting, June 19-22, 1991. 35. Reis A, Hecht W, Gr6ger R, B6hm I, Cooper DN, Lindenmaier W, Mayer H, Schmidtke J. Cloning and sequence analysis of the human parathyroid hormone gene region. Hum Genet 1990;84:119-124. 36. Moallem E, Silver J, Naveh-Many T. Post-transcriptional regulation of PTH gene expression by hypocalcemia due to protein binding to the PTH mRNA 3' UTR. J Bone Miner Res 1995;10(Suppl 1):S142. 37. Moallem E, Kilav R, Silver J, Naveh-Many T. RNA-protein binding and post-transcriptional regulation of parathyroid hormone gene expression by calcium and phosphate. J Biol Chem 1998;273 (9) :5253-5259. 38. Naylor SL, Sakaguchi AY, Szoka P, et al. Human parathyroid hormone gene (PTH) is on short arm of chromosome 11. Somatic Cell Genet 1983;9:609-616. 39. Mayer H, Breyel E, Bostock C, Schmidtke J. Assignment of the human parathyroid hormone gene to chromosome 11. Hum Genet 1983;64:283-285. 40. Zabel BU, Kronenberg HM, Bell GI, Shows TB. Chromosome mapping of genes on the short arm of human chromosome 11: Parathyroid hormone gene is at l lp15 together with the genes for insulin, c-Harvey-ras 1, and [3-hemoglobin. Cytogenet Cell Genet 1985;39:200-205. 41. Antonarakis SE, Phillips III, JA, Mallonee RL, et al. [3-globin locus is linked to the parathyroid hormone (PTH) locus and lies between the insulin and PTH loci in man. Proc Natl Acad Sci USA 1983;80:6615-6619. 42. Schmidtke J, Pape B, Krengel U, et al. Restriction fragment length polymorphisms at the human parathyroid hormone gene locus. Hum Genet 1984;67:428-431.

43. Kittur SD, Hoppener JWM, Antonarakis SE, et al. Linkage map of the short arm of human chromosome 11: Location of the genes for catalase, calcitonin, and insulin-like growth factor II. Proc Natl Acad Sci USA 1985;82:5064-5067. 44. Miric A, Levine MA. Analysis of the preproPTH gene by denaturing gradient gel electrophoresis in familial isolated hypoparathyroidism. J Clin Endocrinol Metab 1991 ;74:509-516. 45. Ahn TG, Antonarakis SE, Kronenberg HM, et al. Familial isolated hypoparathyroidism: A molecular genetic analysis of 8 families with 23 affected persons. Medicine 1986;65:73-81. 46. Suva LJ, Winslow GA, Wettenhall REH, Hammonds RG, Moseley JM, Diefenbach-Jagger H, Rodda CP, Kemp BE, Rodriguez H, Chen EY, Hudson PJ, Martin TJ, Wood WI. A parathyroid hormone-related protein implicated in malignant hypercalcemia: Cloning and expression. Science 1987;237:893-896. 47. Mangin M, Ikeda K, Dreyer BE, Broadus AE. Isolation and characterization of the human parathyroid hormone-like peptide gene. Proc Natl Acad Sci USA 1989;86:2408-2412. 48. Yasuda T, Banville D, Hendy GN, Goltzman D. Characterization of the human parathyroid hormone-like peptide gene: Functional and evolutionary aspects. J Biol Chem 1989;264: 7720-7725. 49. Comings DE. Evidence for ancient tetraploidy and conservation of linkage groups in mammalian chromosomes. Nature 1972;238:455-457. 50. Usdin TB, Hoare SRJ, Wang T, Mezey E, Kowalak JA. TIP39: A new neuropeptide and PTH2-receptor agonist from hypothalamus. Nat Neurosci 1999;2 (11):941-943. 51. Naveh-Many T, Silver J. Regulation of parathyroid hormone gene expression by hypocalcemia, hypercalcemia, and vitamin D in the rat. J Clin Invest 1990;86:1313-1319. 52. Naveh-Many T, Rahamimov R, Livni N, Silver J. Parathyroid cell proliferation in normal and chronic renal failure rats: The effects of calcium, phosphate and vitamin D. J Clin Invest 1995;96:1786-1793. 53. Wada M, Furuya Y, Sakiyama J, Kobayashi N, Miyata S, Ishii H, Nagano N. The calcimimetic compound NPS R-568 suppresses parathyroid cell proliferation in rats with renal insufficiency. J Clin Invest 1997;100(12):2977-2983. 54. Li YC, Pirro AE, Amling M, Delling G, Baron R, Bronson R, Demay MB. Targeted ablation of the vitamin D receptor: An animal model of vitamin D-dependent rickets type II with alopecia. Proc Natl Acad Sci USA 1997;94(18):9831-9835. 55. Li YC, Pirro AE, Demay MB. Analysis of vitamin D-dependent calcium-binding protein messenger ribonucleic acid expression in mice lacking the vitamin D receptor. Endocrinology 1998; 139 (3) :847-851. 56. Nygren P, Larsson R, Johansson H, Ljunghall S, Rastad J, Akerstr6m G. 1,25(OH)zD ~inhibits hormone secretion and proliferation but not functional dedifferentiation of cultured bovine parathyroid cells. CalcifTissue Int 1988;43:213-218. 57. Kremer R, Bolivar I, Goltzman D, Hendy GN. Influence of calcium and 1,25-dihydroxycholecalciferol on proliferation and proto-oncogene expression in primary cultures of bovine parathyroid cells. Endocrinology 1989;125:935-941. 58. Ishimi Y, Russell J, Sherwood LM. Regulation by calcium and 1,25-(OH)zD ~ of cell proliferation and function of bovine parathyroid cells in culture. J Bone Miner Res 1990;5:755-760. 59. Lee MJ, Roth SI. Effect of calcium and magnesium on deoxyribonucleic acid synthesis in rat parathyroid glands in vitro. Lab Invest 1975;33:72-79. 60. Raisz LG. Regulation by calcium of parathyroid growth and secretion in vitro. Nature 1963;197:1115-1117. 61. Brandi ML, Fitzpatrick LA, Coon HG, Aurbach GD. Bovine parathyroid cells maintained for more than 140 population doublings. Proc Natl Acad Sci USA 1986;83:1707-1713.

P T H BIOSYNTHESIS AND METABOLISM 62. Leboff MS, Rennke HG, Brown EM. Abnormal regulation of parathyroid cell secretion and proliferation in primary cultures of bovine parathyroid cells. Endocrinology 1983;113:227-284. 63. Fraser RA, Kronenberg HM, Pang PK, Harvey S. Parathyroid hormone messenger ribonucleic acid in the rat hypothalamus. Endocrinology 1990;127:2517-2522. 64. Manley NR, Capecchi MR. Hox group 3 paralogous genes act synergistically in the formation of somitic and neural crestderived structures. Dev Biol 1997;192 (2):274-288. 65. Manley NR, Capecchi MR. Hox group 3 paralogs regulate the development and migration of the thymus, thyroid, and parathyroid glands. Dev Bio11998;195(1) :1-15. 66. Peters H, Neubuser A, Kratochwil K, Balling R. Pax9-deficient mice lack pharyngeal pouch derivatives and teeth and exhibit craniofacial and limb abnormalities. Genes D e v 1998;12 (17):2735-2747. 67. Gunther T, Chen Z, Kim J, Priemel M, Rueger JM, Amling M, Moseley JM, Martin TJ, Anderson DJ, Karsenty G. Parathyroid hormone secretion in absence of parathyroid glands. J Bone Miner Res 1999;14(Suppl. 1) (Abstract 1008). 68. Baba H, Kishihara M, Tohmon M, et al. Identification of parathyroid hormone messenger ribonucleic acid in an apparently nonfunctioning parathyroid carcinoma transformed from a parathyroid carcinoma with hyperparathyroidism. J Clin Endocrinol Metab 1986;62:247-252. 69. Nussbaum SR, Gaz RD, Arnold A. Hypercalcemia and ectopic secretion of parathyroid hormone by an ovarian carcinoma with rearrangement of the gene for parathyroid hormone. N Engl J Med 1990;323:1324-1328. 70. Motokura T, Bloom T, Kim HG, et al. A BCLl-linked candidate oncogene which is rearranged in parathyroid tumors encodes a novel cyclin. Nature 1991;350:512-515. 71. Russell J, Lettieri D, Sherwood LM. Direct regulation by calcium of cytoplasmic messenger ribonucleic acid coding for preproparathyroid hormone in isolated bovine parathyroid cells. J Clin Invest 1983;72:1851-1855. 72. Brookman JJ, Farrow SM, Nicholson L, O'Riordan JLH, Hendy GN. Regulation by calcium of parathyroid hormone mRNA in cultured parathyroid tissue. J Bone Miner Res 1986;1:529-537. 73. Russell J, Sherwood LM. The effects of 1,25-dihydroxyvitamin D~ and high calcium on transcription of the pre-proparathyroid hormone gene are direct. Trans Assoc Am Physicians 1987;100:256-262. 74. Okazaki T, Zajac JD, Igarashi T, Ogata E, Kronenberg HM. Negative regulatory elements in the human parathyroid hormone gene. JBiol Chem 1991;266:21903-21910. 75. Okazaki T, Ando K, Igarashi T, Ogata E, Fujita T. Conserved mechanism of negative gene regulation by extracellular calcium. J Clin Invest 1992;89:1268-1273. 76. Naveh-Many T, Friedlaender MM, Mayer H, SilverJ. Calcium regulates parathyroid hormone messenger ribonucleic acid (mRNA), but not calcitonin mRNA in vivo in the rat. Dominant role of 1,25-dihydroxyvitamin D. Endocrinology 1989;125:275-280. 77. Yamamoto M, Igarashi T, Muramatsu M, Fukagawa M, Motokura T, Ogata E. Hypocalcemia increases and hypercalcemia decreases the steady state level of parathyroid hormone messenger ribonucleic acid in the rat. J Clin Invest 1989;83:1053-1058. 78. Silver j, Russell J, Sherwood LM. Regulation by vitamin D metabolites of messenger ribonucleic acid for preproparathyroid hormone in isolated bovine parathyroid cells. Proc Natl Acad Sci USA 1985;82:4270-4273. 79. Karmali R, Farrow S, Hewison M, Barker S, O'Riordan JLH. Effects of 1,25-dihydroxyvitamin D~ and cortisol on bovine and human parathyroid cells. JEndocrino11989;123:137-142. 80. Brown AJ, Ritter CR, FinchJL, MorrisseyJ, Martin KJ, Murayama E, Nishii Y, Slatopolsky E. The noncalcemic analogue of vitamin

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D, 22-oxacalcitriol, suppresses parathyroid hormone synthesis and secretion. J Clin Invest 1989;84:728-732. Russell J, Lettieri D, Sherwood LM. Direct suppression by 1,25(OH)zD 3 of transcription of the parathyroid hormone gene. Clin Res 1986;34:726A. Silver J, Naveh-Many T, Mayer H, Schmelzer HJ, Popovtzer MM. Regulation by vitamin D metabolites of parathyroid hormone gene transcription in vivo in the rat. J Clin Invest 1986;78:1296-1301. Sela-Brown A, Russell J, Koszewski NJ, Michalak M, Naveh-Many T, Silver J. Calreticulin inhibits vitamin D's action on the PTH gene in vitro and may prevent vitamin D's effect in vivo in hypocalcemic rats. Mol Endocrinol 1998;12:1193-1200. Shvil Y, Naveh-Many T, Barach P, Silver J. Regulation of parathyroid cell gene expression in experimental uremia. J Am Soc Nephrol 1990;1:99-104. Fukagawa M, Kaname S, Igarashi T, Ogata E, Kurokawa K. Regulation of parathyroid hormone synthesis in chronic renal failure in rats. Kidney Int 1991;39:874-881. Lopez-Hilker S, Galceran T, Chan YL, Rapp N, Martin KJ, Slatopolsky E. Hypocalcemia may not be essential for the development of secondary hyperparathyroidism in chronic renal failure. J Clin Invest 1986;78:1097-1102. Delmez JA, Tindira C, Grooms P, Dusso A, Windus DW, Slatopolsky E. Parathyroid hormone suppression by intravenous 1,25-dihydroxyvitamin D: A role for increased sensitivity to calcium. J Clin Invest 1989;83:1349-1355. Okazaki T, Igarashi T, Kronenberg HM. 5'-flanking region of the parathyroid hormone gene mediates negative regulation by 1,25(OH)z vitamin D3.JBiol Chem 1988;263:2203-2208. Farrow SM, Hawa NS, Karmali R, Hewison M, Walters JC, O'Riordan JLH. Binding of the receptor for 1,25-dihydroxyvitamin D3 to the 5'-flanking region of the bovine parathyroid hormone gene. JEndocrino11990;126:355-359. Demay MB, Kiernan MS, DeLuca HE Kronenberg HM. Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D 3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D 3. Proc Natl Acad Sci USA 1992;89:8097-8101. Liu SM, Koszewski N, Lupez M, Malluche HH, Olivera A, Russell J. Characterization of a response element in the 5'-flanking region of the avian (chicken) parathyroid hormone gene that mediates negative regulation of gene transcription by 1,25dihydroxyvitamin D 3 and binds the vitamin D 3 receptor. Mol Endocrinol 1996;10:206-215. Russell J, Ashok S, Koszewski NJ. Vitamin D receptor interactions with the rat parathyroid hormone gene: Synergistic effects between two negative vitamin D response elements. JBone Miner Res 1999;14(11):1828-1837. Koszewski NJ, Ashok S, Russell J. Turning a negative into a positive: Vitamin D receptor interactions with the avian parathyroid hormone response element. Mol Endocrinol 1999; 13 (3) :455-465. Almaden Y, Canalejo A, Hernandez A, Ballesteros E, GarciaNavarro S, Torres A, Rodriguez M. Direct effect of phosphorus on PTH secretion from whole rat parathyroid glands in vitro. J Bone Miner Res 1996;11 (7):970-976. Slatopolsky E, Finch J, Denda M, Ritter C, Zhong M, Dusso A, MacDonald PN, Brown AJ. Phosphorus restriction prevents parathyroid gland growth. J Clin Invest 1996;97 (11 ) :2534-2540. KilavR, Silver J, Naveh-Many T. Parathyroid hormone gene expression in hypophosphatemic rats. J Clin Invest 1995;96:327-333. Rupp E, Mayer H, Wingender E. The promoter of the human parathyroid hormone gene contains a functional cyclic AMPresponse element. Nucleic Acids Res 1990;18:5677-5683. Peraldi MN, Rondeau E,Jousset V, et al. Dexamethasone increases preproparathyroid hormone messenger RNA in human hyperplastic parathyroid cell in vitro. EurJ Clin Invest 1990;20:392-397.

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99. Naveh-Many T, Almogi G, Livni N, Silver J. Estrogen receptors and biologic response in rat parathyroid tissue and C cells. J Clin Invest 1992;90:2434-2438. 100. Bringhurst FR, Stern AM, Yotts M, Mizrahi N, Segre GV, PottsJT, Jr. Peripheral metabolism of PTH: Fate of biologically active amino terminus in vivo. Am J Physio11988;255:E886-E893. 101. Fox J, Scott M, Nissenson RA, Heath H. Effects of plasma calcium concentration on the metabolic clearance rates of parathyroid hormone in the dog.JLab Clin Med 1983;102:70-77. 102. Martin KJ, Hruska KA, Freitag JJ, Klahr S, Slatopolsky E. The peripheral metabolism of parathyroid hormone. N Engl J Med 1979;302:1092-1098. 103. Segre GV, D'Amour P, Hultman A, Potts JT, Jr. Effects of hepatectomy, nephrectomy, and nephrectomy/uremia on the metabolism of parathyroid hormone in the rat.J Clin Invest 1981;67:439-448. 104. Bringhurst FR, Segre GV, Lampman GW, Potts JT, Jr. Metabolism of parathyroid hormone by Kupffer cells: Analysis by reverse-phase high-performance liquid chromatography. Biochemistry 1982;21:4252-4258. 105. Hilpert J, Nykjaer A, Jacobsen C. Megalin antagonizes activation of the parathyroid hormone receptor. J Biol Chem 1999;274:5620-5625. 106. Rouleau ME Warshawsky H, Goltzman D. Parathyroid hormone binding in vivo to renal, hepatic and skeletal tissues of the rat using a radioautographic approach. Endocrinology 1986; 118:919-931. 107. J/ippner HW, Gardella TJ, Brown EM, Kronenberg HM, Potts, JT, Jr. Parathyroid hormone and parathyroid hormonerelated peptide in the regulation of calcium homeostasis and bone development. In: Degroot LJ and Jameson JL, (eds), Endocrinology, 4th Ed., WB Saunders, Philadelphia, pp. 969-998. 108. Dambacher MA, Fischer JA, Hunziker WH, Born W, Moran J, Roth HR, Delvin EE, Glorieux FH. Distribution of circulating immunoreactive components of parathyroid hormone in normal subjects and in patients with primary and secondary hyperparathyroidism: The role of the kidney and of serum calcium concentration. Clin Sci 1979;57:435-443. 109. Berson SA, Yalow RS. Immunochemical heterogeneity of parathyroid hormone in plasma. J Clin Endocrinol Metab 1968;28:1037-1047. 110. Segre GV, D'Amour P, Potts JT, Jr. Metabolism of radioiodinated bovine parathyroid hormone in the rat. Endocrinology 1976;99:1645-1652. 111. Daugaard H, Egfjord M, Olgaard K. Metabolism of intact parathyroid hormone in isolated perfused rat liver and kidney. Am J Physiol 1988;254:E740-E748. 112. D'Amour P, PalardyJ, Bahsali G, Mallette LE, DeLean A, Lepage R. The modulation of circulating parathyroid hormone immunoheterogeneity in man by ionized calcium concentration. J Clin Endocrinol Metab 1992;74:525-532. 113. Roos BA, Lindall AW, Aron DC, Orf JW, Yoon M, Huber MB, PenskyJ, Ells J, Lambert PW. Detection and characterization of small midregion parathyroid hormone fragment(s) in normal and hyperparathyroid glands and sera by immunoextraction

and region-specific radioimmunoassays. J Clin Endocrinol Metab 1981;53:709-721. 114. Segre GV, Perkins AS, Witters LA, Potts JT, Jr. Metabolism of parathyroid hormone by isolated rat Kupffer cells and hepatocytes. J Clin Invest 1981;67:449-457. 115. MacGregor RR, Jilka RL, Hamilton JW. Formation and secretion of fragments of parathormone. Indentification of cleavage sites. JBiol Chem 1986;261:1929-1934. 116. D'Amour P, LaBelle E LeCavalier L, Plourde V, Harvey D. Influence of serum Ca concentration on circulating molecular forms of PTH in three species. AmJPhysiol 1986;251 :E680-E687. 117. D'Amour P, Lazure C, LaBelle E Metabolism of radioiodinated carboxy-terminal fragments of bovine parathyroid hormone in normal and anephric rats. Endocrinology 1985;117:127-134. 118. Flueck JA, DiBella FP, Edis AJ, Kehrwald JM, Arnaud CD. Immunoheterogeneity of parathyroid hormone in venous effluent serum of hyperfunctioning parathyroid glands. J Clin Invest 1977;69:1367-1375. 119. Hanley DA, Ayer LM. Calcium-dependent release of carboxylterminal fragments of parathyroid hormone by hyperplastic human parathyroid tissue in vitro. J Clin Endocrinol Metab 1986;63:1075-1079. 120. Tanguay KE, Mortimer ST, Wood PH, Hanley DA. The effects of phorbol myristate acetate on the intracellular degradation of bovine parathyroid hormone. Endocrinology 1991 ;128:1863-1868. 121. Goltzman D, Henderson B, Loveridge M. Cytochemical bioassay of parathyroid hormone. Characteristics of the assay and analysis of circulating hormonal forms. J Clin Invest 1980;65:1309-1317. 122. Bringhurst FR, Stern AM, Yotts M, Mizrahi N, Segre GV, PottsJT, Jr. Peripheral metabolism of [35S]parathyroid hormone in vivo: Influence of alterations in calcium availability and parathyroid status. J Endocrinol 1989;122:237-245. 123. Nussbaum SR, Zahradnik RJ, Lavigne JR, Brennan GL, NozawaUng K, Kim LV, Keutmann HT, Wang CA, Potts JT, Jr, Segre GV. Highly sensitive two-site immunoradiometric assay of parathyrin and its clinical utility in evaluating patients with hypercalcemia. Clin Chem 1987;33:1364-1367. 124. Brossard JH, Cloutier M, Roy L, Lepage R, Gascon-Barre M, D'Amour P. Accumulation of a non-(I-84) molecular form of parathyroid hormone (PTH) detected by intact PTH assay in renal failure: Importance in the interpretation of PTH values. J Clin Endocrinol Metab 1996;81 (11) :3923-3929. 125. Lepage R, Roy L, Brossard JH, Rousseau L, Dorais C, Lazure C, D'Amour E A non-(I-84) circulating parathyroid hormone (PTH) fragment interferes significantly with intact PTH commercial assay measurements in uremic samples. Clin Chem 1998; 44 (4) :805-809. 126. John MR, Goodman WG, Gao P, Cantor TL, Salusky IB, Juppner H. A novel immunoradiometric assay detects full-length human PTH but not amino-terminally truncated fragments: Implications for PTH measurements in renal failure. J Clin Endocrinol Metab 1999;84(11 ):4287-4290.

CHAPTER 3

Parathyroid Hormone-Related Protein Gene Structure, Biosynthesis, Metabolism, and Regulation

WILLIAM M. PHILBRICK Section of Endocrinology, Department of Intemal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520

INTRODUCTION

calcium and phosphate metabolism that occur in humoral hypercalcemia of malignancy (1-4,7-9). The similarity between PTHrP and PTH extends to their overall genomic organization as well. Their two genes share a similar organization and similar positioning of i n t r o n - e x o n boundaries (Fig. 1). Furthermore, the h u m a n PTHrP gene maps to the short arm of chromosome 12, where it is flanked by lactate dehydrogenase B and the K-ras protooncogene, and the PTH gene is located on a homologous region of chromosome 11 with lactate dehydrogenase A and H-ras (7-9). These two chromosomes are t h o u g h t to have arisen through a tetraploidization event that occurred some 200 to 300 million years ago (10). Both PTHrP and the P T H / P T H r P receptor sequences have been identified in teleost fish and the respective proteins have been found in the cartilaginous elasmobranchs (11-13), thus it seems quite possible that PTHrP represents the ancestral gene and that PTH evolved subsequent to the gene duplication, as a response to the demands placed on calcium regulation by the emergence of bony skeletons in fish or by the adaptation to a terrestrial envir o n m e n t by early amphibians.

Parathyroid hormone-related protein (PTHrP) was identified in the course of the search for the agent responsible for the paraneoplastic syndrome, humoral hypercalcemia of malignancy. Malignancy-associated hypercalcemia, recognized as a clinical entity since the mid-1920s, was originally assumed to be related to local erosion of the skeleton by resident metastatic lesions (1-4). However, in 1941, Fuller Albright proposed the elaboration of a humoral factor by a neoplasm as an alternative mechanism to explain the reversal of hypercalcemia and hyperphosphatemia after removal of the primary t u m o r in a patient with renal carcinoma (5). It is now recognized that local osteolytic mechanisms account for only approximately 20% of cases of malignancy-associated hypercalcemia and that in the majority of the remaining 80%, hypercalcemia results from elevated circulating levels of PTHrP (1-4,6). The biochemical purification of PTHrP and the subsequent cloning of the cDNA by several laboratories in the late 1980s revealed that PTHrP and parathyroid h o r m o n e shared striking similarity at the aminotermini of the respective mature peptides: 8 out of the first 13 amino acids are identical, a further 3 represent conservative changes, and the two proteins share considerable conformational similarity through residue 34 (Fig. 1). This extensive homology accounts for the ability of PTHrP to bind to and activate classic parathyroid h o r m o n e (PTH) receptors in bone and kidney and consequently to generate the paraneoplastic effects on

The Parathyroids, Second Edition

GENE STRUCTURE: P R O M O T E R ELEMENTS, SPLICE SITES, AND TRANSCRIPTIONAL TERMINATION SITES From their c o m m o n origin, the PTHrP and PTH genes have clearly diverged. In humans the PTHrP gene

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FIG. 1 Comparison of parathyroid hormone-related protein (PTHrP) and parathyroid hormone (PTH). (A) Gene structure. The principal human PTHrP and PTH exons and introns are aligned relative to the coding regions. Exons are boxed, the 5' and 3' untranslated regions (UTRs) are white, the pre segment is hatched, the pro segment is shaded, and the mature peptide is black. Amino acid residues are numbered relative to the first residue of the mature peptide. (B) Amino acid sequence. The sequences of human PTHrP and PTH are aligned from the last two residues of the pro segment (encompassing the main cleavage site) through the first 13 residues of the mature peptide. Identity is indicated by vertical connecting lines and mismatches are scored according to the degree of evolutionary distance, with two vertical dots indicating greater similarity than one dot. In the rat and bovine PTH sequences, the first amino acid of the mature peptide is identical to that in PTHrP (Ala). From data in Mangin et aL (14).

is by far the larger and more complex of the two, spanning more than 15 kilobases (kb) and containing eight exons (Fig. 2; various exon n u m b e r i n g systems have been employed by different research groups; for the sake of simplicity, a sequential 1 through 8 nomenclature is used here). Three promoters have been functionally characterized (14-18). The first (P1) lies just upstream of exon 1 and contains a canonical TATA sequence; a second TATA-containing p r o m o t e r (P2) resides some 2.7 kb downstream within a small, 45-bp intron between exons 3 and 4. An additional p r o m o t e r element has been identified immediately upstream of exon 3, but does not contain a TATA sequence and instead resembles the GC-rich initiator element found in many housekeeping genes. In addition to the use of multiple promoters, the transcriptional diversity of PTHrP gene expression is also mediated by alternative splicing of primary transcripts (19-22). The 5' end of the gene contains four noncoding exons that may be variably incorporated into the mature, processed mRNA and the resultant splicing pattern appears to be largely d e p e n d e n t on p r o m o t e r usage. For transcripts initiating from P2, for example, the first exon (exon 4) is uniformly spliced to exon 5, whereas for transcripts initiating from the initiator element, the first exon (exon 3) is always spliced directly to exon 5, omitting exon 4. For transcripts initiating at P1, however, two possible patterns exist: exon 1 may be spliced to exon 2 (in which case splicing exon 2 to exon 3 and exon 3 to exon 5 is obligatory) or exon 1 may instead be spliced

directly to exon 3 (in which case splicing exon 3 to exon 5 is obligatory and exon 2 is omitted). However, because all of these 5' alternative exons represent untranslated regions (UTRs), the functional consequences of any particular splice choice are presently uncertain. Still further diversity of PTHrP mRNA expression in humans is generated by alternative splicing at the 3' end of the gene, which contains three possible terminal exons, each encoding a distinct carboxy terminus for the PTHrP protein (19-22). The splicing pattern here appears to be generated in large part by the site of transcription termination (Fig. 2). Transcripts terminating immediately 3' of exon 6 possess no 3' splicing alternatives and translation necessarily yields a 139residue mature peptide. Transcripts that terminate following exon 7 have only one possible 3' splice (exon 6 to exon 7), which, if available, will presumably be executed by default during mRNA processing. Translation of this mRNA product then yields a mature peptide of 173 amino acids. Only for transcripts terminating beyond exon 8 does there appear to be a splicing choice (either exon 6 to exon 7 or exon 6 to exon 8, with translation of the latter giving a 141-residue product), although complete removal of intronic sequences would necessarily render obligatory the exon 6 to exon 8 splice. The operative mechanism here, therefore, seems to be one of alternative transcription termination (and polyadenylation), rather than alternative splicing. In theory, the simplest means of generating equal amounts of each of the three alternative

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34

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CHAPTER3

3' mRNA termini (and, therefore, of the three alternative PTHrP peptides as well) would be to employ transcription terminator sequences of graded efficiencies, so that, for example, 33% of transcripts terminate after exon 6, 50% of those remaining terminate following exon 7, and 100% of the rest terminate beyond exon 8. Splicing would then be perfunctory, and not regulatory, in nature, although what regulatory mechanisms for PTHrP gene expression might exist at the level of transcription termination is presently unknown. The complexity in structure and processing evident in the h u m a n PTHrP gene seems to represent a relatively recent p h e n o m e n o n in evolutionary terms, because the organization of the gene is much simpler in lower species. The PTHrP gene in the rodent (both rat and mouse) appears to employ only a single TATA p r o m o t e r and contains only four exons (23-25) (Fig. 3). Exon 1 encompasses most of the 5' untranslated region, exon 2 encodes the "prepro" region from amino acids - 3 6 to - 8 , exon 3 encodes the r e m a i n d e r of the pro sequence (amino acids - 7 to - 1 ) and the bulk of the mature peptide (137 residues in the mouse and 139 in the rat), and exon 4 encodes the final two amino acids, the stop codon, and the 3' untranslated region. As seen in Fig. 3, one possible implication of the a r r a n g e m e n t of exons and introns in the colinear PTHrP genes from rodents and humans is that h u m a n exon 8 (rodent exon 4) represents the ancestral splicing pathway and that the emergence of the splice site defining h u m a n exon 7 occurred at a point in evolution distal to the rodent branchpoint. Interestingly, though the h u m a n exon 7 equivalent is also absent in chickens, the PTHrP gene in this species does display alternative termination 3' to exon 3 (the h u m a n exon 6 equivalent) and consequently generates a 139-residue peptide isoform, a p h e n o m e n o n not seen in rodents (26). This suggests either that the development of this termination site predated mammalian evolution and has been subsequently lost in m o d e r n rodents but retained in the human, or that this mechanism evolved independently in chickens and humans.

PROTEIN STRUCTURE AND POSTTRANSLATIONAL PROCESSING Translation of the three human PTHrP mRNAs, each with alternative 3' termini, generates three protein products with distinct carboxy termini (19-22). Each of these three protein isoforms have 139 amino acids in common; the isoform that undergoes translation termination at a stop codon within exon 6 ends with residue 139, whereas the isoform terminating in exon 8 adds two additional residues for a total of 141 and the isoform terminating in exon 7 extends the 139-amino acid trans-

lated product a further 34 amino acids for a total of 173 (Figs. 2B and 4). Rat PTHrP derives its carboxy terminus from the equivalent of h u m a n exon 8 and is also 141 amino acids in length, whereas the mouse protein, although similarly derived, is only 139 amino acids long due to the deletion of codons 130 and 131 (23-25) (Fig. 4). PTHrP coding sequences are extremely well conserved across species, with the region encompassing residues 1-111 of the mature peptide exhibiting approximately 98% homology between chickens and humans (26). The primary translation products of the human PTHrP gene (and from other species as well) share a c o m m o n 36-amino acid prepro sequence (residues - 3 6 to - 1 , encoded in the h u m a n by exons 3 and 4) that is composed of an N-terminal segment with the typical structural features of a signal peptide (the pre sequence), followed by a short leader peptide (the pro sequence; Fig. 1), the removal of which is required to allow ligand function (i.e., binding to and activation of the P T H / P T H r P receptor) (7-9). By analogy with other secreted proteins, the signal sequence presumably mediates the attachment of the nascent peptide to the endoplasmic reticulum, where it is then cotranslationally removed by signal peptidase as the growing chain is extruded through the membrane. The precise cleavage site is unknown, but is predicted to be in the region of residues - 8 to - 7 . The remaining pro sequence is then presumably cleaved at a tetrabasic site (RRXKR), spanning residues - 5 through - 1 , by the action of furin or a furinlike p r o h o r m o n e convertase within the endoplasmic reticulum or Golgi apparatus (27), thus generating the mature, secreted form(s) of the protein, PTHrP(1-139), PTHrP(1-141), and PTHrP(1-173) (7-9). The function of the pro sequence is unknown, but the sequence could be required for intracellular targeting or proper folding, or may simply serve as an inert spacer.

Amino-Terminal Peptides Early evidence for further posttranslational cleavage or fragmentation of native PTHrP emerged from the contradictory estimates of protein concentration obtained by standardized quantitative immunoassays that used antibodies generated against epitopes on specific regions of PTHrP (amino terminal, carboxy terminal, or midregion) versus two-site immunoassays designed to detect the complete PTHrP molecule. Inspection of the amino acid sequences of the PTHrP isoforms reveals a n u m b e r of potential sites for proteolytic cleavage, many of which appear to be functional in cultured cells or in vivo. There are frequent clusters of basic amino acids (arginine and lysine) across the length of the protein (Figs. 4 and Figs. 5); proteolytic processing at such sites is typical of neuroendocrine peptides

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FIG. 4 Amino acid sequence of the mature form(s) of parathyroid hormone-related protein. The top line is the sequence of the human 141-residue isoform, beginning with the first residue of the mature form and with the pre and pro segments removed. The alternative carboxy termini of the 139- and 173-isoforms are included for comparison. The sequences for mouse, rat, and chicken PTHrP are aligned with the human sequence. Amino acid substitutions are indicated, identity is designated by vertical lines, gaps or deletions are designated by dashes, and insertions are designated by an asterisk. From data in Mangin et aL (19,20,23), Suva et aL (21), Thiede et aL (22,26), Karaplis et aL (24), and Yasuda et aL (25).

such as insulin, proopiomelanocortin, gastrin, calcitonin, chromogranins, and many others (28,29). Cleavage at these sites is commonly carried out by prohormone convertases of the PC2, PC1/3 family located within the Golgi and in secretory granules. Another prominent cleavage site resides within the mature peptide adjacent to a monobasic arginine residue at position 37, thus generating a processed PTHrP peptide that consists of residues 1-36 (30). Although posttranslational processing of peptide hormones occurs more commonly at di- and multibasic sites, there are also numerous instances of cleavage at monobasic sites (31); among hormones processed in this manner are somatostatin, atrial natriuretic peptide, and glucagon. Mutagenesis of Arg-37 to Ala, Phe, or Lys effectively abolishes cleavage at this site (32). Using region-specific antibodies, aminoterminal species of PTHrP that do not contain carboxyterminal or midregion epitopes have been detected in media from cultured cell lines and in sera from hypercalcemic patients (8,9). More importantly, PTHrP(1-36) has been shown to be as potent as PTH (1-34) when analyzed in cell-based assays and in vivo (33). Keratinocytes appear to produce a longer, glycosylated, amino-terminal form of PTHrP (34); the core protein has an apparent molecular mass of 10 kDa; the fully glycosylated form has an apparent molecular mass of 18 kDa. This form has not been observed in other cell types thus far.

Midregion Peptides In addition to generating the 36-residue aminoterminal pepdde, cleavage of PTHrP at Arg-37 also gen-

erates a midregion peptide species with Ala-38 as its amino terminus (30) (Fig. 5). The amino acid sequence of PTHrP extending from residue 38 to 111 is remarkably well conserved among species, with only two substitutions in the h u m a n as compared to the rodent (23-25) (Fig. 4). This midregion peptide has been identified as an endogenous product in cultured cell lines from a n u m b e r of species and has been shown to be produced by rat insulinoma cell lines transfected with cDNAs encoding each of the three human PTHrP isoforms (35). Midregion PTHrP has been shown to be secreted in a regulated fashion by transfected neuroendocrine cells (36) and to be present in the circulation (37). The function of midregion PTHrP has not been well established, but studies in squamous epithelial cells provide evidence for a midregion-specific receptor that mediates the mobilization of cytosolic calcium and the formation of inositol phosphates through activation of the phospholipase C signaling pathway (38). Furthermore, studies in sheep have implicated a midregion peptide in establishing and maintaining the calcium gradient between the mother and fetus (39). This maternal-fetal calcium gradient is also abolished in PTHrP knockout mice, which have a hemochorial placenta as is found in humans. Placental calcium transport can be restored, however, by infusion of full-length or midregion fragments of PTHrP, but not by aminoterminal PTHrP or PTH (40). Finally, the natural secretory form(s) of midregion PTHrP have been only partially characterized; species with an amino terminus at residue 38 and carboxy termini at residues 94, 95, and 101 have been identified thus far (41), although

PTHrP:

G E N E STRUCTURE AND BIOSYNTHESIS

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FIG. 5 Parathyroid hormone-related protein domains. (A) Potential multibasic cleavage sites in the three human PTHrP isoforms. The positions of arginine (R) and lysine (K) residues are marked. Numbering is relative to the first amino acid of the mature peptide. (B) Functional domains and sequence homologies in PTHrP. The signal peptide (SP) and propeptide (P) are removed during intracellular processing. The PTH-like nature of the first 36 amino acids of the mature peptide reflects both the correspondence in primary structure in residues 1 through 13 and the conformational similarity that mediates receptor activation. The region between residues 36 and 111 is highly conserved among all species examined thus far. The carboxy-terminal segment from residues 111 to 139 or 141 is the least conserved region of the peptide and the portion extending beyond residue 141 has thus far only been found in humans. (C) Processed forms of PTHrP. Secreted products include the active, amino-terminal fragment (1-36), a glycosylated amino-terminal fragment found in keratinocytes, and the various midregion fragments defined thus far. Potential carboxy-terminal species include the purported osteostatin peptide and remain largely undefined. Adapted from Broadus AE, Stewart AF. Parathyroid hormone-related protein structure, processing, and physiological actions. In: Bilezikian JP, eds. The parathyroids. New York: Raven, 1994:259-294.

most experimental work testing for effects in cell culture and in vivo has been conducted with a peptide encompassing residues 67-86. Carboxy-Terminal Peptides A n u m b e r of multibasic proteolytic processing sites that may serve as substrates for furin/kexin can also be found in the carboxy terminus of PTHrP (at residues 88-91, 96-98, and 102-106) (42) and there is evidence that a carboxy-terminal species is generated as a native cellular product (Fig. 5). A carboxy-terminal

PTHrP fragment can be detected in the circulation of patients with renal failure (43) and is also found in the urine of normal h u m a n subjects (44). Similar fragments are secreted by h u m a n carcinoma cell lines and by cell lines transfected with PTHrP cDNAs (45). Based on the sequence conservation of PTHrP through amino acid 111 and t h e presence of the potential cleavage sites at residues 102-106, synthetic peptides containing amino acids 107-111 and 107-139 were tested for physiologic effects and were reported to inhibit osteoclastic bone resorption in vitro (46). To date, however, the effects of these

38

/

CHAPTER3

"osteostatin" peptides have not proved to be universally reproducible (47). Carboxy-terminal PTHrP has also been reported to inhibit the proliferation of cultured keratinocytes, whereas amino-terminal PTHrP had the opposite effect (48). Finally, immunoassays with antibodies directed against the unique 141-173 region of PTHrP that is derived from h u m a n exon 7 have suggested the presence of this fragment in fetal cord blood; h u m a n breast milk; cultured keratinocytes; renal, squamous and prostate carcinomas; and h u m a n amniotic m e m b r a n e s (49-51). However, as is the case with all carboxy-terminal species of PTHrP characterized thus far, the precise nature of the secretory form (s) has not been defined. Also contained within the multibasic proteolytic processing region that extends from residue 87 to residue 107 is a consensus bipartite nuclear processing signal, similar to that found in a n u m b e r of steroid h o r m o n e receptors and transcription factors (52-57). This targeting signal has been shown to be both necessary and sufficient for localization of PTHrP to the nucleus and nucleolus and similarly to mediate localization of heterologous peptides. Though the PTHrP signal sequence normally directs the nascent peptide into the secretory pathway via the endoplasmic reticulum, there appear to be circumstances u n d e r which this signal can be overridden and the resultant cytosolic PTHrP can then be directed into the nucleus (52-57). One possible mechanism by which such an "intracrine" pathway might be exercised entails the use of alternative translational initiation sites that effectively prevent the generation of a functional signal sequence. Another potential route by which PTHrP may be directed to the nucleus involves the receptor-mediated internalization of secreted peptide, a pathway that also appears to be d e p e n d e n t on the presence of the nuclear targeting signal and may be mediated by a receptor distinct from the classic type 1 P T H / P T H r P receptor (55-57). Evidence suggests that the biologic effects of nuclear PTHrP can be quite dissimilar to those transduced by interaction with the type 1 receptor, even within the same cell type (53).

POSTTRANSLATIONAL AND SECRETION

MODIFICATION

Although PTHrP contains no consensus sites for N-glycosylation, numerous serine and threonine residues are present that may serve as sites for O-glycosylation. As noted above, a heavily glycosylated fragment of PTHrP has been shown to be secreted by keratinocytes, but has not yet been demonstrated in other cell types (41). PTHrP also contains consensus sites at residues 87-91 and 94-97 for carboxy-terminal amidation by the enzyme peptidyl e~-monooxygenase

(28,29,58). Because peptide amidation is relatively c o m m o n and there is considerable overlap between the tissues that express PTHrP and those that express the amidating enzyme, this particular posttranslational modification of PTHrP seems likely to occur, but has not been formally documented. PTHrP is expressed by a wide variety of cell types, representing both the regulated and constitutive pathways of protein secretion. In n e u r o e n d o c r i n e cell types such as those found in the pancreatic islet, adrenal medulla, pituitary, parathyroid, and central nervous system, PTHrP is packaged into secretory granules, and in the parathyroid gland, evidence suggests that PTHrP is cosecreted with PTH (59). Also, in transfected rat insulinoma cells, midregion and carboxy-terminal fragments, as well as amino-terminal PTHrP, have been shown to colocalize with insulin and their release can be induced by secretagogues such as potassium chloride and leucine (36). In cell types such as keratinocytes, osteoblasts, chondrocytes, renal tubular cells, and smooth muscle cells, however, PTHrP is secreted via the constitutive movement of monensinsensitive secretory vesicles, so that its rate of release is d e p e n d e n t on its rate of synthesis (60). Interestingly, a comparison of the products secreted by cell lines transfected with cDNAs encoding the three h u m a n PTHrP isoforms (1-139, 1-141, and 1-173) and representing either the regulated (rat insulinoma cells) or the constitutive (Chinese hamster ovary cells) secretion pathways suggests that processing of PTHrP peptides can be both cell and isoform specific: though all six cell lines generated an amino-terminal (1-36) product, the CHO cell lines also contained a distinct amino-terminal peptide that was absent in the RIN lines, and both CHO and RIN lines expressing the 1-139 isoform secreted a unique carboxy-terminal peptide that was not secreted from the lines expressing other isoforms (35).

METABOLISM AND DEGRADATION Much is known about the metabolism and degradation of PTH, but this is not the case with PTHrP, although it is likely that there is some overlap in the pathways involved. PTH is predominantly cleared from the circulation by glomerular filtration, uptake by multifunctional endocytic receptors in the renal proximal tubules, and subsequent lysosomal degradation. Given the low circulating levels of PTHrP, this would seem unlikely to be a quantitatively significant disposal route u n d e r normal circumstances, but could well prove critical in conditions of PTHrP overexpression, such as humoral hypercalcemia of malignancy. In n o n h u m a n mammals, exogenous PTHrP fragments encompassing residues 1-34 and 1-86 were rapidly removed from the

PTHrP: GENE STRUCTUREAND BIOSYNTHESIS / circulation, with metabolic clearance rates in the range of 1.25-7.5 m l / m i n per kg, or only slightly slower than that of intact PTH in man (61). There is evidence, however, suggesting that the kidney may play a role in the normal disposal of PTHrE In patients with chronic renal failure, elevated levels of a carboxy-terminal PTHrP fragment can be found in the circulation, presumably due to impaired clearance (43). Further, studies in vitro have implicated the metalloprotease, meprin, in the degradation of carboxy-terminal PTHrP by the kidney (62). Another potential clearance mechanism for PTH is specific binding by the P T H / P T H r P type 1 receptor followed by receptor-mediated endocytosis (55, 57). Because most of the actions carried out by PTHrP appear to be restricted to the microenvironment surrounding its site of secretion, such a mechanism could well be an important means of turning off signal transduction. Unexpectedly, evidence has suggested that although PTHrP is a secreted protein, the precursor propeptide in the endoplasmic reticulum can gain access to the cytoplasm and there serve as a target for ubiquitin conjugation and sub-sequent proteosome-mediated degradation (63,64). Neither the pro sequence nor a carboxy-terminal PEST degradation motif appeared to be required as cis-acting determinants, although carboxy-terminal sequences have been implicated in the intracellular degradation of the fulllength peptide (65). In cell culture studies, both native PTHrP secreted into the medium and added synthetic PTHrP peptides undergo rapid degradation even in the presence of multiple protease inhibitors, suggesting that many cell types secrete proteases capable of progressive cleavage and inactivation of this peptide (66). PTHrP secreted into the extracellular milieu in vivo may also be labile, which would be in keeping with the short half-life of its mRNA. Overexpression of PTHrP at 10 to 20 times the normal level in the epidermis of transgenic mice does not lead to hypercalcemia nor does it generate detectable levels of the peptide in the circulation (67). Proteases known to cleave aminoterminal PTHrP and thus abolish bioactivity include kexin and prostate-specific antigen (68,69).

CONTROL ELEMENTS AND REGULATION OF GENE TRANSCRIPTION

As might be expected, given the size and complexity of the human PTHrP gene, the 5' controlling region contains numerous consensus regulatory sequences in addition to the two TATA-containing promoters and the GC-rich initiator element (14-18) (Fig. 6). Included among these are the following sites: (1) two enhancer sequences (CAAT boxes) within the long intervening sequence between exons 2 and 3, (2) multiple regions (5' to exon 1, within intron 2, and in exons 3 and 4) that contain potential binding sites for the transcription factors, Spl, AP-1 and AP-2, (3) sequences resembling cyclic AMP response elements 5' to exon 1 and within exon 4, and (4) sequences equivalent to glucocorticoid responsive half-sites, located both 5' to and within exon 1, and within intron 1, intron 2, and exon 3. It should be emphasized, however, that many of these sites have been identified on the basis of sequence alone, and not by functional assays. Information about the mechanisms of PTHrP gene regulation has been obtained principally with two complementary approaches: (1) by analysis of biological systems that display aberrant or dysregulated expression, such as cancer cells, and (2) by testing the activity of reporter genes (such as chloramphenicol acetyltransferase or luciferase) under the control of progressive deletions of PTHrP gene promoter regions in transfected cell lines. In adult T cell leukemia/lymphoma (ATLL), infection of T lymphocytes with the HTLV-1 retrovirus results in humoral hypercalcemia of malignancy through the production of PTHrP by tumor cells (70-76). A viral protein required for replication, Tax1, has been shown to transactivate PTHrP gene expression from the P2 promoter by acting at through an upstream binding site for the transcription factor Ets-1 ( - 7 3 to - 6 5 relative to the P2 transcriptional initiation site). Effective up-regulation was dependent on the presence of both Ets-1 and Taxl (70). Subsequent work has also established that P2 basal activity requires binding of the transcription factor Spl at an adjacent site,

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40

/

CHAPTER3

and that Ets-1 interacts directly with both Tax1 and Spl to form a cooperative ternary complex that interacts with the DNA binding site and then transcriptionally activates the promoter (71,72). Transactivation of the PTHrP gene was abolished by deletion of the carboxy terminus of the Tax protein and was also found to be d e p e n d e n t on an interaction between Ets-1 and a site within the amino terminus of Tax (72,73). Similarly, overlapping Ets-1 and Spl sites in the immediate upstream region of the mouse PTHrP gene ( - 8 8 to - 5 8 relative to the transcriptional start site) have been shown to be essential for retinoic acid-induced expression in embryonal carcinoma and embryonal stem cells (74). Additional studies with the h u m a n gene in this system have also shown up-regulation of expression by prostaglandin El, acting via a cAMP-dependent pathway, and by interleukin-2 (IL-2) and the transcription factor AP1/c-jun (73,75,76). Although PTHrP is a normal secretory product of a variety of squamous epithelia, including epidermal keratinocytes, only a subset of squamous carcinomas express the gene at levels sufficient to cause humoral hypercalcemia, raising the possibility that the nature of the transforming events could have an impact on PTHrP gene expression. Comparison of PTHrP expression levels with p53 functional status in a series of squamous carcinoma lines has revealed an association between the loss of p53 function and high levels of PTHrP mRNA (77). Evaluation of p53 isoforms with stabilizing mutations showed them to be capable of repressing PTHrP gene expression in a p53-negative squamous line and, correspondingly, inactivation of an endogenous, stabilized p53 gene product by the introduction of adenoviral E1B genes resulted in an increase in PTHrP expression. Conversely, mutant isoforms of p53 displaying a denatured, rather than stabilized, conformation were found to activate PTHrP gene expression (78). Both repression and activation of the human PTHrP gene by p53 appeared to occur primarily at the two TATA-based promoters. Finally, analysis of a spontaneously immortalized murine keratinocyte line trans-

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formed with adenoviral 12S EIA has shown certain key domains within this E1A gene product to effect marked repression of PTHrP gene expression by acting directly on the mouse TATA promoter (79). Similar analysis with a 13S E1A product identified an additional domain that served as a potent activator of PTHrP gene expression by acting through an Ets-1 site residing approximately 70 bp upstream of the transcriptional start site. Other oncogenes have also been found to affect PTHrP gene expression directly. The direct introduction of activated H- or K-ras oncogenes into a variety of cell lines has been shown to result in overexpression of PTHrP and transfection of cells with a constitutively activated tyrosine kinase oncogene, Tpr-Met, has been shown to enhance PTHrP transcription through the ras signaling pathway (80,81). Furthermore, the induction mechanism appeared to be dependent on ras processing events such as isoprenylation and farnesylation (81,82). In another system, analysis of a panel of renal carcinoma cell lines revealed a series of four CpG dinucleotide sites within the 5' flanking region of the human PTHrP gene that were consistently unmethylated in all PTHrP-expressing lines and methylated in all PTHrP-nonexpressing lines examined (83) (Fig. 7). Together these sites appear to constitute a minimal, methylation-free zone of approximately 550 bp, which resides approximately 1 kb upstream of the GC-rich initiator element and serves as a critical control switch for transcription from all three promoters. Furthermore, treatment of PTHrP-nonexpressing lines with the nucleoside analog, 5-azacytidine, effectively demethylated the identified critical sites and concurrently activated PTHrP gene transcription, thus strongly reinforcing the concept of a regulatory relationship. Also in this system, a 900-bp CpG island was identified in the proximal promoter region overlapping the 5' end of exon 3, which remained methylation-free regardless of PTHrP expression status (83). Work in a squamous carcinoma line from human lung, however, showed persistence of PTHrP gene expression despite

q~

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FIG. 7 Methylation pattern of the human PTHrP gene. Sites were identified in a panel of human renal carcinoma cells by using methylation-sensitive restriction enzymes. The half-filled circles correspond to CpG dinucleotides that were found to be unmethylated in cell lines expressing the PTHrP gene and methylated in cells not expressing the gene. Other CpG dinucleotides were found to be methylated (solid circle) or unmethylated (open circles), regardless of the status of PTHrP gene expression. The CpG island is indicated by a black bar. Modified from Holt et aL (83). Holt, EH., Vasavada, R., Broadus, AE., Philbrick, WM. Region-specific methylation of the PTH-related peptide gene determines its expression in human renal carcinoma lines. J Biol Chem 1993;268:20639-20645.

P T H r P : GENE STRUCTURE AND BIOSYNTHESIS

methylation of the distal two-thirds of the CpG island (84), leaving open the possibility that the methylation status of the 3' portion of the island may have regulatory implications. Control sequences that regulate the degree of basal expression have also been examined. Deletion experiments have identified a n u m b e r of negative regulatory elements operative in both normal and neoplastic cell lines in the regions from 3.8 to 2 kb and 1.1 to 0.35 kb upstream of exon 1 and 1.3 to 0.63 kb upstream of exon 3 (18). An additional regulatory element has been found within a segment from 0.63 to 0.34 kb upstream of exon 3 and appears to act as a repressor of the GC-rich initiator, because deletion of this region leads to increased levels of mRNA transcripts originating from this p r o m o t e r (85-87). Interestingly, this same region has been reported as a positive regulatory element when evaluated in constructs in which reporter gene expression is solely u n d e r the control of the downstream P2 p r o m o t e r (18), suggesting that this one region can differentially regulate two distinct promoters. Relative expression profiles of the three h u m a n PTHrP promoters both in context and in isolation suggest that the sequences within the GC-rich initiator element contribute to the activity of the two TATAcontaining promoters, as well as to the activity of the initiator element (86-88). Further dissection of the sequences within the GC-rich initiator element revealed a prototypical bipartite construction with an internal transcription initiation site s u r r o u n d e d by numerous Spl and AP-2 sites. The two halves of the core element appeared to be functionally equivalent, whereas a region extending from approximately 180 to 340 bp upstream of the GC-element transcriptional initiation site played a positive regulatory role (88). Despite the obvious potential for the generation of discrete, tissue-specific patterns of h u m a n PTHrP gene p r o m o t e r usage or alternative termination, this has not proved to be a c o m m o n finding in vivo. Examination of p r o m o t e r usage (P1 and P2) in a large n u m b e r of benign and malignant tissues (including esophagus, stomach, cecum, liver, pancreas, thyroid, parathyroid, adrenal, and kidney) found no consistent patterns (85,89). Relative differences in PTHrP p r o m o t e r usage do exist, however; in squamous carcinoma cells and keratinocytes, P1 was found to be more active than either P2 or the GC promoter; in renal carcinoma cells, the GC p r o m o t e r appears to be preferred; and in the uterus, P2 and the GC p r o m o t e r are used exclusively (87,88). Similarly, no consistent patterns in 3' exon usage have emerged, although exon 6 appears to be more prevalent in prostate cancer than in normal tissue (90), and has also been reported to be associated with breast cancer metastasis (91). Studies of PTHrP gene regulation have found two primary patterns of response, a rapid, transient

//

41

increase in PTHrP mRNA levels or a delayed, sustained up-regulation. Examples of the former include the response to serum and angiotensin II in smooth muscle cells (92,93), mechanical stimuli in aortic smooth muscle cells (94), serum factors in pancreatic islet cells (95) and keratinocytes (96), endotoxin in mouse spleen (97), and ischemia in rat kidney (98). In this response pattern, steady-state levels of PTHrP mRNA typically display a rapid increase within 1 or 2 hours after stimulation, reach a peak at 4 to 6 hours, and then quickly decline due to the short half-life of the mRNA. Examples of a delayed, sustained induction of PTHrP gene expression include the response to estrogen in rat myometrial cells (99); transforming growth factor [3 (TGF-[3) in h u m a n myometrial and endometrial cells in primary culture (100), mechanical stimuli in the bladder, rat uterus, and avian oviduct (101-104); and cAMP in embryonal cells and trophectoderm (105). The generation of this type of response would require a perpetuation of the transcriptional induction, an increase in the stability of the mRNA, or some combination of the two. A n u m b e r of hormones, cytokines, growth factors, and second messengers have been shown to induce PTHrP gene expression; more often then not, these effects tend to follow the rapid, transient response pattern (Table 1). Examples include estradiol in rat kidney, uterus, pituitary, and hypothalamus (106-108); estradiol and tamoxifen in the MCF-7 breast carcinoma cell line (109); calcitonin in lung carcinoma cell lines (110); TNFot and IL-113 in h u m a n umbilical vein endothelial cells (111); forskolin, cAMP, and IL-2 in HTLV-infected T cells (75,76); bradykinin, serotonin, endothelin, thrombin, and n o r e p i n e p h r i n e in smooth muscle cells (92,94); and prolactin in m a m m a r y tissue (112). As noted above, PTHrP gene expression has been shown to be induced by serum in a n u m b e r of cell types, including rat osteosarcoma (ROS) cells. Induction is rapid (peaking within 4 hours) and is mediated in part via a transcriptional mechanism (113), although a substantial effect on mRNA stability has also been observed in ROS cells (see Posttranscriptional Regulation, below). T h o u g h a prototypical serum response element of the c-fos/[3-actin type is not found in the rat PTHrP gene, the serumresponsive region has been localized to a segment extending from 0.3 to 1.05 kb upstream of the transcriptional start site (113). The effects of serum in this system may be mediated, at least in part, through insulin and epidermal growth factor, because both these factors have been shown to stimulate a rapid induction of PTHrP mRNA. Likewise, inhibition of the angiotensin II receptor greatly attenuated the seruminduced rise in PTHrP gene expression in vascular smooth muscle cells (94), suggesting a primary regulatory role for that factor. Phorbol esters, which activate

42

/

C~a'TF~R3

protein kinase C isozymes, have been reported to strongly up-regulate PTHrP mRNA in several cell types (114-116). Correspondingly, treatment of h u m a n myometrial smooth muscle cells with okadaic acid, an inhibitor of the serine/threonine protein phosphatases 1 and 2A, also effected a marked increase in PTHrP gene expression (116). In contrast to the more prevalent pattern of rapid induction, TGF-[3 has been found to provoke a slow, sustained rise in PTHrP mRNA levels that is maximal by 12 to 24 hours in several cell lines, including h u m a n keratinocytes, renal carcinoma cells, primary endometrial and myometrial cells, and mouse bone organ culture (100,117,118). Conversely, TGF-[3 has also been shown to decrease PTHrP gene expression in immortalized murine endochondral chondrocytes, a property shared by several bone morphogenetic proteins (BMP-2,-5 a n d - 7 ) and opposed by basic fibroblast growth factor (119). There are also examples of negative regulation of PTHrP gene expression (Table 1). The active vitamin D metabolite (1,25-dihydroxyvitamin D~) and two glucocorticoids (dexamethasone and triamcinolone) have been shown to decrease steady-state levels of PTHrP mRNA in a time- and dose-dependent m a n n e r in a human medullary thyroid carcinoma cell line and a lung carcinoid line (120,121). The glucocorticoid effect was completely blocked by the competitive antagonist, RU-486. Nuclear runoff and transcriptional inhibition experiments indicated that neither vitamin D nor glucocorticoids appeared to influence mRNA stability, but that these agents acted by repressing the rate of PTHrP gene transcription. Two noncalcemic analogs of vitamin D, EB1089 and 22-oxacalcitriol, were also found to suppress both basal and s e r u m - o r EGF-stimulated PTHrP gene expression in a lung squamous cancer cell line through a transcriptional mechanism, thus raising the possibility of therapeutic potential (122). In the rat PTHrP gene, two vitamin D-responsive elements (VDREs) have been localized approximately 1 kb upstream of the transcriptional start site; one element resembling a canonical positive regulatory VDRE was identified by both DNA-protein binding studies and functional assays, and a second element bearing similarity to the negative regulatory VDRE in the human PTH gene was identified by binding studies alone (123,124). Treatment with 9-cis-retinoic acid has also been shown to repress the transcriptional activity of the human PTHrP gene and mobility-shift experiments with the rat gene suggest that the effects of vitamin D may be mediated through the binding of a heterodimer composed of the vitamin D receptor and the retinoid X receptor (125). PTHrP has been localized to a n u m b e r of discrete cell populations in the central nervous system and evidence suggests that PTHrP gene expression in these neuronal cell types is up-regulated by excitation

(126,127). In primary cultures of cerebellar granule cells, for example, it has been shown that PTHrP gene expression can be induced by potassium ion-dependent membrane depolarization and the subsequent entry of extracellular C a 2+ into the cell through L-type voltagesensitive calcium channels; depolarization with sodium ionphores o r C a 2+ entry by other routes proved ineffective (128). The induction of PTHrP gene expression in this system appears to be mediated through the C a 2+ calmodulin kinase pathway in a m a n n e r similar to that for the c-fos gene. The L-type voltage-sensitive calcium channels (L-VSCCs) and the C a 2+ calmodulin kinase cascade also appear to be involved in excitation secretion coupling in neuroendocrine cells and in excitation-contraction coupling in skeletal, cardiac, and smooth muscle (129). Interestingly, the role played by PTHrP in the central nervous system appears to be neuroprotective in nature, based on experiments showing that the peptide serves as a highly effective inhibitor of L-VSCC-associated C a 2+ influx and consequent neuronal toxicity (see Chapter 16). PTHrP has also been shown to be expressed in smooth muscle types from a n u m b e r of organs, including the gastrointestinal tract, bladder, myometrium, vasculature, and chicken oviduct. In all of these sites, PTHrP gene expression appears to be induced by a variety of mechanical stimuli, such as balloon angioplasty in the aorta (94), atherosclerotic stenosis in the coronary arteries (130), stretch (131) or shear flow (94) in vascular smooth muscle cells in culture, distension of the uterus by fetal growth or balloon inflation (103), expansion of the chicken oviduct during egg transit (104), expansion of the bladder by urinary volume (101), and distension of the stomach by pyloric ligation and subsequent gastric filling (132) (Fig. 8). Accumulating evidence indicates that this mechanotransduction is mediated through the opening of stretch-activated cation channels, subsequent ion influx and membrane depolarization, and the resultant activation of L-type voltage sensitive calcium channels (133). PTHrP gene expression is thus induced and the secreted peptide then serves as a muscular relaxant and vasodilator, thereby constituting a feedback system to regulate muscular tone (see Chapter 16). The rapid induction-deinduction kinetics that typify PTHrP mRNA responses in many systems are reminiscent of the kinetics associated with so-called primary response or immediate early genes, which include many protooncogenes, cytokines, and growth factors (96,134). The transient nature of the induction serves to limit the translational yield of the resultant proteins and thus to restrict the biologic effects of these powerful regulatory molecules. The induction of PTHrP gene expression by 1713-estradiol in rat GH4C1 pituitary cells has been shown to display many of the features of the

PTHrP: GENE STRUCTURE AND BIOSYNTHESIS //

TABLE 1 Regulation of PTHrP Gene Expression a Stimulus

Physiologic Suckling Uterine occupancy Stretch Stretch Stretch Egg-laying cycle Differentiation Differentiation Differentiation Differentiation Pharmocologic Glucocorticoids Glucocorticoids Glucocorticoids Glucocorticoids 1,25(OH)2D 1,25(OH)2D 1,25(OH)2D 22-oxa-1,25(OH)2D Estrogen Estrogen Estrogen Estrogen Estrogen Serum Serum Serum Serum Growth factors EGF EGF IGF-I TGF-13 TGF-13 TGF-13 TGF-13 Prolactin Cycloheximide Cycloheximide Cycloheximide Tax Forskolin Calcitonin Phorbol ester Phorbol ester Endothelin-I Thrombin Angiotensin II

Tissue/cell type

mRNA/protein

Lactating breast (rat) Myometrium (rat) Myometrium (rat) Urinary bladder (rat) Amnion (human) Oviduct (chicken) Insulinoma (rat) Embryonal carcinoma Keratinocyte (human) Trophoectoderm (mouse)

1" 1" 1" 1" T 1" T 1" T 1"

Carcinoid (human) Insulinoma (rat) Aortic smooth muscle (rat) Keratinocyte (rat) Medullary carcinoma (human) Keratinocyte (human) Keratinocyte (rat) T cell (human) Uterus (rat) Pituitary/hypothalamus (rat) Myometrial cell (rat) Pituitary GH4C1 (rat) Kidney (monkey) Insulinoma (rat) Keratinocyte (human) Keratinocyte (rat) Aortic smooth muscle Keratinocyte (human) Keratinocyte (rat) Mammary epithelial (human) Mammary epithelium (human) Renal carcinoma (human) Myometrial (human) Endometrial (human) Keratinocyte (rat) Mammary gland (rat) Multiple (rat and human) Osteosarcoma (human) Insulinoma (rat) T cells (human) T cell MT-2 (human) Squamous carcinoma (human) Osteosarcoma (human) T cells (human) Aortic smooth muscle (rat) Aortic smooth muscle (rat) Aortic smooth muscle (rat)

,I, $ $ $ J, $ ,1, $ T T 1" T T 1" 1" T 1" T 1" 1" 1" T T 1" T 1" T T 1" T 1" T T 1" 1" T T

aModified from Broadus AE, Stewart AF. Parathyroid hormone-related protein structure, processing, and physiological actions. In: Bilezikian The parathyroids. New York: Raven, 1994:259-294.

43

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CHAPTER3

FIG. 8 Induction of PTHrP mRNA during gestation in rat uterus. Little PTHrP gene expression is detected in the nongravid uterus by Northern blot analysis. With progressive distention of the uterus during gestation, steady-state levels of PTHrP mRNA are markedly induced. After parturition on day 21, PTHrP gene expression declines precipitously and continues to fall throughout the postpartum period. When pregnancy is prolonged by the administration of progesterone, PTHrP mRNA levels continue to rise. Reproduced from Thiede et aL (103). Thiede MA, Daifotis AG, Weir EC, Brines ML, Burtis W J, Ikeda K, Dreyer BE, Garfield RE, Broadus AE. Intrauterine occupancy controls expression of the parathyroid hormone-related peptide gene in pre-term rat myometrium. Proc Natl Acad Sci USA 87:6969-6973, 1990.

primary response pattern (134). Steady-state mRNA levels peaked at 1 to 2 hours due to a burst of transcription that was maximal at 20 to 40 minutes and declined thereafter. The 30-minute half-life of PTHrP mRNA in these cells, although unaffected by estradiol, was sufficiently short to mediate the rapid decline in steady-state mRNA levels. Inhibition of protein synthesis by treatm e n t with cycloheximide had no effect when used alone and failed to block the estradiol-mediated induction of PTHrP gene expression, but eliminated the transcriptional arrest, thus implicating the action of a labile transcriptional repressor protein that is estrogen inducible (134). As is the case for other primary response genes, the combination of a rapid post stimulation repression of gene transcription with a short mRNA half-life is essential to the generation of the transient response.

POSTTRANSCRIPTIONAL

REGULATION

A further measure of control over PTHrP gene expression appears to be exerted at the level of mRNA stability. The steady-state mRNA levels of all genes represent the p r o d u c t of both the rate of transcription and the rate of degradation. T h o u g h the transcription rate of the PTHrP gene is similar to that of the actin gene, steady-state levels of PTHrP mRNA are extremely low in most tissues (estimated to be from 0.001 to 0.01% of mRNA), as a result of rapid turnover (19,23,135-137). M e a s u r e m e n t of the half-life of PTHrP mRNA in a variety of tissues has ranged from 30 minutes to several

hours, kinetics that are similar to those for a n u m b e r of cytokines and protooncogenes. For such factors, a rapid rate of degradation serves to allow a rapid response to the transcriptional down-regulation of the gene, both in terms of mRNA levels and consequent biologic effects. The sequences responsible for mediating mRNA instability in many of these genes are AU-rich elements (AREs) typically found in the 3' untranslated regions of these genes (138). These motifs are also present in all PTHrP mRNAs characterized thus far; multiple iterations of the core element, AUUUA, can be found in all three alternative h u m a n PTHrP 3' transcriptional termini (exons 6, 7, and 8), as well as in the 3' untranslated regions (UTRs) of PTHrP mRNAs from the mouse, rat, and chicken. There is also some functional evidence to suggest that the relative stability of h u m a n PTHrP mRNAs is mediated through the 3' UTRs, with transcripts containing exon 8 typically displaying a more rapid degradation rate than those containing exons 6 or 7 (139,140). Transfection of fibroblasts or keratinocytes with each of the three alternative h u m a n PTHrP 3' UTRs fused to a luciferase reporter gene also showed transcripts containing exon 8 to be the most unstable, although this was d e p e n d e n t on the cell line used (141). A n u m b e r of studies looking at AU-rich elements in other genes, however, have indicated that the core AUUUA motif is insufficient by itself to mediate instability and that the m i n i m u m consensus element is an octamer or n o n a m e r (142,143), neither of which is found in PTHrP mRNAs. This implies that instability in this system may be mediated by non-AUUUA AREs or by elements other than AREs. Finally, there are data to suggest that the degradation rate of PTHrP mRNA is a regulated p h e n o m e n o n , because a n u m b e r of factors, including serum, TGF-[3 and epidermal growth factor (EGF), have been reported to affect stability (139,144-146). Preliminary evidence indicates that the TGF-[3-dependent stabilization of h u m a n PTHrP mRNAs may be mediated through cis-acting sequences that are not contained in the 3' UTR, but rather reside within the coding region (146). The inhibition of protein synthesis has been shown to result in the superinduction of PTHrP mRNA expression in a n u m b e r of h u m a n and rat cell lines (95,96,114,147). T h o u g h there is often a transcriptional c o m p o n e n t in this p h e n o m e n o n , the effect is mediated in large part at the posttranscriptional level and is presumed to reflect the reduced synthesis of critical components in the mRNA degradation/instability pathway.

DEVELOPMENTAL REGULATION The pattern of PTHrP gene expression in the adult is widespread and is even more so in the fetus (Table 2).

PTHrP: GENE STRUCTURE AND BIOSYNTHESIS /

TABLE 2

45

PTHrP Gene Expression during Embryogenesisa

Chicken

Mouse and rat

Human

Days 3-10 Viscera Allantois Yolk sac Chorioallantoic membrane Day 15 Brain Heart Lung Liver Gizzard Intestine Skeletal muscle

Day 3mcompacted morula Day 7.5mtrophoblast Days 8-12mplacental decidua Days 13-14 Epidermis, skin appendages Skeletal, cardiac muscle Vascular smooth muscle Liver parenchyma Renal tubular epithelium Bronchiolar epithelium Gastrointestinal epithelium Choroid plexus, spinal cord, dorsal root ganglia, and eye Days 15-16 Lung epithelium Perichondrium Dental lamina, inner ear Day 18 Salivary ducts Pancreatic ducts, islets Day 20.5--keratinocytes

Weeks 7-8 Trophoblastic layers of chorionic villi Lung epithelium Liver parenchyma Pancreatic acini Stomach epithelium Hindgut epithelium Kidney Perichondrium Epidermis Otic placode Tooth bud Choroid plexus, spinal cord, dorsal ganglia Weeks 18-20 Cardiac, skeletal muscle Vascular smooth muscle Endochondral and intramembranous bone

aModified from Broadus AE, and Stewart AF. Parathyroid hormone-related protein structure, processing, and physiological actions. In: Bilezikian JP, eds. The parathyroids. New York: Raven, 1994:259-294.

Cumulative data from a large n u m b e r of localization studies in fetal tissues allow the following generalizations: (1) PTHrP mRNA a n d / o r peptide can be found in almost every embryonic tissue examined, but are restricted to certain cell types within those tissues, (2) the types of tissues that express PTHrP encompass derivatives of all three germ layers, as well as extraembryonic sites, such as the amnion and trophoblast (148,149), (3) the levels a n d / o r locations of PTHrP gene expression are not static, but change as a function of developmental stage, and (4) at most sites of expression, both PTHrP mRNA and peptide are present in low abundance and require sensitive methods for detection. Indeed, it was precisely this pattern of near ubiquitous fetal expression, coupled with both spatial and temporal specificity, that first suggested that PTHrP was likely to be a factor involved in the regulation of growth and differentiation. The P T H / P T H r P type 1 receptor is also widely expressed during fetal life (150) and studies in the mouse suggest that PTHrP and the PTH1 receptor represent one of the earliest h o r m o n e receptor pairs operative in development (105). Mso, in most tissues, PTH and PTHrP are coordinately expressed in adjacent

cell layers (typically, PTHrP displays focal expression in the surface epithelium, but the receptor is expressed diffusely in the underlying mesenchyme), a pattern that is consistent with our emerging understanding of their paracrine interactions (150). Localization studies in preimplantation mouse embryos have shown that PTHrP can be detected as early as the compacted morula stage before the onset of e n d o d e r m a l differentiation (105) (Fig. 9) and that the peptide serves as an early marker for cells of the trop h e c t o d e r m lineage. PTHrP gene expression is also induced on differentiation of F9 embryonal carcinoma stem cells into parietal endoderm-like cells (137). Other studies with cultured cells, including keratinocytes and pancreatic islet tumor cells, have also shown up-regulation of PTHrP mRNA or protein when differentiation is stimulated (95,151). The temporal sequence of the tissue-specific acquisition of PTHrP gene expression during the fetal development of the rat has been carefully examined by in situ hybridization (150) (Fig. 10). By El5 to El6, PTHrP mRNA was found to be most highly expressed in the epidermis, hair follicles, and the enamel epithelium of the tooth

46

/

CHAPTER3

i~f ~ ~ ' "~....

~:~ ....

~:.:...~:~:~:,~...~a,,,

% ~ ~.......... ..

FIG. 9 Acquisition of PTHrP gene expression in preimplantation mouse embryos. Whole embryos were subjected to immunofluorescent staining with an anti-PTHrP antibody. (A) The 8-cell stage; (B) compacted morula; (C) blastocyst. Left panels: Nomarsky image. Right panels: fluorescent image. Preparations were viewed with confocal scanning laser microscopy. Reproduced from Van de Stolpe et aL (105), The Journal of Cell Biology, 1993, Vol. 120, pp. 235-243, by copyright permission of The Rockefeller University Press.

buds. Lower levels of expression were apparent in the epithelia of the inner ear and nasal cavity, the bronchial epithelium of the lung, the ependymal cells of the choroid plexus, and the endocardial cushion region of the heart. Diffuse mesenchymal expression could also be detected in some developing organ systems at this stage, but this expression diminished over time. Expression of PTHrP along the border of the intestinal epithelium could not be detected until E20. In the skeleton at E15-E16, hybridization was seen in the membranous bone forming within the mandible, in early chondrocytes of the cartilage primordia of endochondral bones such as the ribs and digits, and in the perichondrium of the sternum and the periosteum of the clavicle. By El8, expression could also be detected in the hypertrophic chondrocytes of the developing growth plates (150).

FIG. 10 Expression of PTHrP and the PTH/PTHrP receptor during development of the rat. Tissue sections were subjected to in situ hybridization and emulsion autoradiography. Darkfield views are shown. (Top) Whole rat fetus from day E15. (Bottom) Craniofacial portion of day E19 fetus. Labels: rP, PTHrP; R, PTH/PTHrP receptor; CP, choroid plexus; E, inner ear; TB, tooth bud; L, lung; H, heart; I, intestine; MC, Meckel's cartilage; Md, mandible; Mx, maxilla. Reproduced from Lee et aL (150). Lee K, Deeds JD, Segre GV. Expression of parathyroid hormone-related peptide and its receptor messenger ribonucleic acids during fetal development of rats. Endocrinology 1995; 136:453-463.

Longitudinal surveys have revealed a number of instances in which developmental progression is accompanied by changes in either the quantitative levels or the spatial patterns of PTHrP gene expression (150,152-158). In both the human and rat kidney, early expression of PTHrP is evident in the glomeruli and in the tubular epithelium of the mesonephros and metanephros. Soon after, however, glomerular expression begins to decline, and by midgestation, expression is limited to the proximal and distal tubules, the collecting duct, and the urothelium (152-155,158). As noted above, in the developing skeletal system of the rat, PTHrP is expressed in the early mesenchyme and cartilage primordia of the ribs, limbs, and vertebrae. As maturation proceeds, however, expression of the pep-

P T H r P : GENE STRUCTURE AND BIOSYNTHESIS

tide becomes increasingly restricted to hypertrophic chondrocytes, osteoblasts, and areas of the perichondrium and bone collar (150,152,154). Similar changes in PTHrP expression patterns have been observed to occur in the lung, liver, and dental lamina. The stage-specific acquisition of PTHrP gene expression in a number of organ systems, especially the developing epidermis, mammary gland, placenta, endochondral bone, tooth, and central nervous system, has been shown to correlate with distinct developmental functions, many of which are now becoming increasingly well understood. These varied roles of PTHrP in regulation of programmed differentiation and physiologic responses are detailed in succeeding chapters.

ACKNOWLEDGMENTS This work was supported by National Institutes of Health grants DE12616, AR46032, and DK45735.

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hormone-related protein expression by oral cancer cells (HSC-3). J Endocrino11998;156:349-35 7. Weaver DR, Deeds JD, Lee K, Segre GV. Localization of parathyroid hormone-related peptide (PTHrP) and PTH/PTHrP receptor mRNAs in rat brain. Mol Brain Res 1995;28:296-310. Weir EC, Brines ML, Ikeda K, Burtis WJ, Broadus AE, Robbins RJ. Parathyroid hormone-related peptide gene is expressed in the mammalian central nervous system. Proc Natl Acad Sci USA 1990;87:108-112. Holt EH, Broadus AE, Brines ML. Parathyroid hormone-related peptide (PTHrP) is produced by cultured cerebellar granule cells in response to L-type voltage-sensitive Ca 2+ channel flux via a CaZ+/calmodulin-dependent kinase pathway. J Biol Chem 1996;271:28105-28111. Moreno Davila H. Molecular and functional diversity of voltagegated calcium channels. Ann NYAcad Sci 1999;868:102-117. Nakayama T, Ohtsuru A, Enomoto H, Namba H, Ozeki S, Shibata Y, Yokota T, Nobuyoshi M, Ito M, Sekine I, Yamashita S. Coronary atherosclerotic smooth muscle cells overexpress human parathyroid hormone-related peptides. Biochem Biophys Res Commun 1994;200:1038-1045. Noda M, Katoh T, Takuwa N, Kumada M, Kurokawa K, Takuwa Y. Synergistic stimulation of parathyroid hormonerelated peptide gene expression by mechanical stretch and angiotensin II in rat aortic smooth muscle cells. J Biol Chem 1994;269:17911-17917. Ito M, Ohtsura A, Enomoto H, Ozeki S-I, Nakashiima M, Nakayama T, Shichijo K, Sekine I, Yamashita S. Expression of parathyroid hormone-related peptide in relation to perturbations of gastric motility in the rat. Endocrinology 1994;134:1936-1942. Calaghan SC, White E. The role of calcium in the response of cardiac muscle to stretch. Prog Biophys Mol Bio11999;71:59-90. Holt EH, Lu C, Dreyer BE, Dannies PS, Broadus AE. Regulation of parathyroid hormone-related peptide gene expression by estrogen in GH4C1 rat pituitary cells has the pattern of a primary response gene. J Neurochem 1994;62:1239-1246. Ikeda K, Mangin M, Dreyer BE, Webb AC, Posillico JT, Stewart AF, Bander NH, Weir EC, Insogna KL, Broadus AE. Identification of transcripts encoding a parathyroid hormonelike peptide in messenger RNAs from a variety of human and animal tumors associated with humoral hypercalcemia of malignancy. J Clin Invest 1988;81:2010-2014. Ikeda K, Weir EC, Mangin M, Dannies PS, Kinder B, Deftos LF, Brown EM, Broadus AE. Expression of messenger ribonucleic acids encoding a parathyroid hormone-like peptide in normal human and animal tissues with abnormal expression in human parathyroid adenomas. Mol Endocrino11988;2:1230-1236. Chan SDH, Strewler GS, King KL, Nissenson RA. Expression of a parathyroid hormone-like protein and its receptor during differentiation of embryonal carcinoma cells. Mol Endocrinol 1990;4:638-646. Sheng M, Greenberg ME. The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron 1990;4:477-485. Heath JK, Southby J, Fukumoto S, O'Keeffe LM, Martin TJ, Gillespie MT. Epidermal growth factor-stimulated parathyroid hormone-related protein expression involves increased gene transcription and mRNA stability. BiochemJ 1995;307:159-167. SouthbyJ, Heath JK, O'Keeffe LM, Martin TJ, Gillespie MT. The 3' untranslated sequence of the human PTHrP gene confers differential mRNA stability. J Bone Miner Res 1995;10 (Suppl. 1):$279. Campos RV, Zhang L, Drucker DJ. Differential expression of RNA transcripts encoding unique carboxy-terminal sequences

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of human parathyroid hormone-related peptide. Mol Endocrinol 1994;8:1656-1666. Knlys V, Huez G. Translational control of cytokine expression by 3' AU-rich sequences. Biochimie 1994;76:862-866. Zubiaga AM, Belasco JG, Greenberg ME. The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation. Mol Cell Bio11995;15:2219-2230. Zakalik D, Diep D, Hooks MA, Nissenson RA, Strewler GJ. Transforming growth factor beta increases stability of parathyroid hormone-related protein messenger RNA. J Bone Miner Res 1992;7:S118. Kirayama T, Gillespie MT, Glutz JA, Fukumoto S, Moseley JM, Martin TJ. Transforming growth factor beta stimulation of parathyroid hormone-related peptide: A paracrine regulator? Mol Cell Endocrinol 1993;92:55-62. Sellers RS, Tannehill-Gregg SH, Capen CC, Rosol TJ. Cis-acting elements in the 3' untranslated and coding regions of parathyroid hormone-related protein mRNA mediate transforming growth factor-J3 induced stability. J Bone Miner Res 1999; 14 (Suppl. 1) :$546. Ikeda K, Lu C, Weir EC, Mangin M, Broadus AE. Regulation of parathyroid hormone-related peptide gene expression by cycloheximide. J Biol Chem 1990;265:5398-5402. Beck E Tucci J, Senior PV. Expression of parathyroid hormonerelated protein mRNA by uterine tissues and extraembryonic membranes during gestation in rats. J Reprod Fertil 1993;99:343-352. Karperien M, Lanser P, DeLaat SW, Boonstra J, DeFize LHK. Parathyroid hormone-related peptide mRNA expression during murine postimplantation development: Evidence for involvement in multiple differentiation processes. Int J Dev Biol 1996;40:599-608. Lee K, Deeds JD, Segre GV. Expression of parathyroid hormonerelated peptide and its receptor messenger ribonucleic acids during fetal development of rats. Endocrinology 1995;136:453-463. Kremer R, Karaplis AC, Henderson J, Gulliver W, Banville D, Hendy GN, Goltzman D. Regulation of parathyroid hormonelike peptide in cultured normal human keratinocytes. J Clin Invest 1991;87:884-893. Burton PBJ, Moniz C, Quirke P, Malik A, Bui TD,J/ippner H, Segre GV, Knight DE. Parathyroid hormone-related peptide: Expression in fetal and neonatal development. JPath 1992;167:291-296. Campos RV, Asa SL, Drucker DJ. Immunocytochemical localization of parathyroid hormone-like peptide in the rat fetus. Cancer Res 1991;51:6351-6357. Moniz C, Burton PBJ, Malik AN, Dixit M, Banga JP, Nicolaides K, Quirke P, Knight PE, McGregor AM. Parathyroid hormonerelated peptide in normal human fetal development. J Mol Endocrinol 1990;5:259-266. MoseleyJM, HaymanJA, DanksJA, Alcorn D, Grill V, SouthbyJ, Horton MA. Immunochemical detection of parathyroid hormone-related protein in human fetal epithelia. J Clin Endocrinol Metab 1991;73:478-484. Senior PV, Heath DA, Beck E Expression of parathyroid hormonerelated protein mRNA in the rat before birth: Demonstration by hybridization histochemistry. J Mol Endocrino11991 ;6:281-290. Schermer DT, Chan SDH, Bruce R, Nissenson RA, Wood WI, Strewler GS. Chicken parathyroid hormone-related protein and its expression during embryo loci development. JBone Miner Res 1991;6:149-155. Dunne FP, Ratcliffe WA, Mansour P, Heath DA. Parathyroid hormone-related protein (PTHrP) gene expression in fetal and extra-embryonic tissues of early pregnancy. Hum Reprod 1994;9:149-156.

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CHAPTER 4

Interactions of Parathyroid Hormone and Parathyroid Hormone-Related Protein with Their Receptors

MICHAEL CHOREV, JOSEPH M. ALEXANDER, AND MICHAEL ROSENBLATT

Division of Bone and Mineral Metabolism, Charles A. Dana and Thorndike Laboratories, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215

INTRODUCTION

plex protein structure occupying three distinct phases: the intracellular loops and carboxyl terminus (located in the cytoplasm), the seven hydrophobic membranespanning helices, and the extracellularly oriented N terminus and loops. The receptor is posttranslationally modified by N-glycosylation and disulfide bond formation, and u n d e r certain circumstances it can also be phosphorylated. Heterotrimeric G proteins are composed of a unique ot subunit, which binds GDP or GTP with high affinity, and which is associated with a [3y complex. H o r m o n e binding to the GPCR increases its affinity for the GDP-bound heterotrimeric G protein complex, which in turn activates it and causes a "GTP for GDP" exchange. The GTP-bound G protein separates from the receptor, and the Got-GTP subunit dissociates from the [3y complex. Both the G0t-GTP and the [3y dimer are able to interact with effectors such as adenylyl cyclase and phospholipase C. Hydrolysis of GTP to GDP by the GTPase activity of the e¢ subunit results in the dissociation of the oL subunit from the effector molecule, allowing its reassociation with the [3y dimer. G protein signaling is thus governed by the rates of GTP binding (catalyzed by the receptor) and GTP hydrolysis. This system is highly dynamic. The hormone has more diversified conformations when in solution than when membrane bound, and its conformation changes on interaction with the receptor. On ligand binding, the receptor may change conformation to allow global movements of transmembrane domains that lead to changes in the conformation of the cytoplasmic portions of the receptor. This, in turn, increases affinity

Obtaining a detailed understanding of structurefunction relations of a h o r m o n e - r e c e p t o r complex at the most fundamental level currently requires an interdisciplinary approach; state-of-the-art techniques in peptide and protein biochemistry, as well as cellular and molecular biology, must be utilized. Traditionally, most or all of the insights regarding the h o r m o n e - r e c e p t o r complex have been obtained by correlating the effects of structural modifications on function of either the hormone or the receptor molecule alone. The detailed mechanisms that explain how structural changes in either hormone or receptor can alter the h o r m o n e - r e c e p t o r bimolecular interaction or later in signaling events are only beginning to be understood. The complexity of the parathyroid hormone ( P T H ) - r e c e p t o r system presents a significant challenge, but investigations of this system have yielded novel insights, some of which may be generalizable to many members of the superfamily of G protein-coupled receptors (GPCRs). The P T H - r e c e p t o r system is composed of at least three major constituents: the linear peptide hormone, heptahelical transmembrane receptors, and heterotrimeric guanine nucleotide binding proteins (G proteins). PTH, a fully active form of which is a linear peptide 34 amino acids long, is a highly flexible molecule. Therefore, an assortment of low-energy conformations exists in fast dynamic equilibrium. Only a subset of these conformations is thought to be acceptable to the receptor for binding. The receptor is acomThe Parathyroids, Second Edition

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Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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toward the GDP-bound G protein. The formation of the hormone-receptor-G-protein ternary complex represents the initiation of the intracellular signaling cascade. In addition to h o r m o n e - r e c e p t o r - G protein interactions, there are important interactions of these components with other molecules, such as [3-arrestins, regulators of G-protein signaling (RGS) proteins that act as GTPase-activating proteins (GAPs), G proteincoupled receptor kinases (GRKs), and receptor activitymodifying proteins (RAMPs). Formation of GPCR homodimers and heterodimers may represent an additional mechanism for modulating receptor function. Several receptors, including the type 1 P T H / PTH-related protein (PTHrP) receptor (PTH1-Rc), can interact with more than one G protein (e.g., the PTH1Rc interacts with e i t h e r Gq o r G s proteins), but other GPCRs interact directly via their C-terminal domain with proteins containing primary decidual zones (PDZs) and Enable/vasodilator-stimulated phosphoprotein (VASP) homology-like domains. The complexity of the GPCR signaling system requires that studies of structure-function relations include direct analysis of multicomponent systems, such as the ligand-receptor complex, in order to achieve useful level of resolution. Analyzing the system at this level may provide insight into partially understood physiologic and pathophysiologic processes associated with the PTH/PTHrP-receptors system. These studies have the potential to identify new therapeutic targets and generate paradigms useful for the development of novel agents directed at ligand-receptor interactions. This chapter summarizes recent findings obtained from studies of either PTH ligands or receptors as single entities and the most recent data emerging from direct study of the complexes formed when PTH or PTHrP interact with the PTH1-Rc or PTH2-Rc. To provide a framework for understanding the most recent approach developed to study the PTH/PTHrP-receptors system, namely the "ligand-receptor-centered" approach, we review first the "ligand-centered" and "receptor-centered" approaches. Studying ligands and receptors separately also generates important insights. However, these concepts can only be validated when the hormone-receptor bimolecular interaction is examined directly.

LIGAND-CENTERED APPROACH Recent Advances in Structure-Activity Relations Extensive reviews have been published covering early work on structure-activity relations of PTH and PTHrP (1-3). It has been established for both these calciotrophic hormones that the N-terminal 1-34 amino

acid sequence of either is sufficient to induce the entire spectrum of in vitro and in vivo PTH1-Rc-mediated activities (4,5). PTH(1-34) and PTHrP(1-36) are equipotent for binding to the PTH1-Rc and for stimulating adenylyl cyclase and intracellular calcium transients in cells expressing the PTH1-Rc. Significant sequence homology is shared by residues 1-13 of both hormones (8 identical residues), though sequence homology is negligible for the 14-34 sequence. The assignment of "activation domain" to the homologous N-terminal sequences is based on demonstration that this region has a functional role in intracellular signaling, and that truncation of 2-6 residues from the N terminus converts an agonist peptide to an antagonist (6-8). The divergent mid- and C-terminal amino acid sequences contain the "binding domain" assigned to residues 14-34 (9-12). Based on these observations, it was hypothesized that N-terminal sequences comprising the activation and binding domains of both hormones share similar conformations despite their sequence differences (10,13). The early work on structure-activity relations of PTH and PTHrP has been reviewed previously (1,2); provided below is a summary of the more recent progress in this field.

Truncated Sequences Although amino-terminal fragments of PTH and PTHrP shorter than 1-27 were initially reported to be devoid of biological activity (14-17), recent efforts of Gardella and co-workers have focused on the activation domain represented by the amino terminus PTH(1-14) (18-20). In the search for small peptide and nonpeptide molecules with PTH activity as potential therapies for metabolic bone disorders, the marginally active PTH(1-14) was used as the starting point for structure-activity studies. The rationale was based on site-directed mutagenesis and chimera studies of PTH1-Rc (21-24), functional analysis of structural complementary between PTH1-Rc/calcitonin (CT) receptor chimera and P T H / C T hormone hybrids (25), and photoaffinity cross-linking studies between photoreactive PTH and PTHrP analogs and PTH1-Rc (26-30) or PTH2-Rc (31). These studies suggest that the activation domain of PTH interacts with the extracellular loops (ECLs) and the juxtamembrane portions of the transmembrane (TM) domains of receptor. These receptor sites are different than those involved in interacting with the binding domain of PTH, which is primarily within the receptor's N-terminal extracellular domain (N-ECD). Similar observations were reported for secretin (32,33), vasoactive intestinal peptide (VIP) (33,34), CT (35), and CT/glucagon chimera (36) receptors, all belonging to class II (or group B) of the GPCRs.

PTH/PTHrP/REcF~eTOR INTERACTIONS / PTH (1-14) stimulated cAMP levels with equipotency (EC50 ~ 100 IzM) via the intact rat (r) PTH1-Rc and the N-terminal truncated (missing residues 26-181 of the N-ECD) receptor (rANt), both transiently expressed in COS-7 cells. In contrast, PTH(1-34) was two orders of magnitude less potent in stimulating cAMP accumulation in the rANt than in the intact rPTH1-Rc (18). In addition, "Ala-scan" of PTH(1-14) revealed that the first nine N-terminal residues form the critical activation domain and are involved in ligand-receptor interaction rather than an intramolecular interaction with the C-terminal domain PTH(15-34), as was previously suggested (37-39). This study concludes that the N terminus of PTH interacts with binding determinants within the ECLs and the juxtamembrane portions of the TM domains of PTH1-Rc, Interestingly, some substitutions in the 10-14 sequence of the hormone were not only compatible with function, but also resulted in more potent peptides , such as [Ala3,10,12, Arg ~1] PTH (1-14) 1 and [Ala ~'1°, Arga1]PTH(1-11), which were 100- and 5-fold more active than PTH(1-14), respectively (19). In addition, increases in cAMP levels were observed following the insertion of His, a "Zn 2+ switch," into positions in the 10-13 sequence of PTH(1-14) (20). Taken together, Gardella and co-workers suggest that the C-terminal portion of PTH(1-14) contributes important interactions with the ECLs and TM domains, which are stabilized by complex formation with Zn 2+ salts. However, in the absence of demonstrable specific binding, the extremely small increases in cAMP production do not provide a high degree of confidence that the reported cAMP increases are mediated by a specific interaction between PTH(1-14) and PTH1-Rc. Substitutions within the Intact Sequence

Several independent studies comprehensively scanned either the entire or limited segment of PTH(1-34/36) by a multiplicity of substitutions. Extensive corroborating data were generated, as well as some new insights into the tolerance and significance of certain residues with regard to bioactivity (40-42). Both Gardella and Oldenburg and their co-workers used recombinant DNA methodologies to generate analogs either randomly mutated at codons 1-4 in hPTH (40) or with individual codon replacement with [ (A/G) (A/G)G] (coding for lysine, arginine, glummine, or glycine) (42). In addition, Gombert et al. used a parallel multisynthesis approach to generate D-Ala, L-Ala, and D-Xxx scans of hPTH (1-36) (41). 1To simplify the reference to amino acid residues in the ligand and the receptor, the amino acids of the ligand are denoted using the three-letter code, whereas the one-letter notation is used for the residues of the receptor.

55

For the D-Ala scan, the largest decrease in binding affinity was observed for the segments 2-8 and 20-28, accompanied by the largest loss in efficacy for the latter segment, especially for Arg-20, Trp-23, Leu-24, and Leu-28 substitutions (41). A high correlation between binding and adenylyl cyclase activation was observed in the L-Ala scan, with the segment 2-8 suffering the largest loss in activity (41). Only substitutions of Lys-13, Asn-16, and Glu-19 yielded slightly more active analogs. The D-Xxx scan resulted in an overall loss of affinity and efficacy, with the greatest loss at the putative amphiphilic helical domain (residues 23-29), and a slightly better tolerance at the C-terminal segment (32-36) (41). Mutations of the evolutionarily conserved first four N-terminal residues in PTH were carried out by Gardella and co-workers (40). Residues Glu-4 and Val-2 were less tolerant of substitution, suggesting that they contain important determinants for receptor binding and activation (40). Conversely, Ser-1 and Ser-3 were more tolerant of substitution, suggesting that they play less critical roles in h o r m o n e activity. The most intriguing finding of this study was the divergent activity displayed by [Arg2, Tyr~4]PTH(1-34)NH2 in two different cell lines, both expressing the wild-type PTH1-Rc (40). This analog binds to ROS 17/2.8 cells, a rat osteosarcoma cell line, with twofold higher affinity than do OK cells, an opossum kidney cell line (40). Nevertheless, it is a weak partial agonist for stimulation of adenylyl cyclase in ROS 17/2.8 cells, whereas it is a full agonist for cAMP increases in the OK cell system (40). It remains to be determined whether the differences in activity were related to potential tissue-and speciesspecific effects across the two cell types. A latter study addressed some of these questions and will be discussed below (43). The highly conserved residues Ser-3 and Gln-6 in PTH and PTHrP contribute importantly to binding and activation (39). Substitution of Ser-3 by either Phe or Tyr and of Gln-6 by Phe and Ser generated partial agonists. Both [Phe3]hPTH(1-34) and [Phe6]hPTH(1-34) were found to inhibit competitively bPTH(1-34)- and PTHrP(1-34)-stimulated adenylyl cyclase activity. Taken together, the findings that substitutions within the "activation domain" may convert full agonists into partial antagonists provide new tools to design potent full-length antagonists (1-34). It also suggests that structural perturbation of the ligand-receptor bimolecular interactions at the N terminus of PTH(1-34) may interfere with the conformational changes required for coupling the ligand-occupied receptor to G proteins, thus inhibiting induction of intracellular signaling. Some provocative observations were reported by Oldenburg and co-workers following an extensive

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CI-IAeTWk4

mutational study of h P T H ( 1 - 3 4 ) in UMR-106 rat osteosarcoma cells (42). Single nonconservative substitutions spanning the 11-30 sequence, Leu 11 Arg, Asn TM Lys, Glu 19 Arg, Glu 22 Arg, Asp 3° Arg, as well as a conserved one, Lys26 Arg, resulted in e n h a n c e m e n t of bioactivity. The C-terminal amino acid substitutions Asn 3~ Glu/Gly and Tyr 34 Lys/Glu also resulted in e n h a n c e d potency. One of the most interesting analogs is [Arg 19'22'3°, Lys 20, Hse34]hPTH(1-34), a peptide carrying a net charge of + 7 concentrated in the C-terminal amphiphilic helix. The analog is equipotent (EC50 --0.9 and Kd --- 1.5 nM) to bPTH. One possible explanation for the observation is that the amphiphilic nature of this helix is not critical per se, but rather the presence and disposition of the positive charges in this helix are critical for either intramolecular, ligand-receptor, or ligand-lipid interactions (42). Taken together, these results suggest that some favored paradigms, such as salt bridges stabilizing the bioactive conformation of PTH a n d / o r intramolecular stabilization between the amino- and carboxyl-terminal regions of PTH forming a U-shaped conformation, now need to be reexamined.

Receptor Subtype Specificity Switch The two homologous PTH receptors, PTH1-Rc and PTH2-Rc, display differing specifications for ligands: PTH1-Rc binds both PTH and PTHrP, whereas PTH2Rc binds only PTH. These two receptors provide a platform for probing the basis of molecular recognition of ligand and receptor (44,45). The finding that the N-truncated sequence, P T H r P ( 7 - 3 4 ) , can bind and weakly activate the PTH2-Rc suggests that the Nterminal sequence 1-6 of PTHrP must contain a structural element that disrupts P T H r P ( 1 - 3 4 ) - P T H 2 - R c interaction (45). Swapping the nonconserved residues in position 5 between PTH(1-34) and PTHrP(1-34) generates the single-point mutants [His 5, Nle s'ls, Tyr~4]bPTH(1-34)NH2 and [Ile5]PTHrP(1-34)NH2 (45). Indeed, in HEK293 cells stably transfected with

either hPTH1-Rc or hPTH2-Rc, the receptor specificity of these point hybrids is reversed when compared with their parent compounds. Therefore, His-5 is the specificity "switch" between these two highly homologous receptor subtypes (45). Gardella and co-workers conducted studies in COS-7 cells transiently expressing the PTH1-Rc and PTH2-Rc and reached a somewhat different conclusion (46). According to their study, two sites are responsible for the divergent specificity: position 5 determines signaling and position 23 determines receptor binding affinity. Swapping the residues in positions 5 and 23 between PTH and PTHrP results in [His 5, Phe 2~, Tyr34]PTH(1-34)NH2 (IC50 > 10,000 n M f o r both PTH1Rc and PTH2-Rc, and EC50 - 1.18 and > 1000 n M for PTH1-Rc and PTH2-Rc, respectively) and [Ile 5, Trp 2s, Tyr36]PTHrP(1-36)NH2 (IC50 = 16 and 10 nM, and EC50 = 0.21 and 0.5 n M for PTH1-Rc and PTH2-Rc, respectively). In that study [Trp 2~, Tyr36]PTHrP (1-36) NH 2 is an antagonist for the PTH2-Rc but a full agonist for the PTH1-Rc. The discrepancies between the two studies (45,46) may be related to differences in the experimental systems employed, such as stable versus transient transfections or homologous versus heterologous receptor-cell systems, and the use of different radioligands, e.g., rat- versus bovine-derived peptides. An endogenous ligand selective for PTH2-Rc, tuberoinfundibular peptide of 39 amino acids (TIP39), has been purified from bovine hypothalamic extracts (47). A homology search reveals that 9 out of the 39 residues of TIP39 are identical to b P T H (Fig. 1). Interestingly, TIP39 did not activate adenylyl cyclase in COS-7 cells transfected with either h u m a n or rat PTH1Rcs (47). The physiologic role of the TIP39-PTH2-Rc system remains to be established.

Search for the Putative Bioactive C o n f o r m a t i o n Identification of the peptide ligand conformation responsible for the recognition by, binding to, and activation of the GPCR is a major objective in structural

1

34

hPTH

SVSEIQLM

HNLGKHLNSM

~.RVEWLPKKL

QDVHNF ~~~

hPTHrP

AVSEHQLL

HDKGKSIQDL

RRRFFLHHLI

AEIHTA ~~~

SLALADDAAF

RERARLLAAL

~.RRHWLNSYM

HKLLVLDAP

TIP39

FI6. 1 Peptide Homology for human PTH, PTHrP, and TIP39. The functional N termini of hPTH and hPTHrP are shown [264-266], and are aligned with the complete human tuberoinfundibular peptide of 39 amino acids (TIP39) (47). Boldfaced amino acid residues shown in TIP39 have direct homology to the boldfaced ones shown in either PTH or PTHrP. The numbering at the top refers to PTH and PTHrP, and at the bottom, to TIP39.

PTH/PTHrP/REcEPTOR INTERACTIONS / biology. The ligand-receptor complex is the definitive system for study of the putative bioactive conformation. Unfortunately, for GPCRs this is currently an unattainable goal because no h o r m o n e - G proteincoupled receptor complex has been crystallized, probably because the receptor is embedded in the cell membrane. Based on GPCRs being embedded in the membrane, the hypothesis formulated by Schwyzer proposes that the initial conformation adapted by a ligand is induced by nonspecific interactions with the membrane (48-50). This membrane-induced conformation is the one recognized by the membrane-embedded GPCR. Therefore, study of conformations in the presence of membrane-mimetic milieu, like the micellar environment, is probably the best available approximation of the natural state. Secondary structure prediction methods (51-54) suggest that the N-terminal 1-34 sequences of both PTH and PTHrP assume helical structures at their N and C termini (39,55,56). These helical domains span residues 1-9 and 17-31 in PTH and 1-11 and 21-34 in PTHrP (39). Correlation between the receptor binding affinity and the extent of helicity was determined by circular dichroism (CD), a method that can assess the global conformational nature of a peptide (57). The same spectroscopic method estimated PTH(1-34) in water to have, on average, less than eight residues in the helical conformation. This n u m b e r was even smaller for PTHrP(1-34) (39,55,58-60). In the presence of 45% trifluoroethanol (TFE), a solvent that promotes secondary structure, the total helical content of bPTH(1-34) and hPTHrP(1-34) is 73% (39). Nevertheless, there is much controversy about the relevance of the conformation in TFE to the bioactive conformations. Early ~H nuclear magnetic resonance (NMR) studies in water demonstrate that PTH (1-34) is mostly random in structure, except for a short ordered region encompassing residues 20-24 (61-63). According to our recent findings, hPTH(1-34) in water is highly flexible, with some evidence of transient helical loops spanning the sequence 21-26 and 7-8 (64). Cohen and co-workers suggest that in TFE the amphiphilic helices located at the N and C termini of bPTH(1-34) and hPTHrP(1-34) interact to form a U-shaped tertiary structure with the hydrophobic residues facing inward to form a hydrophobic core. The hydrophilic residues orient outward and are exposed to the polar solvent (39). However, the lack of long-range interactions between the two helices in both hPTH(1-34) (65-67) do not support the notion of a U-shaped tertiary structure. Interestingly, the longrange p r o t o n - p r o t o n correlations between the two N-terminal helices (sequences 1-10 and 17-27) in full-

57

length recombinant hPTH(1-84) in aqueous TFE are d e p e n d e n t on interactions provided by the middle and C-terminal portion of the molecule (sequences 30-37 and 57-62, respectively) (68). In TFE, the low dielectric constant, which helps to stabilize helices, is also supposed to shield the side chains from hydrophobic interactions between the helices and, therefore, destabilizes alleged U-shaped tertiary structures. Marx and co-workers suggest that hPTH(1-37) in aqueous solution containing high salt concentration assumes a U-shaped structure (37). However, their reported long-range p r o t o n - p r o t o n correlations are limited to side chains of Leu-15 and Trp-23 located close to the bend forming the putative U-shaped structure, therefore leaving too much flexibility to define a stable U-shaped structure. The same researchers identified the loop region around Hisl4-Ser 17 and longrange p r o t o n - p r o t o n correlations between Leu 15 and Trp 23 found in hPTH (1-37) and in N-truncated analogs hPTH(2-37), hPTH(3-47), and hPTH(4-37) but did not interpret it to stabilize a U-shaped structure (69). Other studies of PTHrP analogs described interactions between the N- and C-terminal helical domains, in the presence of TFE, thus offering support for the U-shaped structure (70-74). However, current established understanding runs counter to the U-shaped structure as the predominant bioactive conformation of PTH(1-34) and PTHrP(1-34). In our studies (13,75) of PTHrP(1-34)-related analogs in aqueous solutions and in the presence of TFE, we could not confirm the presence of long-range helix-helix interactions (76). In addition, we studied a series of side chain to side chain-bridged monocyclic and bicyclic lactam-containing PTHrP analogs. These analogs are cyclized through side chain pairs AsplS-Lys17 and Lys26-Asps°, located at the putative N- and C-terminal helical domains, respectively (13,75,77-80). The i to i + 4 side chain to side chain cyclization is known to stabilize helical structures. Bioactivity in the agonist (1-34) and antagonist (7-34) series of lactamcontaining analogs was found to require well-defined N- and C-helical domains that are linked by two flexible hinges located around residues 12-13 and 19-20, the latter being associated with high bioactivity (Fig. 2) (13,75). Similar conclusions were reached by Gronwald and co-workers studying PTHrP(1-34) in water and in 50% TFE (81). In the presence of TFE, they observe two stable or-helical regions spanning residues 3 to 12 and 17 to 33, which are connected by a flexible linker. Their observations clearly exclude the possibility of any significant tertiary structure (81). Although Barden and Kemp mention the presence of a hinge at ArglO-Arg2° in [Ala°]PTHrP(1-34)NH2 and attribute to it a functional role in signal transduction, they also postulate

58 / CHAr'TR4 V, Antagonist C-Terminus

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FIG. 2 Superposition, using the heavy backbone atoms of residues 22-32, of agonist and antagonist containing (top) the midregion lactam, c[Lys13-Asp1~]PTH(1-34)NH 2 and c[Lys13-AsplT]PTH(7-34)NH2, respec26 30 26 30 tively, (middle) C-terminal region lactams, c[Lys -Asp ]PTH(1-34)NH 2 and c[Lys -Asp ]PTH(7-34)NH 2, 13 17 26 30 respectively, and (bottom) bicyclic mid- and C-terminal region lactams, c[Lys-Asp ,Lys-Asp ]13 17 26 30 PTH(1-34)NH 2 and c[Lys -Asp ,Lys -Asp ]PTH(7-34)NH 2, respectively. The structures were taken from the molecular dynamic trajectories to illustrate the consequences of the flexible hinge centered around Arg-19. Reprinted with permission from Ref. 13. Copyright 1997 American Chemical Society. (See color plates.)

PTH/PTHrP/Pd~CEPTOk INTERACTIONS / long-range interactions between side chains located on both sides of the turn Gln16Arg 19, implicating the presence of a tertiary structure (72). The CD and NMR studies of hPTH(1-34) in aqueous solutions (variable pHs and salt concentrations) and dodecylphosphocholine (DPC) micelles, coupled with distance geometry calculations, generate conformations that were refined by molecular dynamic simulations explicitly incorporating solvent (H20) (64). This study generated high-resolution conformational preferences of hPTH(1-34), which were then used in our building of an experimentally based model of the hormone-receptor complex. In benign aqueous solution and in the presence of DPC micelles, we observe conformational averaging that is fast on the NMR time scale. As anticipated, the two helical domains observed in aqueous solution, the N-terminal helix, comprising residues 6-14, and the C-terminal helix, comprising residues 19-23, are extended (4-17 and 21-33, respectively) and stabilized in presence of DPC micelles. A region of flexibility, which is centered around residues 15-16 in aqueous solutions and around residues 18-19 in the micellar system, separates both helices (Fig. 3). Therefore, in solution the helices adopt a range of orientations, none of which corresponds to a tertiary structure in which helix-helix inter-

59

actions can be observed (64). This observation is in complete accord with conformational studies of lactam-containing PTHrP analogs (13), PTH-PTHrP point-mutated hybrids (82), and a model amphiphilic oL-helix-containing PTHrP analog (83). The more hydrophobic and amphiphilic C-terminal sequence in PTHrP has a higher propensity for helicity than does the N-terminal domain, as demonstrated by the higher percentage of TFE needed for nucleation of an N-terminal helix (13). Similar conclusions were reached on the addition of lipids (55,58,84) or DPC micelles (64,83) to PTH- and PTHrP-derived sequences. Taken together, the conformational analyses allow us to postulate a dynamic model for ligand-receptor binding. Binding is initiated by complementary hydrophobic interactions between the hydrophobic face of the amphiphilic C-terminal helical domain of the ligand, including the principal binding domain, and the hydrophobic membrane. This hydrophobic ligand-membrane interaction allows the propagation of the C-terminal helix and formation of specific interactions with the extracellular portions of the PTH receptor. In the membrane environment, nucleation of the N-terminal helix occurs, either cooperatively with (in the antagonist) or independently from (in the agonist) the previously formed C-terminal

FIG. 3 Ribbon diagram of the two conformations resulting from the ensemble-based calculations. Averaging over this twomember ensemble fulfills all of the experimental observations. The different locations and extents of the (x-helices are highlighted in gray; the side chain of Trp-23, used to align the conformations, is shown in ball-andstick conformation (64).

60

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CHAPTER4

helical domain. Consequently, in the case of P T H / P T H r P agonists, the flexibility around hinges 12-13 and 19-20 allows the membrane-induced "message domain," to be correctly positioned within the receptor. Thus, specific message-receptor interactions leading to the conformational changes required for signal transduction can occur. In the case of P T H / PTHrP antagonists, which lack most of the "message" sequence, no conformational change in the receptor occurs and no signal transduction event is triggered. Absence of the critical hinge around position 19-20 or a shift in register of the hinge region results in reduced binding affinity and efficacy, as observed for the cyclic PTHrP analogs (13) and point-mutated PTH/PTHrP hybrids (82). This proposed dynamic model for ligandmembrane-receptor interaction provides a testable paradigm for future experiments.

cations that stabilize an element important in the putative bioactive conformation (13,77-79,85-87). Much attention has been drawn to the amphiphilic nature of the C-terminal helix comprising residues 20-34 of hPTH(1-34) and its role in receptor-binding (11,58). A Lys 27 to Leu substitution in PTH(1-34)NH 2 and PTH (1-31) NH 2 improved the amphiphilic character of the C-terminal helical sequence, resulting in about a fivefold and twofold increase in adenylyl cyclase activity over the corresponding nonsubstituted sequences (85,86). The substitution in PTHrP(1-34)NH 2 of the sequence 22-31 with a model amphiphatic peptide (MAP; Glul-Leu-Leu-Glu-Lys-LeuLeu-Glu-Lys-Leu-Lysl°), which is highly e~-helical when incorporated into short peptides (88), generates [ (MAP~_10)22-31]hPTHrP(l-34) NH 2 (RS-66271) (Fig. 4) (89). Important structural features, such as Leu-24 and Leu-27, are maintained in this analog; Ile-22 and Ile-31 are substituted conservatively by Leu. In aqueous buffer, RS-66271 displays eight- to ninefold higher helicity compared to the parent peptide. Detailed conformational analysis of RS-66271 in water, employing CD and 1H NMR spectroscopy, confirms the presence of an extensive helical structure encompassing residues 16--32 (83). The absence of a hinge element around Arg-19, considered to contribute to high biological activity, may explain the 6- and 10-fold lower adenylyl cyclase and binding affinity, respectively, in ROS 17/2.8 cells of RS-66271 as compared to the more flexible and less helical PTHrP(1-34) (88,90). Importantly, the preservation of significant in vitro potency despite multiple substitutions validates the rationale behind the design of RS-66271. Despite reduced binding affinity and efficacy in vitro, RS-66271 has significantly greater activity than either PTH (1-34) or PTHrP (1-34) in restoring lost trabecular and cortical bone in vivo in ovariectomized osteopenic

Conformation-Guided Design of PTH and PTHrP Analogs According to a generally accepted working hypothesis, only a small fraction of the ensemble of dynamically equilibrating conformations presented by linear sequences of native bioactive peptides display the putative bioactive conformation. Therefore, introduction of conformational constraints may stabilize the ligand in receptor-favored bioactive conformations by precluding a wide range of nonproductive conformations. Alternatively, enhancement of the amphiphilicity of a helix will increase potency if this structural feature is indeed important for stabilizing a favored secondary structure a n d / o r providing an important productive interaction with the receptor. Such modifications may favor conformations with the highest affinity for the receptor, resulting in enhancement of bioactivity. To this end, several studies incorporated structural modifi-

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the

PTH/PTHrP/RECF~PTOR INTERACTIONS rats (89,90). Another study confirmed the reduced in vitro activity of RS-66271, but observed anabolic activity in vivo that was similar to that of PTH (1-34) (91 ). Usdin and co-workers have suggested a different explanation to resolve the discrepancy between the low binding affinity of RS-66271 in vitro and its potent anabolic activity in vivo (92-94). Using a membrane-based binding assay, they demonstrated that hPTH(1-34) binds with high affinity (IC50 < 10 nM) to the PTH1-Rc whether it is coupled or uncoupled to G-protein (93). By contrast, RS-66271 binds with high affinity (IC50 = 16 pM) to the coupled receptor but with much lower affinity (IC50 > 100 nM) to the uncoupled receptor (94). Therefore, the uncoupled PTH1-Rc in the cell-based binding assay binds RS-66271 with lower affinity compared to hPTH(1-34). The lower affinity to the uncoupled receptor may result from either less favorable interactions between the MAP sequence and the receptor or a compromised conformation of RS-66271. It is generally accepted that PTH(1-34) and PTHrP(1-34) contain two helical domains spanning sequences 13-18 and 20-34 (66,67,95,96). Introduction of side chain to side chain cyclizations between residues that are four amino acids apart and located across a single helical pitch (residue i to residue i + 4) has been demonstrated to be an effective way to stabilize a helical structure (97-101). Therefore, we undertook replacement of a potential ion pair, participating in or-helical stabilization, by a covalent lactam bridge, in an attempt to further stabilize the helices in these regions. The initial application of this approach generated c[Lysl~-Asp17]PTHrP(7-34)NH2, which was about 10-fold more potent than the linear parent antagonist (Kb = 18 and 170 nM, Ki = 17 and 80 nM, respectively, in SaOS-2/B10 cells) (77). Rigidification of the C-terminal helix in c[LysZ6-Asp3°]PTHrP(7-34)NH2 did not improve antagonist potency (79). However, combination of two 20membered lactam bridges, in both the N- and C-terminal helices, generated c[Lysl~-Asp17, Lys 26Asp~°]PTHrP(7-34)NH2, a potent (Kb = 95 n M and Ki = 130 nM, in SaOS-2/B10 cells) (79) highly conformationally constrained PTHrP-derived antagonist and a valuable tool for conformational studies (75). The same approach applied to the agonist PTHrP(1-34)NH 2 yielded the mono- and bicyclic analogs, 13 17 26 c[Lys13-Asp17]PTHrP(1-34)NH2 and c[Lys-Asp , L y s 3O Asp ]PTHrP(1-34)NH 2, which were equipotent to the linear parent compound (Kb = 3.2, 2.1 and 1 nM, Km = 0.17, 0.22, and 0.57 nM, respectively, in SaOS-2/B10 cells) (79). A similar approach was also applied to the signalingselective analogs, hPTH (1-31)NH 2 and the more potent [Leu 27]hPTH (1-31 ) NH 2. Both of these analogs stimulate

/

61

adenylyl cyclase but not the PLC/PKC signaling pathway (85). Though i to i + 4 lactam bridge formation between Glu-22 and Lys-26, as in c[Glu 22LysZ6,Leu27]hPTH (1-31) NH 2, results in about a fourfold increase in adenylyl cyclase activity, as compared to the linear parent peptide (EC50 = 3.3 and 11.5 nM, respectively, in ROS 17/2 cells), similar cyclization between Lys-26 and Asp-30 or i to i + 3 lactam bridge formation between Lys27 and Asp-30 results in cyclic analogs that are less potent than the corresponding linear parent peptides (85). Interestingly, the higher adenylyl cyclase activity in vitro observed for c[ Glu22-LysZ6,Leu 27] hPTH (1-31 ) ~qH2 compared to the linear peptide results in greater anabolic effect on trabecular bone growth in ovariectomized rats (102) and affords more effective protection than hPTH(1-34) against loss of femoral trabeculae in the same animal model (103). The retention of full ability to activate PKC (in ROS 17/2 cells) by the extensively N-terminally truncated linear fragment, [LysZ7]hPTH(20-34)NH2, and the structurally related lactam-bridged analog, c[Lys 26Asp~°]hPTH(20-34)NH2, was consistent with the stabilization of the amphiphilic helix at the C-terminus, implicating the helix as an important functional motif for binding to the PTH1-Rc (104). Taken together, the above studies provide important insights regarding the structural nature of the hormones PTH(1-34) and PTHrP(1-34) and help to better characterize conformational features important for PTH binding and bioactivity.

Signaling-Selective Ligands Activation of PTH1-Rc evokes dual signaling pathways, increasing both adenylyl cyclase/PKA via GsOLand PLC/IP~-DAG/cytosolic transients of [CaZ+]i/PKC via Gq (43,105-111). Dual signaling is observed in homologous and heterologous receptor/cell systems, which include rat, opossum, mouse, porcine, and h u m a n receptors and cells. In general, maximal signaling intensity through both pathways increases with receptor density. However, a larger n u m b e r of PTH1-Rcs per cell is needed to activate the PLC-associated pathway than is needed to stimulate adenylyl cyclase. PTH modulates downstream activities in osteoblasts, leading to regulation of cell growth, proliferation, and differentiation (112,113). PTH affects osteoclasts indirectly through its direct action on osteoblasts (114). Subcutaneous administration of PTH results in an immediate and transient expression of c-fos mRNA in PTH1-Rc-bearing cells (chondrocytes, osteoblasts, and spindle-shaped stromal cells), followed by a delayed expression in the majority of stromal cells and osteoclasts (115). This observation provides further support

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for the indirect action of PTH on osteoclasts, which may be mediated by osteoblasts, a n d / o r a subpopulation of stromal cells. In UMR cells, PTH rapidly and dose-dependently induces transcription of c-fos (116,117). Pearman and co-workers reported that the cAMP response element (CRE) in the c-fos p r o m o t e r is required for PTH-induction of c-fos in UMR cells and that the CRE binding protein (CREB) binds to this site, apparently as a homodimer, and is phosphorylated in a PTH-inducible fashion at Ser-133 (118). Therefore, c-fos appears to have pleiotropic and essential effects in bone, including mitogenesis a n d / o r differentiation in the skeletal system, as well as inhibition of osteocalcin expression by binding to the AP-1 site in the osteocalcin promoter, thereby suppressing the mature osteoblast phenotype (119). PTH-induced c-fos p r o m o t e r activity was completely inhibited in a concentration-dependent m a n n e r by transfection of a heat-stable inhibitor of PKA (120). This finding provides strong evidence that PKA is the enzyme responsible for phosphorylation of CREB at Ser-133 in response to PTH and that PKA activity is required for PTH-induced c-fos expression. Nevertheless, the relationship between these signaling pathways and cellular and in vivo responses to PTH has not been completely elucidated. Understanding the role of cellular processes such as receptor inactivation, internalization, trafficking, and recycling in bone metabolism is only beginning to be elucidated. One of the major questions in the PTH field focuses on the mechanism responsible for catabolic versus anabolic actions of PTH induced by continuous versus intermittent administration of hormone, respectively. The linkage of one or both the signaling pathways to the anabolic activity of PTH remains to be established.

Search for the Ligand-Based Signaling Specificity Switch Much attention is directed toward identifying a signaling pathway that is specifically associated with the anabolic activity of PTH. One hypothesis is that this pathway or portions of it will be c o m m o n to all agents and treatments that have anabolic effects on bone. Studies carried out on bone cell and organ cultures suggest that residues 1-7 in PTH constitute the cAMP/PKA activation domain (121), whereas residues 28-34 in PTH comprise the PKC activation domain (122,123). The latter encompasses the region also associated with PTH mitogenic activity on cultured osteoblast-like cells (residues 30-34) (124,125). Cyclic AMP appears to be involved in the bone formation (126) and resorption activities (16) of PTH. PTH analogs that stimulate increases in cAMP levels have been shown either to inhibit (127-129) or stimulate (129-131) osteoblastic cell proliferation, d e p e n d i n g on species, the cell models used, and the experimental

conditions. However, N-terminally truncated fragments of PTH, which selectively activate PKC without affecting cAMP (121,132,133), are also mitogenic for osteoblastic cells (134). Because these truncated fragments do not stimulate bone resorption (16), they may be more effective "anabolic" analogs than are peptides with an intact N terminus. Truncation of two amino acids from the N terminus of PTH(1-34), e.g., PTH(3-34), reduces adenylyl cyclase activation without significantly affecting PKC activation or the mitogenic response in vitro (132). Similarly, the stimulation of TE-85 h u m a n osteosarcoma cell proliferation by PTH(1-34) is not associated with an increase in intracellular cAMP (135). Therefore, if stimulation of bone formation in vivo is related only to the mitogenic response in vitro, the bone formation response should be retained in the aminotruncated PTH fragments. Although PTH stimulation of bone resorption in vitro is mediated primarily through cAMP-dependent activation of PKA (136), it may not be the sole second messenger pathway involved in this activity (137,138). One of the current working hypotheses holds that dissociation between the two signaling pathways of PTH, adenylyl cyclase and PLC, will result in separation between the anabolic and catabolic activities of PTH in bone (14,126,132,139-143). If the stimulation of bone resorption in vivo is related to the bone resorption response in vitro, the in vivo response should be diminished in amino-terminal-truncated PTH fragments. However, neither PTH(3-34) nor PTH(3-38) (both PKC-selective, N-terminal-truncated analogs of PTH) are active in vivo as bone anabolic agents (4,122,126,143-146). Furthermore, desaminoPTH(1-34), which has drastically reduced ability to stimulate adenylyl cyclase but is equipotent to h P T H ( 1 - 3 4 ) in stimulating PKC, does not stimulate cortical or trabecular bone growth in ovariectomized rats (126). Surprisingly, hPTH (1-31 ) NH 2 (Ostabolin), an adenylyl cyclase-selective PTH agonist equipotent to PTH(1-34) in stimulating cAMP production in ROS 17/2 (57,123), strongly stimulates cortical and trabecular bone growth in ovariectomized rats (126,143,145-147). In this analog the putative PKC-signaling motif GlnZS-His32 is compromised by the elimination of His-32 (123). A second generation of adenylyl cyclase-selective analog, 22 26 27 c[Glu -Lys ,Leu ] hPTH (1-31) NH2, in which the helical nature of the C terminus was enhanced by the formation of a side-chain to side-chain lactam ring and the introduction of a hydrophobic residue at position 27, was 1.4to 2-fold stronger than the linear parent analog as a stimulator of femoral trabecular bone growth (102). Both 22 26 27 hPTH (1-31) NH 2 and c[Glu -Lys ,Leu ] hPTH (1-31)NH 2 were reported to prevent loss of vertebral trabecular bone in ovariectomized rats and to increase vertebral tra-

PTH/PTHrP/RECEPTOR INTERACTIONS / becular volume and thickness over those of control vehicle-injected sham-operated rats (147). The action of these analogs on vertebral bone was as effective as that of h P T H ( 1 - 3 4 ) N H 2. However, unlike h P T H ( 1 - 3 4 ) N H 2, their effects on pelvic BMD were equivocal. An alternative view has been offered regarding the structural determinants associated with signaling pathway activation. Replacement of Glu 19 --) Arg, a receptorbinding affinity-enhancing modification, generated [Arg19]PTH(1-28) as a potent and full stimulator of adenylyl cyclase and PLC. Interestingly, substituting 1 19 Gly-1 for Ala generated [Gly ,Arg ] h P T H ( 1 - 2 8 ) , which is an adenylyl cyclase-selective agonist (148). This study concluded that the extreme N terminus of h P T H constitutes a critical activation domain for coupling to PLC. The C-terminal region, especially h P T H ( 2 8 - 3 1 ) , contributes to PLC activation through receptor binding, but the domain is not required for full PLC activation. The N-terminal determinants for adenylyl cyclase and PLC activation in h P T H ( 1 - 3 4 ) overlap but are not identical; subtle modifications in this region may dissociate activation of these two effectors. Another approach attempted to design target organspecific PTH analogs on the assumption that a boneselective analog would be a better bone anabolic agent. To this end, [HisS] - and [Leu~]hPTH(1-34) were generated and found to be partial agonists of adenylyl cyclase in a kidney cell line (50 and 20%, respectively), but full agonists in UMR-106 rat osteosarcoma cells (149). However, both analogs were less potent than native PTH(1-34) in vivo in the induction of bone formation. In the course of designing photoreactive PTHrP analogs for mapping the bimolecular ligand-receptor interface, we generated trB p a 1,Ile 5,Ar g 1113 ' ,T ry 361jPTHrP(1-36)MH 2 (29). This analog binds and stimulates adenylyl cyclase equipotently to the parent analog [Ile5,Arg11'a~,Try~6]PTHrP (1-36) NH 2 in HEK293/C-21 cells overexpressing the h u m a n PTH1-Rc (--400,000 receptors/cell), but does not elicit intracellular calcium transients. Moreover, it does not stimulate translocation of [3-arrestin2-green fluorescent protein (GFP) fusion protein, an effect that is PKC d e p e n d e n t (150). In summary, development of an effective and safe therapeutic modality that would stimulate the formation of new, mechanically competent bone and possibly reconstitute trabecular architecture in osteoporotic patients continues to be a worthy goal. This goal may be approached by analogs that interact with the PTH1-Rc in a signaling-selective manner.

Nuclear Localization o f PTHrP PTHrP has been shown to function in a second mode of action: as an intracrine factor with direct intracellular effects following translocation into the nucleus

63

a n d / o r nucleolus of the cell. Exogenous h u m a n PTHrP(1-108) is internalized specifically by UMR106.01 osteogenic sarcoma cells that express PTH1-Rc. The h o r m o n e accumulates in the nucleus and nucleolus (151). PTHrP contains a putative nuclear localization sequence (NLS) (residues 61-94) homologous to SV40 T antigen. Deletion of the NLS, or mutation of the conserved GxKKxxK motif within the NLS, effectively prevents both cell surface binding and n u c l e a r / n u c l e o l a r accumulation of PTHrP(1-141) (152). In contrast to proteins containing conventional NLS motifs, which are actively transported by importinet[3 heterodimers (members of a family of structural molecules that mediate nuclear import of proteins containing NLS motifs), PTHrP is recognized exclusively by importin-[3 and the small GTPase, Ran, which together actively transport PTHrP to the nucleus i n d e p e n d e n t of importin-e~ (151). Thus, PTHrP appears to be actively transported to the nucleus via a novel mechanism that is i n d e p e n d e n t of importin-ot, although the biologic significance of this alternate nuclear targeting pathway is currently not understood. Synchronized cell culture studies have demonstrated that PTHrP localizes to the nucleus at the G 1phase of the cell cycle and is transported to the cytoplasm on initiation of mitosis (153). Scanning mutagenesis reveals that T-85 adjacent to the NLS of PTHrP was phosphorylated by CDC2-CDK2 in a cell cycle-dependent manner. Mutation of PTHrP, [As5]PTHrP, results in nuclear accumulation of PTHrE Mutation to [E85], which mimics a phosphorylated threonine residue, results in localization of PTHrP predominantly to the cytoplasm. This study concludes that phosphorylation of T-85 results in decreased nuclear accumulation of PTHrP, whereas the unphosphorylated state (e.g., [A-85] mutant) is preferentially nuclear localized. However, a potential role for PTHrP in regulating cellular phenotype in a cell cycled e p e n d e n t m a n n e r is currently not known. Although the precise role of PTHrP translocation to the nucleus is currently unknown, it may participate in the regulation of cell proliferation, differentiation, and apoptotic cell death during development. Future studies that characterize the nuclear actions of PTHrP would add significantly to our understanding of the role of PTHrP during embryonic skeletal development and as an oncoprotein whose expression in ~ many tumors may correlate with increased tumor aggressiveness and metastatic potential.

RECEPTOR-CENTERED A P P R O A C H The physiologic effects of PTH and PTHrP are largely mediated through the glycosylated PTH1-Rc receptor. PTH1-Rc is encoded by a single-copy gene that

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CHAPTER4

is expressed in PTH target tissues, including kidney, intestine, and bone, where it is essential for maintaining proper mineral ion homeostasis (154). In kidney, PTH1Rc mediates PTH-induced calcium resorption directly by the distal nephrons (155,156). It also inhibits phosphate resorption in the brush border membrane of proximal tubules by targeted lysosomal degradation of the sodium-dependent cotransporter Npt2 (157). In kidney and intestine, PTH1-Rc indirectly mediates vitamin D-dependent calcium absorption by regulating 10t-hydroxylase activity as well as vitamin D receptor biosynthesis (158,159). In bone, PTH1-Rc mediates acute release of calcium from mineralized matrix by activation of osteoclasts. It also plays a major role in modulating more long-standing calcium metabolism by osteoblasts and indirectly by osteoclasts. PTH1-Rc is expressed on osteoblasts as well as in many osteosarcoma cell lines, where it has been demonstrated to signal PTH-mediated changes in gene expression for a number of critical factors in bone homeostasis, including osteocalcin and osteoprotegerin (160). PTH1-Rc has been cloned from several diverse species, including human, rat, mouse, opossum, Xenopus, and zebrafish (27,110,154,161-163). It is a m e m b e r of the class II G protein-coupled receptors that include heptahelical transmembrane receptors for peptide hormones such as secretin, glucagon, and calcitonin (Fig. 5) (164). The 85-kDa PTH1-Rc contains many of the hallmark structural features of class II GPCRs, including a large extracellular domain amino terminus (N-ECD), eight conserved extracellular cysteine residues, and a large (150-190 amino acid residues) cytoplasmic C terminus (Fig. 6). Class II receptors are also identified by conserved cysteines in the first and second extracellular loops as well as by several homologous N-glycosylation sites within the N-ECD. However, though highly conserved, N-glycosylation of PTH1-Rc appears to have little or no influence on receptor expression, ligand binding, and intracellular signaling (165). Like several characterized class II GPCRs, PTH1-Rc is capable of activating multiple intracellular signaling cascades. On binding ligand, PTH1-Rc rapidly up-regulates activity of two distinct intracellular pathways, G, ot/adenylyl cyclase/protein kinase A (PKA) and Gq/phospholipase C/protein kinase C (PKC) (111,166-169). The activation of these signaling cascades gives rise to increased cytosolic cAMP or calcium, respectively. In addition to multiple PTH receptor genes, differential mRNA exon splicing is another cellular mechanism for generating receptors that have altered ligand specificity a n d / o r signaling capacity. For example, Northern blot analyses of human squamous cell lines and keratinocytes demonstrate expression of multiple PTH1-Rc mRNA transcripts that differ in size from the cloned human receptor mRNA (170). Further analysis using a

GIP-Rc Gluc-Rc GLP1-Rc PTHR1 PTHR2 PACAP-Rc VIP2-Rc Sec-Rc VIP1-Rc GHRH-Rc

CRH-Rc

t

msDH-Rc CTR-Rc

FIG. 5 Phylogenetic dendrogram of the human class II GPCR gene family. Thirteen related receptors are shown. GIP-Rc, Gastric inhibitory polypeptide receptor; Gluc-Rc, glucagon receptor; PACAP-Rc, pituitary adenylate cyclase activiating peptide receptor; VIP1- and VIP2-Rc, vasoactive intestinal peptide type 1 and type 2 receptor; Sec-Rc, secretin receptor; GHRH-Rc, growth hormone releasing hormone receptor; CRH-Rc, corticotropin releasing hormone receptor; msDH-Rc, Manduca sexta diuretic hormone receptor; CTR-Rc, calcitonin receptor.

polymerase chain reaction (PCR)-based strategy in human kidney as well as SaOS-2 osteoblast cell lines detected two variants of the PTH1-Rc mRNA that are created by alternative splicing of exons coding for the N-terminal receptor domain (171). One alternatively spliced receptor, designated the S-N3-E2 isoform, juxtaposes exon 1 encoding the signal peptide (S) to an inframe alternative 3 acceptor site within the N3 intron. This splicing event produces a novel receptor with an additional 12 amino acids in the N-terminal extracellular domain of the receptor. In a second characterized PTH1Rc isoform, S-E2, an entire exon is deleted, causing a shift in the reading frame and premature translational truncation of receptor protein. However, an N-terminal truncated receptor may be produced by reinitiation of translation at a downstream initiation codon. A recombinant cDNA encoding the S-N3-E2 alternatively spliced receptor isoform exhibited weak signaling, inducing a two- to threefold increase in cAMP content, but not intracellular calcium, after stimulation with human PTH(1-34). A recombinant cDNA encoding the truncated S-E2 isoform failed to activate either signaling

PTH/PTHrP/R~cF~pTOR INTERACTIONS /

1

S i gnu_!. Sequence ..........................

....

PGLALLLCCP PSLALLLCCP WGWLMLGSCL CGWLILRSCL

60

hPTHR1 rPTHRI hPTHR2 rPTHR2

~~~MGTARIA .....MGAARIA MAGLGASLHV MPWLEALPYI

VLSSAYALVD VLS SAYALVD L.. .ARAQLD L ....VGAQLD

hPTHRI rPTHRI hPTHR2 rPTHR2

61 RPASIMESDK GWTSASTSGK P ~ K A S G K L YPESEEDKEA PTGSRYRGRP TAANIMESDK GWTPASTSGK PRKEKASGKF YPESKENKDV PTGSRRRGRP ITAQLQEGE ...................................... GN ...... ITAQFQEGE ...................................... GN .......

120 ~LP~HIL~ | t I l ~LP~NIV~ ~FPEWDGLI~ ~FPE~GLI~

hPTHRI rPTHR1 hPTHR2 rPTHR2

121 WPLGAPGEVV WPLGAPGEVV WPRGTVGKIS WPRGTAGKTS

180 SE~VKFLTNE S KFMTNE S LQPD SD~..FLQPD

AVP~PDYIYD I I AVP~PDYIYD I ! AVP~PPYIYD ~ P S ~

ADDVMTKEEQ ADD~~EQ SDGTITIEEQ SDGTITIEEQ

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iii

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rPTHRI TiP, ER~"~tFD h P T H ~ I S IGKQEFFE rPTHR2 INIGKQEFFE

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rPTHR2

36z hPTHR1 s

PGHNRTWANY PGhq~RTWANY HSLNK~ HGS~qK~ANY

TMI

rPTHR1 ~

hPT.RI

IFLLHRAQAQ ~EKRLKEVLQ IFLLHRAQAQ ~DKLLKEVLH IVLVLKAKVQ ~ ......~ IVLVMKAKMQ

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rPTHR1 S G ~ i ~ i ii~~ iiii~T~LRETNAGRCDTRQQYr~ii hPT~ AGDI~~ i~ iii~i ~I~=AV GHDTRKQ~i rP T H ~ A G D . ~ i i i i ~ ~ i ~ ~ I ' ~ T N A V GHDMRKQYN~

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FIG. 6 Alignment of amino acid sequences and assignment of TM domains (shaded segments) of human ' (h) and rat (r) PTH1-Rc and PTH2-Rc.

65

66

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CHAPTER4

pathway in response to ligand. However, studies utilizing either iodinated PTH or receptor-specific antibodies to evaluate the cell surface expression of receptor isoforms indicate that the low or absent responses to PTH stimulation for these alternatively spliced receptors were in part due to low surface expression of the S-N3-E2 and S-E2 isoforms. Therefore, these data suggest that exon E1 is critical for cell surface expression of PTH1-Rc, but the S-N3-E2 recombinant isoform lacking this exon is capable of PTH binding and ligand-induced intracellular signaling, albeit at low levels.

PTH1-Rc and Ligand Binding A great deal of information is available detailing the current model of PTH1-Rc-ligand interaction and the important regions of receptor that facilitate recognition and binding. Experimental approaches utilizing site-specific mutagenesis, receptor chimeras with other class II GPCRs, PTH and PTHrP ligand alterations as functional probes, and photoaffinity cross-linking of receptor-ligand interfaces all have contributed to the most current understanding of PTH1-Rc structure and function. Though each of these approaches have inherent limitations, the "receptor-centered" approach to PTH1-Rc structure/function continues to refine an overall working model of the receptor domains critical for both mediating ligand binding, pharmacology, and intracellular G protein activation. Moreover, structure-function analysis of PTH1-Rc using these techniques is especially important given that X-ray crystallography, which has been instrumental in dissecting enzyme-substrate interactions, is not available for GPCR transmembrane molecules because of the lack of suitable crystals for analysis.

Chimera Receptors and a General Model of Ligand-Receptor Interactions Studies examining the structure-function of recombinant chimeric receptors, cloned from two class II members, support a general model that relies on distinct extracellular interaction domains that act in concert to affect G protein binding and activation on the intracellular receptor surface. Cognate class II members are thought to be derived from a single ancestral precursor receptor, and therefore share a general mechanism for ligand binding and activation. In this model, the N terminus of the ligand binds to the extracellularjuxtamembrane regions of the transmembrane and extracellular loop regions of TM5, TM6, and TM7 as well as ECL3 and is responsible for G protein activation; the C terminus of the ligand is critical for specific binding to the receptor N terminus. Though class II GPGR receptor structure has diverged to allow for specificity of ligand binding and

receptor activation, chimeric receptor studies have revealed that the overall pattern of ligand-receptor interactions has remained similar for many members of this large receptor family of molecules. This "cognate receptor" model was directly tested using the porcine calcitonin receptor (CTR) and rat PTH1-Rc (25). Though CT and PTH share little homology, the N termini of both ligands have been shown to be critical for receptor activation, and the C termini for receptor-binding specificity. Though similar in structure, the CTR and PTH1-Rc class II receptor glycoproteins share only 42% homology and are selectively activated only by their respective ligands. Bergwitz et al. created reciprocal CT/PTH1-Rc chimeras in which the N-ECD was exchanged between the two receptors (25). Similarly, chimeric ligands were synthesized in which the ligand activation and binding domains of each ligand were exchanged to create C T / P T H hybrid peptides. Using a COS-7 mammalian expression system to assess ligand binding and cAMP accumulation, it was demonstrated that reciprocal hybrid ligands (CTl-11/PTH 15-34 and PTHl-13/CT12-32), which do not activate the normal CT or PTH1-Rc receptors, could activate P T H / C T and C T / P T H receptor chimeras, respectively. This interaction was dependent on the receptor N-ECD binding the appropriate ligand C terminus. Chimeric receptor was then activated by the common N terminus on each hybrid ligand. Similar studies using interspecies PTH1-Rc chimeras have defined receptor domains critical for ligand binding (21). The recombinant human PTH1-Rc binds several PTH ligands that lack the N-terminal activation domain, including bPTH(7-34), bPTH(15-34), and hPTH(10-34), with at least 50-fold higher affinity than does the rat PTH1-Rc homolog, whereas binding affinities for bPTH(1-34) are similar for both receptor homologs. Applying a similar approach to the CT/PTH1-Rc chimeric receptors, recombinant chimeric r a t / h u m a n PTH1-Rc receptors were cloned and expressed in COS-7 cells. All chimeras bound bPTH (1-34) with normal affinity. However, chimeras encoding the N-ECD of the hPTH1-Rc bound bPTH (7-34), bPTH ( 15-34), and hPTH (10-34) with high specificity, whereas chimeras expressing the rat N-ECD failed to bind those ligands. As in humans, the opossum PTH1-Rc homolog binds bPTH(7-34) with high specific affinity. Studies of rat/opossum PTH1-Rc chimeras confirm the importance of the N-ECD for bPTH (7-34) binding. Thus, studies utilizing chimeric receptors that are designed to exploit the differential binding of PTH analogs demonstrate consistently that a domain within the N-ECD region of the PTH1-Rc is a critical r e c e p t o r region in determining the binding affinity of amino-terminally truncated PTH analogs.

PTH/PTHrP/RECEPTOR I N T E R A C T I O N S Reciprocal receptor chimera studies have implicated the amino-terminal portion of each receptor in having a major role in observed differences in ligand binding affinity by specifically interacting with the C terminus of PTH(l-34). Another series of experiments investigating rat/opossum chimeras helped to elucidate the mechanism by which the N-terminally modified analog, [Arge]PTH(l-34), is an antagonist with rat PTH1-Rc but is an agonist when bound to opossum PTH1-Rc (24). Here, ligand activity was associated with extracellular juxtamembrane residues of TM5 and TM6 in the carboxyl terminus of PTH1-Rc. Site-specific mutagenesis further refined the residues critical for rat PTH1-Rc interactions with the Arg-2 sidechain a s S 370 and V~71 (TM5) and L 427 (TM6). Mutagenesis studies that replaced these residues in rat PTH1-Rc with corresponding residues in the opossum PTH1-Rc (S~7°A, V371I, and LazwT) resulted in an alteration in [ArgZ]PTH(1-34) binding toward that seen with wild-type opossum receptor, yet had no effect on the binding of PTH(1-34). One of these mutations in rat PTH1-Rc, $37°A, also conferred agonist activity to [ArgZ]PTH(1-34) in cAMP assays, whereas V371I 427 and L T failed to alter receptor activation by [ArgZ]PTH(1-34). Thus, these reciprocal mutations of specific residues confirmed results from chimeric receptors. In addition, specific mutagenesis pinpoints potential residues that are critical for local direct interactions with the amino terminus of the ligand as well as for the pharmacologic profile of [Arg 2]PTH(1-34). Thus, chimeric receptor studies have pointed to at least two distinct, independently functioning domains on the extracellular surface of the PTH1-Rc receptor: (1) the N-ECD, which largely determines binding specificity of ligand by interactions with the C terminus of PTH(1-34), and (2) the TM5/ECL3/TM6 region of the receptor, which interacts with the N-terminal activation domain in PTH. Taken together, receptor chimera-based studies indicate that these class II receptors share a similar overall structure with multiple functionally independent, ligand-specific domains. These domains are sufficiently different to permit synthetic hybrid ligands to bind and efficiently activate the complementary receptor chimeras.

Site-Specific Mutagenesis Identifies Residues Critical for Receptor-Ligand Interactions Chimera studies have provided a general model of PTH1-Rc-ligand interactions. Mutagenesis studies with intact receptors have provided more detailed insights into PTH1-Rc-ligand interactions (Fig. 7). Though receptor chimera studies have identified the juxtamembrane region composed of residues on TM5 and

/

67

TM6 as an important interaction domain for the extreme amino terminus of PTH and PTHrP, scanning mutagenesis of the N-ECD has identified an important binding region in the rat PTH1-Rc (residues 182-190). Specifically, F184A, Rla6A, L187A, and I19°A located at the base of the N-ECD were demonstrated to be important determinants for maximum binding of 125I-labeled bovine PTH(l-34) and 125I-labeled bovine PTH(3-34) (172). Homologous substitutions further revealed that hydrophobicity at positions occupied by F TM and L 187 in the PTH1-Rc plays an important role in determining functional interaction with the 3-14 portion of PTH. Conversely, deletion or epitope tag substitutions of more distal N-ECD domains are welltolerated by PTH1-Rc in terms of expression efficiency, ligand-binding affinity, and specificity. Mutagenesis strategies have also identified polar residues within the hydrophobic transmembrane domains of PTH1-Rc as important determinants of

IT33A,Q37A [

Extracellular

R233H, I234N R227A,

Intracellular

12

T33A, Q37A P132L R186A H223R R227A, R230A, R233H I234N L289I, I363Y $370A, V371I T410P M425L L427T W437L Q440L Q451K I458R

PTH COOH-terminus binding Bloomstrand chondrodysplasia PTH Bpzl3 crosslinking site Jansen's metaphyseal chondrodysplasia Disruption of ligand binding, signaling Critical for ligand specificity PTH position 5 (I/H) selectivity [Arg2]PTH binding Jansen's metaphyseal chondrodysplasia PTH Bpal crosslinking site [Arg2]PTH binding PTH[1-34] binding PTH[1-34] binding Disruption of binding, signaling Jansen's metaphyseal

FIG. 7 Overview of mutational analysis of residues involved in ligand binding and receptor activation.

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CHAPTER4

receptor function (22,173). Random mutagenesis of two highly conserved polar sites resulting in conservative polar residue substitutions in the TM regions of the rat PTH1-Rc, R 233 in TM2 and Q451 in TM7, causes 17- to 200-fold reductions in the binding affinity of the agonist peptide PTH(1-34). Yet these residue changes failed to alter the binding affinity of the antagonist/partial agonist PTH (3-34). Furthermore, the double-mutant receptor (R23~/Q451) displays a binding affinity for PTH (1-34) nearly equal to that of the wild-type receptor. However, the double-mutant receptor fails to activate either GsoLor G e~ signaling. Mutation of three residues, S227, R 230, and S2%, predicted to be aligned on the same face of TM2, resulted in blunted PTH(1-34)-stimulated adenylyl cyclase response and lower binding affinity for the agonist despite efficient cell surface expression (22). The same mutation at the corresponding sites in the secretin receptor, another member of the class II GPCRs, resulted in a similar reduction in adenylyl cyclase activity. Taken together, Turner and co-workers suggest that this region in TM2 participates in the mechanism of signal transduction that is common to the class II subfamily (22). Another series of experiments confirms the important role of TM regions in ligand recognition as well as receptor structure. Mutation of a single amino acid (N~92I) in the second TM of the secretin receptor to the corresponding residue in the PTH receptor led to PTH binding and functional signaling by secretin receptor (174). The reciprocal mutation in the PTH1-Rc (I2~4N) led to a PTH1-Rc that was responsive to secretin. Neither mutation significantly altered the response of the receptors to their own ligands. The results suggest a model of specificity wherein TM residues near the extracellular surface of the receptor function as selectivity filters that block access of the wrong ligands to sites involved in receptor activation (174). Recombinant expression of portions of PTH1-Rc has also offered insights into the molecular mechanisms of ligand recognition and receptor activation. The studies described above offer a model of PTH(1-34) receptor binding whereby the extreme N terminus of the peptide interacts with binding determinants within the extracellular loops/juxtamembrane region of the receptor, but more C-terminal residues of the ligand interact with the amino-terminal extracellular domain of PTH1-Rc. Studies by Luck et al. of binding of PTH(1-14) also support this paradigm (18). Other receptor deletion mutant studies have eliminated the second extracellular loop without affecting receptor function and have shown that PTH1-Rc can accommodate a heterologous epitope tag replacement of a portion of that region and retain full binding and signaling capacity (175).

PTH2-Rc

A second class II GPCR, designated PTH2-Rc, selectively binds PTH, but not PTHrE It has been cloned from rat and h u m a n cDNA libraries (176). Immunocytochemical and in situ hybridization studies have identified a n u m b e r of endocrine cells expressing PTH2-Rc, incuding thyroid parafollicular cells, pancreatic islet D cells, and a subset of gastrointestinal peptide-synthesizing cells (177). However, little is known about the endocrine role of PTH2-Rc in these tissues. Though its tissue distribution, in particular its lack of expression in kidney and bone, suggests it has a limited physiologic role in mineral metabolism, its ligand specificity has provided insight into the current model of PTH ligand-receptor interactions. Several lines of evidence suggest that PTH is unlikely to be a physiologically important endogenous ligand for PTH2-Rc: (1) different ligand rank order of intrinsic activity of a series of PTH analogs in the human and rat PTH2-Rcs, (2) considerable lower intrinsic activities and relative potencies of PTH-like ligands at the rPTH2-Rc than at the hPTH2Rc, and (3) the partial agonist effect of PTH-based peptides when compared to bovine hypothalamic extracts (178). Receptor chimera studies, in which the extracellular domains of PTH2-Rc are selectively replaced with homologous portions of PTH1-Rc, have identified several binding domains that regulate selectivity between PTH and PTHrE For example, Turner et al. demonstrated that chimeras in which the N-ECD of PTH1-Rc was fused to the remaining PTH2-Rc at the TM1 extracellular surface permitted binding and cAMP accumulation by PTHrP (177). Similarly, PTH2-Rc N-ECD fused to PTH1-Rc altered ligand specificity and disrupted binding of PTHrE In addition, mutational analysis of PTH2-Rc residues within TM3 and TM7 demonstrated that residue changes of I244L in TM3 and both C3°7Yand F4°°L in TM7 altered specificity of PTH2-Rc and increased binding of PTHrE Based on these data, it was postulated that the extracellular juxtamembrane portion of the transmembrane domain bundle functions as a selectivity filter or barrier that prevents PTHrP from interacting with the PTH2-Rc. In addition, another study using PTH2-Rc demonstrated that the N-ECD and the ECL3, specifically residues R3O4Q and Q44°R of human PTH2-Rc and PTH1-Rc, respectively, interact similarly with PTH and that both domains contribute to differential interaction with PTHrP (179). Other chimeric studies have identified three single amino acids in PTH2-Rc, I TM in TM3, y318 in TM5, and C 307 in TM7, as being involved in the specificity switch for PTH and PTHrP (180).

PTH/PTHrP/RECEPTOR INTERACTIONS / A Third PTH Receptor Subtype Three PTH receptor genes, including a novel PTH3Rc, were cloned by genomic PCR from zebrafish (z) DNA. The zPTH1-Rc and zPTH3-Rc receptors exhibited 69% similarity (61% identity), but less homology with zPTH2-Rc. Zebrafish PTH1-Rc and zPTH3-Rc showed 76 and 67% amino acid sequence similarity with hPTH1-Rc, respectively; but similarity with hPTH2-Rc was only 59% for both teleost receptors. Recombinant zPTH1Rc bound a variety of PTH and PTHrP ligands with a high apparent affinity (IC50, 1.2-3.5 nM), including [Tyr34] hPTH- (1-34) NH2 (hPTH), [Tyr36]hPTHrP(1-36) NH 2 (hPTHrP), and [AlaZ9,Glu3°,Ala~4,Glu35,Tyr36] fugufish PTHrP(1-36)NH 2 (fuguPTHrP). In addition, zPTH1-Rc was efficiently activated by all three peptides (EC50, 1.1-1.7 nM). PTH3-Rc exhibited higher affinity for hPTHrP and fuguPTHrP (IC50, 2.1-11.1 nM) than for hPTH (IC50, 118.2-127.0 nM) and adenylyl cyclase was more efficiently stimulated by fugufish and human PTHrP (ECs0 = 0.47 _+/0.27 and 0.45 _+0.16 nM, respectively) than by hPTH (EC50 = 9.95 _+1.5 nM). Finally, total inositol phosphate accumulation by zPTH1-Rc was observed to increase after agonist administration; however, zPTH3-Rc failed to activate this signaling pathway. These studies suggest that PTH and PTH-like peptides may exert their effects via as yet uncharacterized receptors.

Receptor Mutations and Human Disease

Jansen'sMetaphysealChondrodysplasia Jansen's metaphyseal chondrodysplasia (JMC) (also see Chapter X) is a rare form of short-limb dwarfism associated with abnormalities in endochondral skeletal development, hypercalcemia, hypophosphatemia, and normal levels of PTH and PTHrP. Originally, two missense mutations in the PTH1-Rc coding region were discovered in patients with the disease (181,182). These mutations, H223R and T41°P, resulted in constitutive activation of the cAMP signaling pathway and are both located at the cytoplasmic base of TM2 and TM6, respectively. A third novel missense mutation was found (I458R) in anotherJMC patient, and is located at the cytoplasmic juxtamembrane region of TM7 (183). In COS-7 cells expressing the human I458RPTH1-Rc, basal cAMP accumulation was approximately eight times higher than in cells expressing the recombinant normal receptor. Furthermore, the I458Rmutant showed higher activation by PTH than by the normal receptor in assays measuring accumulation of downstream effectors, adenylyl cyclase and phospholipase C. Like the HZZ3R and the T41°p mutants, the I458Rmutant does not constitutively activate basal inositol phosphate accumulation. Interestingly,

69

these mutations all occur at TM regions near the intracellular loops of PTH1-Rc that are hypothesized to interact and activate intracellular G proteins and the subsequent signaling cascade. These mutations in PTH1Rc also have been utilized to screen for identification of PTH and PTHrP analogs with inverse agonist activity. Two peptides, [Leu 11, D-Trp12]hPTHrP(7-34)NH2 and [D-Trp12,Tyr34]bPTH (7-34)NH 2, exhibited inverse agonist activity in COS-7 cells expressing either mutant receptor (H22~Rand the T41°P), and reduced cAMP accumulation by 30-50% with an EC50 of approximately 50 nM (184). Such inverse agonist ligands someday may be useful tools for exploring the different conformational states of the receptor as well as leading to new approaches for treating human diseases with an underlying etiology of receptor-activating mutations.

Blomstrand Chondrodysplasia Blomstrand osteochondrodysplasia (BOCD) (also see Chapter 44) is a rare lethal skeletal dysplasia characterized by accelerated endochondral and intramembranous ossification. The phenotype of BOCD is strikingly similar to PTH1-Rc knockout mice in which PTH1-Rc-ablated mice display prominent pathology in the growth plate (185). In both human disease and the PTH1-Rc-ablated mouse model, the growth plate is reduced in size due to a lack of columnar architecture of proliferating chondrocytes, as well as a greatly reduced zone of resting cartilage. This overall similarity of phenotype suggests an inactivating mutation of PTH1-Rc as a possible underlying genetic defect causing BOCD. To date, two types of inactivating mutations have been documented in BOCD (186,187). The first is a single homozygous nucleotide exchange in exon E3 of the PTH1-Rc gene. This alteration changes a proline residue to leucine at position 132 in the receptor's amino-terminal extracellular domain. Proline 132 is conserved in all mammalian class II G proteincoupled receptors. COS-7 cells expressing a green fluorescent protein-tagged mutant receptor do not accumulate cAMP in response to PTH or PTHrP and do not bind radiolabeled ligand, despite being expressed at levels comparable to GFP-tagged wild-type PTH1-Rc. Thus, while full-length PTH1-Rc is being synthesized, it lacks binding of ligand and is functionally inactive. At least one mutation in PTH1-Rc has also been detected in BOCD that causes a shift in the receptor mRNA open reading frame and thus generates truncated receptor fragments (188). Sequence analysis of all coding exons of the PTH1-Rc gene identified a homozygous point mutation in exon EL2 in which one nucleotide (G at position 1122) was absent. The

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CI-IAPTWR4

missense mutation produces a shift in the open reading frame, leading to a truncated protein after amino acid 364 in the second extracellular loop. The mutant receptor, therefore, lacked transmembrane domains 5, 6, and 7. These are precisely the regions thought to be critical for (1) interaction of PTH1-Rc with the activation domain at the extreme N terminus of PTH and PTHrP and (2) the activation of coupled G proteins at the intracellular surface of the receptor. Functional analysis of the m u t a n t receptor in COS-7 cells and of dermal fibroblasts obtained from the patient demonstrated that the mutation was inactivating. Neither the transiently transfected COS-7 cells nor the dermal fibroblasts increased cAMP accumulation in response to PTH or PTHrE

LIGAND-RECEPTOR BIMOLECULAR COMPLEX-CENTERED APPROACH One of the most effective ways of characterizing any ligand-acceptor system is to study the intact bioactive bimolecular complex under conditions that will not perturb their active conformation. Routinely, X-ray crystallography and NMR spectrometry are the tools of choice. These methods yield very detailed structures that have been utilized in rational drug design and have generated unprecedented leads for the development of novel therapeutic agents. Enzyme-substrate/inhibitor such as cathepsin K-inhibitor (189) and HIV protease-inhibitor (190), and soluble protein acceptor-ligand systems, such as the h u m a n growth h o r m o n e (hGH)-extracellular domain of the hGH receptor (191), erythropoietin (EPO)-EPO receptor (192), and ligand-FK506 binding protein (193) are just a few of a long list of successes demonstrating the power of studying the bimolecular complex and identifying intermolecular interfaces. Unfortunately, membrane-embedded proteins like the G protein-coupled receptors are not amenable for either NMR or X-ray analysis because of their large molecular weights and inability to form crystals. Two approaches, one ligand-centered and the other receptor-centered, have been pursued to further the understanding of ligand-PTH-Rc interactions and each has made important contributions (see preceding sections). The hormone-centered approach succeeded in mapping functional domains within the h o r m o n e for receptor-binding and activation. In some cases, structural features responsible for biologic properties have been identified down to the level of a single amino acid. However, this approach cannot be used to deduce the domains of the Rc that are in contact with the horm o n e across the interface. Furthermore, in many cases, the consequences of modifying the primary structure of

the h o r m o n e cannot be assumed to alter Rc interaction unambiguously. Although structural modifications of the h o r m o n e may alter directly the interaction with an important complementary structural feature of the Rc, some substitutions in the h o r m o n e may produce their effect on bioactivity through either local or global conformational changes within the h o r m o n e that prevent adaptation of an optimal "bioactive conformation." In essence, the hormone-centered approach is "blind" to the structure of Rc. The receptor-centered approach also has succeeded in providing valuable insights. PTH receptors with amino acid substitutions or chimeric structures (interspecies of PTH1-Rc, such as rat with opossum Rc, or interhormone receptors, such as PTH1-Rc with calcitonin or secretin Rcs) have been created, and information has been obtained regarding the importance of specific Rc domains and single amino acids necessary for Rc function. However, analysis of the consequences of modification of Rc structure alone cannot be used to deduce interacting complementary elements in the hormone. Furthermore, one usually cannot determine unambiguously whether a modification in the Rc disrupts function as a result of either a local change in an important "contact site," which affects directly the interaction with a site in the hormone, or an internal global conformational change in Rc leading to modified Rc topology, and thereby altered interaction with hormone, or both. Hence, despite the attractiveness of both lines of investigation and the importance of the contributions each makes, conclusions drawn from both the hormone-centered and the Rc-centered approaches have inherent limitations and are inferential at best. Therefore, the most appealing m e t h o d for identifying hormone-Rc interacting domains is a direct one, based on the analysis of cross-linking sites. Photoaffinity labeling has emerged as an effective methodology for studying interactions of biologic macromolecules with their ligands (194-197). It is now feasible to use a photocross-linked conjugate as a starting point for mapping "contact domains," and even "amino acid-to-amino acid contact points," between a biologically active comp o u n d and an interacting macromolecule (198-210). We and others have embarked on a challenging program to map the bimolecular interface between a large peptide h o r m o n e and a seven-transmembrane-spanning Rc. The approach, using photoaffinity scanning (PAS) to identify directly contact sites in the hPTH1-Rc responsible for h o r m o n e binding and signal transduction, relies on six parallel efforts: (1) the design and synthesis of bioactive PTH analogs that are resistant to certain kind of cleavage agents and enzymes, and that incorporate a photoreactive moiety and a radionucleide; (2) production of sufficient high quantities of functional

PTH/PTHrP/RECEPTOR INTERACTIONS /

tify at a highly localized level the structural elements critical for h o r m o n e - r e c e p t o r interaction. The mapping effort is interdisciplinary. Site-directed mutagenesis is used to generate new specific cleavage sites or to eliminate existing ones in an attempt to validate the digestion map generated from the wild-type receptor. Alternatively, mutagenesis is important to reduce the size of a cross-linked fragment in order to further delineate the contact site. Synthesized or expressed receptor sequences that include a contact site are conformationally analyzed, in the presence of micelles to mimic the m e m b r a n e milieu, to provide insight on the bioactive conformation of these domains. Finally, homology searches, computer modeling employing distance geometry, and molecular dynamics are used to merge the various inputs in an effort to generate a unified experimentally based model of the bimolecular ligand-GPCR complex. Indeed, this is an iterative process in which every new finding added to the data base results in the modification or confirmation of the emerging bimolecular model. Because of the nature of this approach it cannot yield molecular structures of the same resolution as those obtained by X-ray crystallography or NMR analysis. Nevertheless, u n d e r the current circumstances and with the technology available at h a n d it yields the best approximation of the actual ligand-receptor complex.

native and mutant hPTH-Rcs to permit cross-linking, exhaustive digestion, purification (epitope-tagged hRc), and subsequent analysis of fragments generated; (3) devising a strategy for a cascade of cleavages that identifies unambiguously hormone-binding sites within the hPTH-Rcs; (4) production of antibodies to various hRc extracellular epitopes for use in purification and analysis; (5) expressing receptor domains that contain the contact sites for conformational studies; and (6) integration of the cross-linking data with our Rc mutagenesis data, and eventually with conformational analysis and molecular modeling data to generate an experimentally based model of h o r m o n e - R c complex. Special design, and synthesis of PTH and PTHrP analogs containing photophores that are strategically and uniquely inserted along the h o r m o n e sequence permit the identification of h o r m o n e - r e c e p t o r interaction sites. These could be either interactions between an amino acid in the ligand and a contact site in the receptor, or more precisely between an amino acid in the ligand and an amino acid in the receptor, namely point-to-point interactions (Fig. 8). A radiolabeled h o r m o n e - r e c e p t o r photoconjugate thus generated is fragmented using enzymatic or chemical cleavage methods. The radiolabeled h o r m o n e or its fragment is covalently linked to a segment of the receptor containing a binding domain, and is subsequently isolated and characterized, thereby identifying small regions of horm o n e and receptor that are in contact with or in proximity to each other. By moving the photoreactive cross-linking moiety along the peptide sequence to certain discrete positions in PTH or PTHrP where bioactivity can be maintained, it will be possible to map precisely the binding sites of the receptor and to iden-

Radiolabe!ed Photoreactive

Receptor

Ligand

Ligand-Receptor Complex

71

Photoreactive Analogs The initial efforts to generate a photoreactive, radiolabeled, and biologically active analog of PTH aimed to identify the receptor as a distinct molecular entity (211-214). All of these studies used poorly character-

Ligand-Receptor Ligand-Receptor Conjugate ConjugatedFragment

2A

7

=/m i

_u

Binding

UV

3

• '

Radiolabef ~ - - Photoreactive moiety

j

FI6. 8 Schematic approach to photoaffinity scanning of PTH receptors. Photocross-linking is followed by fragmentation of the resultant radiolabeled hormone-receptor photoconjugate. Comparison of the fragmentation pattern elucidated by SDS-PAGE analysis with the theoretical restriction digestion map of the receptor identifies the putative contact site. Mass spectroscopic and microsequence analysis will identify the cross-linked residue in the receptor. (See color plates.)

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CI-IAeTWI~4

ized nitroarylazide-based photophores and reported molecular masses ranging between 28 and 95 kDa for the hormone-receptor complex. Shigeno and coworkers carried out a careful synthesis and characterization of the nitroarylazide-based photoligand and identified it to be [Nle8'18,Lysla (N~-(4-N~-2-NO2-phenyl),Tyra4] PTH(1-34)NH 2, a fully active analog in ROS 17/2.8 cells (Kd = 2.8 nM compared to 0.8 n M for the corresponding photoinactive radiolabeled PTH) (215). Using this photoaffinity ligand, they were able to identify in the same cells a plasma membrane glycoprotein corresponding to the PTH receptor that had the apparent molecular mass of 80 kDa (215,216). Nevertheless, more extensive characterization of the cross-linking site could not be carried out beyond this level with the arylazide-based photoreactive PTH analogs. Our strategy for the ligand/receptor-based approach was to introduce arylketone-based PAS methodology (27,217-220) into the field of calciotrophic hormones and their corresponding receptors (26,28,29,31, 220-222). In this section we summarize the major achievements in the design and development of benzophenone-containing PTH and PTHrP ligands, and their contribution to the mapping of the bimolecular ligand-receptor interface. The benzophenone moiety (which cross-links with >50% efficiency and has greater specificity than arylazides) was employed successfully in other systems (198-210). But in the mid-1990s the methodology was still at an early stage of development and was never applied to any of the calcium-regulating h o r m o n e / G proteincoupled Rc systems. The photogenerated triplet state of the benzophenone is capable of inserting into many types of C-H bonds, provided close proximity is achieved (223). This is in contrast to most other photoaffinity labels, e.g., azidoaryl functions, which generate highly reactive electrophilic species and therefore interact preferentially with nucleophilic groups on proteins (194). There are several advantages of benzophenones over other photophores. Relatively low-energy UV radiation is needed for photoactivation (224) and the reactive biradical is nearly nonreactive in water (225). Therefore, during cross-linking experiments, a large excess of photoaffinity label is unnecessary: efficiency of cross-linking is high because only a small amount is lost to hydrolysis. In addition, the photolabile moiety is compatible with solid-phase peptide synthesis methodology. Furthermore, synthesis, purification, and biological evaluation can be conducted in the laboratory under normal ambient light conditions. Radioiodination was chosen as the tagging method of choice because of its high specific radioactivity translating into high sensitivity of detection of the radiolabeled conjugated ligand-receptor complex and the fragments derived from it. The drawbacks are the regulatory constraints imposed on working with radioactive material

and the relatively short half-life of the radiolabeled material. Needless to say, radioiodination is not necessarily an innocuous modification. In several cases, radioiodination of otherwise bioactive benzophenone-containing PTH analogs resulted in a radiolabeled analog devoid of the binding affinity required for efficient photocross-linking. Because current technology has not optimized substitution of the benzophenone moiety with a radioiodine (226), these two features must be presented on two different amino acid moieties in the ligand. Therefore, successful PAS analysis requires maintaining the connectivity between the radiotag and the photophore throughout the controlled degradation of conjugated ligand-receptor complex. Modifications in PTH (1-34), which include Met 8 and 18 _...) Nle 8 and 18, Lysl~,26, and 27 .__) Lys13,26, and 27, and Yrp 2~ --+ 2-naphthylalanine 2~ (Nal), render the ligand resistant to the various chemical and enzymatic cleavage agents [i.e., CNBr, lysyl endopeptidase (Lys-C), and BNP-skatole, cleaving at the carboxyl side of Met, Lys, and Trp, respectively]. The premise of any photoaffinity cross-linking study is that analogs with similar pharmacologic profile share with the parent peptide hormone similar bioactive conformation and generate topochemically equivalent ligand-receptor complexes. The photoreactive benzophenone-containing analogs of PTH and PTHrP were designed specifically for PAS studies aimed at investigating the bimolecular interactions of the activation and binding domains of PTH and PTHrP with either the PTH1-Rc or the PTH2-Rc subtypes. Table 1 summarizes all bezophenone-containing analogs of PTH and PTHrP reported to date.

Identification o f Contact Sites We and others have identified contact sites for positions 1, 13, and 27 in PTH and positions 1, 2, and 23 in PTHrP using the PAS methodology (26-29,222,227). Two different photophores were used in different studies; p-benzoylphenylalanine (Bpa) (28,29,227) and Lys(N~-p-benzoylbenzoyl) (Lys(N~-pBz2)(26,27,29,222). The former has the benzophenone moiety attached to the peptide backbone through a [3-carbon while the latter is presented on a relatively long side chain removed by six atoms from the backbone. The differential positioning of the benzopheneone moiety relative to the backbone may play a limited role in selecting the cross-linking sites. Cross-Linking to Position 1 in PTH 1 Photocross-linking of [Bpa 1 ,Nle'8 1 8 ,Arg~'26'Z7,NalZ~,Tyr34]bPTH(1-34)NH2 (Bpal-PTH) to the human PTH1-Rc stably overexpressed (---400,000 Rcs/cell) in human embryonic kidney cell line 293 (HEK293/C-21)

PTH/PTHrP/R~cF.PToR INTERACTIONS / TABLE 1

Analog I

II III IV V Vl VII VIII IX X Xl Xll XlII XlV XV XVl XVll XVlII XlX XX XXl XXll XXlII XXiV XXV XXVl XXVll XXVlII XXlX XXXl XXXll XXXlII XXXlV XXXV

73

Benzophenone-containing analogs of PTH and PTHrP Analog (Ref.)

Position

[Bpa', Nle8'~8,Arg~3'26'2z,Na123,Tyr34]bPTH( 1-34)NH 2 (28) [Bpa2,Nle8'~8,Arg'3'26'27,Na123,Tyr34]bPTH(1-34)NH2 (28) [Bpa3, Nle8'~8,Arg~3'26'27,Na123,Tyr34]bPTH(1-34)N H2 (28) [Bpa4,Nle8'~8,Arg~3'26'27,Na123,Tyr34]bPTH(1-34)NH2 (28) [Bpa5, Nle8'~8,ArgO3'26'27,Na123,Tyr34]bPTH(1-34)N H2 (28) [Bpa6, Nle8"8,Arg'3'26'27,Na123,Tyr34]bPTH(1-34)NH2 (28) [Bpa7,NleS"8,Na123,Tyr34]bPTH(1-34)NH2 (217) [Arg2, Lys7(N'-pBz2),Tyr34]bPTH(1-34)NH 2 (217) [NleS"8,BpaZ,a-Nal'2, Na123,Tyr34]bPTH(7-34)NH2 (217) [Nle8'~8,Bpa~2,Na123,Tyr34]bPTH(1-34)NH2(217) [Nle8"8,Bpa'2,Na123,Tyr34]bPTH(7-34)NH2 (217) [NleS,,8,Lys,3(N,pBz2),Na123,Tyr34]bPTH(1_34)NH2 (217) [NleS,,8,D.Nal,2,Lys,3(N,.pBz2), Na123,Tyr34]bPTH(7_34) N H2 (217) [Nle8,,8,Lys,3(N,_p(3_l_Bz)Bz),Na123,Arg26,27,Tyr34]bPTH(1_34)NH2 (26) [Nle8,,8,Lys,3(N,.pBz2), Na123,Arg26,27,Tyr34]bPTH (1_34) N H2 (263) [Arg2, Lys '3(N'-pBz2),Tyr34]bPTH(1-34)N H2 (217) [NleS"8,Bpa23,Tyr34]bPTH(1-34)NH2 (217) [Nle8"8,D-NaI~2,Bpa23,Tyr34]bPTH(7-34)NH2 (217) [Nle8"8,D-NaI'2,Na123,Lys26(N'-pBz2),Tyr34]bPTH(7-34)NH2 (217) [Nle8"8,Na123,Lys26(N'-pBz2),Tyr34]bPTH(1-34)NH2 (217) [Nle8'~8,Arg'3'26,L-2-Na123,Lys27(N'-pBz2),Tyr34]bPTH(1-34)NH2 (222) [Bpa~, Ile~,Arg'l"3,Tyr36]PTHrP(1-36)NH 2 (29) [Bpa',IleS,Trp23,Tyr36]PTHrP(1-36)NH2 (227) [Bpa2,11e~,Arg~'~3,Tyr36]PTHrP(1-36)NH2 (29) [Bpa2, IleS,Trp23,Tyr36]PTHrP(1-36)NH2 (227) [Bpa3,11eS,Arg~"~3,Tyr36]PTHrP(1-36)NH2 (29) [Bpa3,11eS,Trp23,Tyr36]PTHrP(1-36)N H2 (227) [Bpa4,11e~,Arg~"3,Tyr36]PTHrP(1-36)NH2 (29) [Bpa4, IleS,Trp2~,Tyr36]PTHrP(1-36)N H~ (227) [BpaS,Arg~"3,Tyr36]PTHrP(1-36)NH2 (29) [Bpa5,Trp23,Tyr36]PTHrP(1-36) N H2 (227) [Bpa6,11e5,Arg'~"3,Tyr36]PTHrP(1-36)NH2 (29) [Bpa6, Ile~,Trp~3,Tyr36]PTHrP(1-36)NH2 (227) [lleS,Bpa23,Tyr36]PTHrP(1-36) N H2 (30)

1 2 3 4 5 6 7 7 7 12 12 13 13 13 13 13 23 23 26 26 27 1 1 2 2 3 3 4 4 5 5 6 6 23

generates an 87-kDa photoconjugate (28). Chemical digestions by CNBr and BNPS-skatole, which cleave at the carboxyl end of Met and Trp, respectively, and enzymatic digestions by lysyl endopeptidase (Lys-C) and endoglycosidase F/N-glycosidase F (Endo-F), which cleave at the carboxyl end of Lys and deglycosylate the aspargines at the consensus glycosylated sites, respectively, generate a digestion restriction map of the photoconjugated receptor. Although the resolving power of polyacrylamide gel electrophoresis is limited, the combination of consecutive cleavages (e.g., Endo-F followed by Lys-C followed by CNBr) carried out in

reversed order (e.g., Lys-C followed by BNPS-skatole and BNPS-skatole followed by Lys-C) is extremely powerful. It generates a reproducible pattern of digestions and produces a set of fragments delimited by specific end residues and the presence or absence of glycosylation sites. Comparing the putative digestion map of the hPTH1-Rc with the actual fragments identifies 125 the sequence of the smallest I-radiolabeled 1 Bpa-PTH-PTH1-Rc conjugated fragment (---4 kDa). This fragment includes the ligand (4489 Da) modified by a moiety contributed by a Met residue belonging to

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CHAPTER 4

the receptor (28). Two Met residues, 414 and 425, present at the midregion and the extracellular end of TM6, emerged as potential contact sites for position 1 in PTH. Contact between residue 1 in PTH and M 414 requires the N terminus of PTH to protrude into the seven helical and hydrophobic transmembrane domain bundle. In contrast, contact with M 425 can be achieved while the N terminus is dipping superficially into the transmembrane domain bundle. These biochemical methods can be supplemented by molecular biology to provide additional resolving power to the PSA. Transient expression of two point-mutated hPTH1-Rcs, [M414L] and [M425L], in COS-7 cells, generated fully active receptors (28). The 125I-labeled Bpa ~PTH lost its ability to photocross-link to [M425L] but not to [M414L], suggesting that position 425 is the putative contact site for position 1 in PTH. Cross-Linking to Position 13 in PTH

Biochemical analysis of the photocross-linking product of radiolabeled [NleS'lS,Lysl~(Aff-p(3-I-Bz)Bz) ,NalZ3,ArgZ6'ZW,Tyr~4]bPTH (1-34) NH 2 [Lys 13(PBz2)PTH] with hPTH1-Rc expressed in HEK293/C-21 cells identifies a glycosylated radioactive band of---6 kDa, which is delimited by Lys-C and CNBr cleavage sites at the N and C termini. The theoretical cleavage restriction map of hPTH1-Rc reveals the minimal 125 13 radiolabeled I-Lys (PBz2)-PTH-hPTH1-Rc conjugated fragment, corresponding to hPTH1-Rc(173-189) located at the C-terminal region of the extracellular N terminus (26). Site-directed mutagenesis within the 17 amino acid residues comprising hPTH1-Rc(173-189) combined with subsequent biochemical analysis further delineates the boundaries of the contact site for 125I-Lys13(pBz2)PTH to hPTH1-Rc(182-189), an 8-amino acid sequence (263). Several single site-mutated receptors were generated, which include a new Lys-C-susceptible cleavage site. The mutant [RlSlK]hPTH1-Rc was stably expressed in HEK293 cells (---200,000 Rcs/cell) and was fully functional. Compared to the wild-type receptor, Lys-C cleavage of the 125I-Lys13(pBz~)-PTH-[RlS1K] photoconjugate produces a smaller conjugated fragment (---18 vs. --~9 kDa, respectively), corresponding to a cleavage site upstream to the N-glycosylated Asn 176. Interestingly, the only functional mutations that failed to cross-link to 125I-Lys13(pBzz)-PTH were the [RlS6K/A] mutants (263). However, [R~S6K]hPTH1-Rc stably expressed in HEK293 cells cross-links effectively to 125IBpal-PTH and displays wild-type receptor-like cyclase activity and binding affinity similar to that in HEK293/C-21 cells. These findings suggest that R 186 participates in an interaction with the ligand that either

provides a contact site for position 13 in the ligand or provides an interaction that brings the ligand into the close spatial proximity required for cross-linking within the hPTH1-Rc(182-189) contact site (263). This interaction does not appear to be essential for a productive ligand-receptor interaction because [RlS6K] is fully functional and cross-links effectively with a25I_Bpaa_PTH. Cross-Linking to Position 27 in PTH

Though the previously mentioned studies address interaction between residues in the extended activation domain of PTH comprising residues 1-13, a similar approach was directed toward the principal binding domain (sequence 24-34). We have analyzed • • • 8 18 13 26 the blmolecular Interaction between [Nle' ,Arg ' ,L-223 27 34 27 Nal ,Lys (N~-PBz2),Tyr ]bPTH(1-34)NH 2 [Lys (pBzz)PTH], modified by a benzophenone-containing photophore at position 27, and hPTH1-Rc by employing a combination of biochemical analysis of the photoconjuate and site-directed mutagenesis (222). Analysis of the 5 27 I-Lys (pBzz)-PTH-PTH1-Rc photoconjugate by CNBr/ Endo-F and BNPS-skatole/Endo-F degradation pathways produced an overlapping sequence corresponding to L232-W 298. This contact domain includes part of TM2, ECL1, and the entire TM3. Secondary digestions of the CNBr- and BNPS-skatole-derived fragments by endoproteinase Glu-C, which predominantly cleaves at the carboxyl side of Glu, converged on an overlapping 38-amino acid sequence corresponding to L261-W298, which includes part of ECL1 and the entire TM3 (222). To further delineate and validate the sequence containing the cross-linking site for position 27 in PTH, three mutated receptors were generated and transiently expressed in COS-7 cells. All three receptors, [R262K], [LZ61M] a n d [LZ61A], were expressed and displayed characteristic binding affinity and PTHstimulated adenylyl cyclase activity compared to wildtype receptor. [RZ62K] a n d [LZ61M] were designed to modify the Lys-C and CNBr cleavage pattern, respectively. The [L26~A] was introduced to eliminate a favorable insertion site at position 261. Restriction digestion 125 27 262 analysis of the ' I-Lys (pBzz)-PTH-[R K] photoconjugate delineated the contact site to hPTH1-Rc(232-262). Taken together, the minimal contact sites (sequence 261-298) and (sequence 232-262) obtained from the analysis of the wild-type and m u t a n t [RZ62K] receptors, respectively, suggest either L 261 or R 262 as the contact site for Lys27 . Treatment of the 125I-Lys27(pBz2)-PTH-[LZ61M] photoconjugate with CNBr generated a conjugated fragment similar in size to the ligand. This result suggests position 261 in the receptor to be the contact

PTH/PTHrP/RECEPTOR INTERACTIONS / site for position 27 in the ligand. This was further confirmed by the elimination of effective cross-linking of I125-LysZ7(pBzz)-PTH to the mutated receptor [L261A] i n which a reactive insertion site such as Leu is replaced by Ala, a poor insertion site for the photoactivated benzophenone-derived biradical. Position 261, the contact site for position 27 in PTH, is located near the center of ECL1 (222). The identification of L 261 in hPTH1-Rc as a contact site in f o r Lysz7 in PTH provides important information for mapping the PTH-PTH1-Rc interface. The remoteness of position 27 from positions 1 and 13 in PTH, and that of L 261 f r o m R 186 and M e t 425 in hPTH1-Rc, generates an important additional structural constraint that can be used to refine the emerging experimentally based model of the PTH-PTH1-Rc complex. Based on conformational analyses and structure-activity studies of PTH(1-34) and PTHrP(1-34), the prevailing view argues that these two hormones interact very similarly if not identically with the PTH1-Rc. In line with this assumption, radioiodinated [Bpa1,Ile5,TrpZ3,Tyr36]PTHrP (1-36) NH 2 [lZ5IBpal-PTHrP] photocross-links to M 425 in hPTH1-Rc in the same fashion as the corresponding PTH analog, lz5I-Bpal-PTH (29). Cross-Linking to Position 23 in PTHrP

Another photoreactive analog of PTHrP, [Ile5,Bpa 2~, Tyr~6]PTHrP(1-36)NH2 [Bpa2~-PTHrP], modified by a benzophenone moiety incorporated at position 23, was reported by Mannstadt and co-workers to cross-link to Y23-L4°, located at the very N-terminal end of rat PTH1Rc (30). CNBr analysis of the 125I-Bpa2~-PTHrP-rPTH1Rc photoconjugate suggests that the contact site resides at the N terminus of the receptor, rPTH1-Rc(23-63). A combination of site-directed mutagenesis (single point mutation [M63I] and the double mutants [M63I,L4°M] and [M6~I,L41M]) and CNBr cleavages further delineates the contact site to span the sequence 23-40. Earlier findings demonstrated that the two mutant rPTH1-Rcs with deletions of residues 26-60 or 31-47 transiently expressed in COS-7 cells had little or no capacity to bind 125I-labeled PTH, therefore suggesting these regions to be important for ligand binding (228). Only two of the 31 32 33 four cassette mutant receptors ([V A,F A,T A, 35 36 37 38 K34A,Ea5A] and [E A,Q A,I A,F A]) spanning the 31-47 sequence displayed diminished 125I-labeled PTH binding capacity. Finally, in an Ala-scan of the 31-38 region, mutants [Ta3A] and [Q~WA] exhibited the largest loss in binding affinity of 125I-labeled PTHrP and complete loss of binding affinity toward the antagonist 11 12 [Leu ,D-Trp ]PTHrP(7-34)NH 2. Relying primarily on mutagenesis-based analysis, Mannstadt and co-workers

75

suggest that the first 18 amino acid residues of the PTH1Rc comprise the contact site for position 23 in PTH, and T 33 a n d Q37 are functionally involved in binding of the 7-34 region in PTH rather than the 1-6 region (30). The location of contact sites for two closely spaced residues in PTH/PTHrP (23 and 13) at both ends of the extracellular amino terminus of the receptor (within 23-40 and in proximity to R 186,respectively) is consistent with the current model of the ligand-receptor binding interface. The extensive length of the putative extracellular amino terminus of PTH1-Rc (---167 residues) allows for assumption of secondary and tertiary structures by the receptor that can accommodate simultaneously the above-mentioned bimolecular interactions. Cross-Linking of Position 1 of Agonist vs. Antagonist

A very interesting observation that directly distinguishes between the nature of the bimolecular interaction of an agonist versus antagonist with PTH1-Rc was recently reported by Behar and co-workers (29). Photoconjugation of radiolabeled [Bpa2,Ile5,Arg 11'13, Tyra6]PTHrP (1-36) NH 2 [BpaZ-PTHrP], a highly potent antagonist, to hPTH1-Rc was carried out in HEK293/ C-21 cells (29). Unlike the analog [BpaZ,Nle s'18, Ar g 132627 ' , , NalZ3,Tyra4]bPTH(1-34)NH2 [BpaZ-PTH], which is a full agonist (28) and cross-links t o M 425 in PTH1-Rc in a manner similar to 125I-Bpa1-PTH o r 125IBpal-PTHrP, 125I-BpaZ-PTHrP also cross-links to a proxi' mal site within the receptor domain p415-M425 (29). These results may reflect either differences between the binding modes of agonist and antagonist or differences in the interaction between the two consecutive positions in the PTHrP(1-36) sequence and PTH1-Rc. In an attempt to distinguish between these two possibilities, we utilized the agonist analog BpaZ-PTH, which carries the same photoreactive moiety at the same position as the antagonist BpaZ-PTHrE Analysis of 125I-Bpa2-PTH photoconjugates with wild-type [MalaL] and [M4Z5L] mutated hPTH1-Rcs indicates that this ligand cross-links only to the e-methyl of Met 4z5, similar to Bpal-PTHrP and to Bpal-PTH cross-linking (28). These results, therefore, provide strong support for the hypothesis that the differences observed between 2 the cross-linking of 125I-Bpa1- and 125 I-Bpa-PTHrP may reflect different interaction modes of an agonist versus an antagonist with the PTH1-Rc. Interestingly, two additional Bpa-containing PTHrP(1-36) analogs, [Bpa 2 ,Ile 5 ,Trp 23 ,Tyr36 ]-and [Bpa 4, 5 23 36 Ile ,Trp ,Tyr ]PTHrP(1-36)NH 2, were reported to preferentially antagonize and cross-link to hPTH1-Rc and hPTH2-Rc stably expressed in LLC-PK1 cells, respectively (227). However, in homologous systems composed of hPTH1- and hPTH2-Rcs expressed in a human cellular

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/

C~TF~k4

background (HEK293/C-21 and HEK293/BP-16, respectively), Bpa2-PTH is a full agonist and Bpa4-PTH is a very weak agonist with a slightly better affinity for the hPTH2-Rc (28,227). Similar to [Bpa4,Ile5,Trp2~,Tyr~6]PTHrP (1-36) NH 2 (227), [Bpa4,Ile5,Arg 11'13,TyrS6]PTHrP(1-36)NH 2 displays poor binding affinity and negligible efficacy in HEK293/C-21 cells expressing the hPTH1-Rc (29). Although PTH2-Rc may not be the physiologic target for PTH or PTHrP, its structural resemblance to PTH1-Rc, its high binding affinity, specific cross-linking, and effective coupling to the PTH-induced intracellular signaling pathways make it an attractive target for exploring structure-function relations in the P T H / PTHrP-PTH1-Rc system. Analysis of the photoconjug ates obtained on cross-linking of '25I-BpaI-PTH and 25I-Lys'~(PBz2)-PTH to hPTH2-Rc stably expressed in HEK293 cells (HEK293/BP-16, ---160,000 Rcs/cell) revealed that both hPTH1-Rc and hPTH2-Rc use analogous sites for interaction with positions 1 and 13 (31). The PAS methodology offers the only readily available experimental approach to study directly the bimolecular ligand-GPCR interface. To practice this methodology we introduce b e n z o p h e n o n e moieties, radioiodine, and substitutions that provide resistance to specific chemical and enzymatic cleavages. These modifications are tolerated as long as the modified photoreactive ligand binds to the receptor specifically and with high affinity, and stimulates adenylyl cyclase in a PTH-like manner. The photoinsertion site of the benzophenone moiety is dictated by spatial proximity. But it is also biased toward the more reactive insertion sites within its reactivity sphere. Last but not least, the cleavages employed and the level of resolution allowed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis limit analysis of the photoconjugate. The validation of a putative contact site by site-directed mutagenesis is not necessarily benign. It generates some degree of perturbation, which we accept as long as the mutated receptor is expressed and functions similarly to the wild type. Taken together, PAS is not the perfect method, but we believe it is the best available. Future improvement in separating the photoconjugated receptor a n d / o r fragment from the nonconjugated species, and the elimination or replacement of the radioactive tag with a nonradioactive tag will be very helpful in terms of allowing access to high-resolution instrumental techniques. The currently labor-intensive PAS methodology will become less time consuming and more robust as the technology evolves.

Experimentally Based Molecular Modeling The contact sites identified in the cross-linking studies described above are only a small fraction of the large

ensemble that forms the bimolecular interface. The cumulative effect of these multisite bimolecular interactions results in specific ligand recognition, binding affinity, and eventually a conformational change in the receptor that leads to specific intracellular signal transduction. In general, not all contact sites revealed by PAS methodology will have the same functional significance. However, all these contact sites will be part of the ligand-receptor interface and are therefore indispensable targets in mapping efforts. The objective of the PAS studies is to generate a series of constraints, which will be used in mapping the bimolecular interface. To this end, merging the information generated by the PAS studies with information about the conformation of the ligand and receptor domains, as well as molecular modeling, generates an integrated approach that results in an experimentally based ligand-receptor model. This integrated approach based on experimental data is in contrast to the more c o m m o n approaches that predict conformation and molecular models solely on a theoretical basis. The model for the PTH-PTH1-Rc complex is steadily evolving as new bimolecular contact sites are identified (28,229,230). The combination of hydrophobicity profile analysis and search of the Brookhaven Protein Data Bank (PDB) employing the Basic Logic Alignment Search Tool (BLAST) can identify and refine, respectively, the location of the TM helices (231,232). The identification of the TM domains of the PTH1-Rc is in good agreement with respect to the location of peptides containing TM helical regions as determined by high-resolution NMR (233,234). The arrangements of the TM heptahelical bundle in rhodopsin and bacteriorhodopsin (235-239) were used as templates for the initial arrangement of the putative TM helical domains of the PTH-Rcs. Rotating the helices about their long axis to orient the hydrophobic m o m e n t toward the membrane environment and optimize their helix-helix, helix-core, and helixmembrane interactions generated the putative core of our receptor model (Fig. 9) (234). The modeling of the extracellular and intracellular domains responsible for binding the ligand and coupling to the G protein and other adapter molecules is not as straightforward as that of the membraneembedded core. Unlike the high structural similarity for the arrangement of the seven TM domains bundle (240), the cytoplasmic and ectopic domains of the GPCR are extensively variable and no a pr/0r/ structure is available. The loops are constrained to some extent by the TM helical domains to which they are attached. Additional constraints are imposed by the three disulfide bridges at the extracellular N terminus and the disulfide bridge connecting the first and the second ECL. All of these cysteines are highly conserved in the class II GPCRs, of which PTH1-Rc is a member.

PTH/PTHrP/RECEPTOR INTERACTIONS /

TM1 TM5

IC3 FIG. 9 Depiction of the PTH1-Rc from a molecular dynamic simulation. The transmembrane oL-helices are depicted as cylinders. The regions of the receptor that have been experimentally determined are depicted as ribbons. The regions of the receptor that have been shown to crosslink with PTH analogs, PTH1-Rc(173-181) (26,27) and M42s (28), are depicted in gray (241). (See color plates.)

Unfortunately, the pattern of disulfide bridge formation in the ectopic part of the class II GPCRs is not known and therefore cannot be used in building the receptor model. Homology search with BLAST (232) helps to assign putative elements of secondary structure to regions in the receptor that are homologous to regions of protein for which a secondary structure is available. Once a secondary structure element is identified, it can be incorporated into the model. Practically, we focus only on the identification of ahelices, which can be studied in isolation from other secondary structural elements. Indeed, sequence homology searches identify conformational preferences of the C-terminal portion of the extracellular amino terminus proximal to TM1 of PTH1- and PTH2Rcs and the third extracellular loop of PTH1-Rc (28,229). These homology searches indicate that the third ECL adopts a helical conformation that is highly p r o n o u n c e d for T435-Y443 (229), and amphiphatic helices for K172-M 189 and L129-E 139 in PTH1- and PTH2Rcs, respectively (28,229). Unfortunately, such homology searches may not always result in the assignment of distinct secondary structure to a specific receptor sequence.

77

The modeling procedure for the receptor and receptor-ligand complex is described in detail by Mierke and Pellegrini (241). Briefly, the primary structure of the PTH1-Rc is embedded into a three-phase, H z O / d e c a n e / H 2 0 (40 A each) simulation cell. A multistep simulation is carried out in which either the heptahelical bundle a n d / o r cytoplasmic and extracellular domains are allowed to move freely. PTH, in its membrane-associated conformation, is added to the receptor model, applying the ligand/receptor distance constraints elucidated from the cross-linking experiments, and additional simulations are carried out. Indeed, additional constraints obtained via sitedirected mutagenesis, and chimera studies, PTH2-Rc and other class II GPCRs can be incorporated into the development of the model. The most direct way to identify the conformational features of the cytoplasmic and ectopic domains of the GPCR is by generating these receptor fragments and examining them by NMR in a membrane-mimetic system. Adding a small portion of the corresponding TMs to the otherwise flexible receptor-derived termini or loops provides anchors that partially reproduce the native orientation of the receptor domain relative to the membrane-mimicking milieu. Another design element useful in restraining an excised loop sequence from assuming extended conformations is the covalent binding of both ends of the sequence by a linker of---12 A, the approximate distance between two consecutive TM domains (235,237). To this end, we have characterized the conformational features of the third intracellular loop (ICL) of PTH1-Rc, which was constructed as a 29-amino acid peptide with the side chains of Cys residues in positions 1 and 28 bridged by an octamethylene linker (233,234). This linker also assists in the association of the peptide termini with the membranemimicking micelle. In the presence of either SDS or dodecylphosphocholine (DPC) micelles, the peptide assumes two helical domains composed of residues L e u 4 L e u 8 and Yyr22-Leu26, and [3-turns at Glul°-Ala 13 and Glya4-Asp 17. The hydrophobic residues of the N-terminal amphiphilic helix are embedded in the hydrophobic portion of the micelle, whereas the polar side chain protrudes into the aqueous phase. The two [3-turns point away from the membrane and are exposed to the aqueous solvent. The structure of the bridged peptide in the presence of micelles is distinct from the same peptide in the absence of micelles as well as from the linear form of the peptide. A similar approach was applied to characterize the conformational features of two ectopic domains found to photocross-link to Lys13 a n d Lys27 in PTH(1-34) (28,221,222). Position 13 in PTH was found to cross-link within an 8-amino acid domain,

78

/

C~a'TER4

PTH1-Rc(182-189), located at the C-terminal portion of the N-ECD proximal to the first TM helix (28,221). The combination of homology search and molecular dynamic calculations, using a two-phase simulation cell consisting of H20 and CC14 (to mimic a membrane-water interface), suggests that the segment R179-E-R-E-V-F-D-R-L-G-M189forms an amphipathic oL-helix whose axis is parallel to the membrane surface and points away from the heptahelical bundle (28,242). 1H NMR analysis of the synthetic peptide hPTH1-Rc(168-198) in presence of micelles (to provide the membranelike environment) was carried in combination with distance geometry and molecular dynamic simulation (Fig. 10) (242). The analysis identifies a C-terminal helix, hPTH1-Rc (190-196), corresponding to the ectopic portion of the first TM helix, which was perpendicularly embedded in the micelle. Two oL-helices, (180-188) and (169-176), lie on the m e m b r a n e surface. The polar residues in the linker, E 177 and R 170, and in the middle helix, R TM, E 182, D 185, and R 186, are exposed to the solvent while the hydrophobic residues, F 173, F TM,and L 187, are projecting

toward the hydrophobic membrane (242). Based on the finding that the contact site for Lys 1~ in PTH is within residues (182-189), which includes negatively charged amino acids, coulombic interaction between these charges and the positive charge on Lys is may function as one of the ligand/receptor-specific interactions. Nevertheless, this may not be an essential interaction because analogs in which the e-amino on Lys 1~is blocked by acylation maintain high affinity and efficacy. At this point one can bring together findings obtained from the cross-linking studies of position 13 in hPTH(1-34) with the putative bioactive conformation of PTH (64), and the conformational analysis of the receptor domain containing the contact site for position 13 in order to generate the first generation of an experimentally based model of the PTH-hPTH1-Rc complex (28). Using the cross-linking data as a docking cue to position the ligand (in its putative bioactive conformation) places its C-terminal amphiphilic helix parallel to the membrane-aligned portion of the receptor-derived peptide. This allows the formation of complementary coulombic interactions between the

A 8o

G188

¸¸¸i i Cytoplasm

B

FIG. 10 Structural features and topological orientation of PTH1-Rc (168-198) located at the C-terminal region of the extracellular N terminus followed by the ectopic portion of the first TM domain (241,242). (A) Schematic representation of the experimentally determined conformation. The structure consists of three o~-helices, two of which have been determined to lie on the surface of the membrane; the third, at the top of TM1, is membrane embedded. (B) The orientation of this peptide is shown with respect to the surface of the dodecylphosphocholine micelles used in the NMR study. The hydrophobicity of the molecule is indicated (blue, polar; red, hydrophobic). The decane molecules of the water/decane simulation cell used in the structure refinement are shown in green as CPK space-filling spheres. (See color plates.)

P T H / P T H r P / R E c E P T O R INTERACTIONS /

polar residues in the helix comprising the principal binding domain of the ligand and the polar residues E 177, R 179, R TM, E 182, D 185, and R 186 in the receptorderived peptide. Interestingly, this docking procedure brings only M 425, a n d not M 414, into sufficient proximity to permit cross-linking to position 1 in 125I-Bpa1-PTH. Therefore, these observations are in complete agreement with the results obtained through cross-linking studies (Fig. 11) (28). The structural features of the first ECL in the presence of dodecylphosphocholine micelles were revealed from the high-resolution NMR study, followed by distance geometry calculations and molecular dynamic simulations of a peptide, hPTH1-Rc(241-285), comprising the loop and few residues from the ectopic portions of TM2 and TM3 of the receptor (230). This peptide contains L 26], which was found to cross-link to Lys27 in hPTH(1-34) (222). The structure of this receptor fragment includes three or-helices, (241-244), (256-264), and (275-284). The first and the last helices correspond

79

to the ectopic pordons of TM2 and TM3, respectively. The topological orientation of the helices relative to the membrane surface was examined in the presence of 5and 12-doxylstearic acids, nitroxide radical-containing molecules that serve as reporters for the localization of amino acid residues in the membrane. The amino acids corresponding to the ectopic portion of the TMs are more strongly associated with the lipid micelle and may serve as membranal anchors. All of the hydrophobic residues in the partially ordered central helical portion (terminated by the unique helix-breaking sequence p258_p_p_p261) are projecting toward the lipid surface (230). The conformational analysis of the first ECL is very helpful in gaining important insights into the bimolecular ligand-receptor interaction revealed by crosslinking studies. The benzophenone moiety o n Lys27 in 125I-KZT(N~-pBzz)-PTH cross-links to L 261 in hPTH1-Rc (222). The long, amphiphilic C-terminal helix, which i n c l u d e s Lys27, was found to lie on the surface of the

W457

Extraeellu!ar

M414 s~

Intraeellu!ar Receptor

B TM3 ~.

TM2

i~:

N-Terminus .....

~

TM7

TM5 C.Te~in~

N~Te~ni:~s PTH Ligand

FIG. 11 Model for the binding of hPTH(1-34) to hPTH1-Rc. For clarity, only portions of the TM helices, N terminus, and the third extracellular loop are shown in blue (non-cross-linked domains) and green (contact domains hPTH1-Rc(173-189) and hPTH1-Rc(409-437)) (A, side view; B, top view). The amphipathic oL-helix of the extracellular N terminus of the receptor is projecting to the right, lying on the surface of the membrane. The high-resolution, low-energy structure of hPTH(1-34) determined by NMR in a micellar environment is presented in pink. Residues in cross-linking positions 1 and 13 of hPTH(1-34) are denoted in yellow. The C-terminal amphipatic oL-helix of hPTH(1-34) is aligned in antiparallel arrangement with the amphipatic cx-helix of the extracellular N-terminus hPTH1-Rc(173-189), contiguous with TM1 and encompassing the 17-amino acid contact domain (in green), to optimize the hydrophilic interactions. Side chains of residue M414 and M425 within the "contact domain" TM6-third extracellular loop (hPTH1-Rc(S"°9-W"37)) are shown (28). (See color plates.)

80

/

CHAPTER4

micelle with its hydrophobic face projecting into the lipid layer (64). We therefore propose that these two helices, the C-terminal helix in PTH and the central helix in the first ECL in PTH1-Rc, interact in an antiparallel fashion allowing exposed charged residues on both helices to form numerous intermolecular interactions. Integrating these findings into the PTH-PTH1-Rc model results in the enhancement and refinement of the overall bimolecular topology by positioning the C-terminal helix of PTH between the first ECL and the C-terminal helix of the N-ECD of hPTH1-Rc. This topological organization is consistent not only with the individual bimolecular contact sites between positions 1, 13, and 27 in PTH and the respective sites in PTH1-Rc (namely, M 425, a site in the proximity of R 186, and L261), but also accommodates the contact site between position 23 in PTHrP and YZ~-L4° in PTH1-Rc (Fig. 12). R61z and co-workers constructed the PTH1-Rc and PTH2-Rc as described previously and used the membrane-bound conformation of hPTH(1-34) (64) and the contact sites identified for positions 1 and 13 in PTH (26,28,221) to dock hPTH(1-34) to the receptors (229). Using these models, they identify interresidue contacts within the seven-transmembrane helical bundle (Fig. 13) and suggest explanations for ligand specificity (46,243), site-directed mutagenesis (23,173,177,179,180), constitutively activated receptors (182,244), cross-linking outcomes (28), substitutions

N-terminus 261

C-terminus FIG. 12 Schematic representation of the binding of PTH to its G protein-coupled receptor, PTH1-Rc. The locations of the contact points in PTH1-Rc identified by photoaffinity crosslinking are indicated (SerlmM 42s, Lys13--R 186,Trp23--T33/Q37, Lys2L--L261). The structural features of the PTH and fragments of PTH1-Rc are indicated (230).

C281-C351

. , a t e , TM2

TM1 TM4

TM7 TM6 FIG. 13 Illustration of some key residue-residue contacts within the seven-transmembrane helix bundle of PTH1-Rc. These contacts provide support that the model contains the correct topology of the seven-TM helices. Reprinted with permission from Ref. 229. Copyright 1999 American Chemical Society.

within the ligand (39,245), and signal transduction. These authors also suggest some mutations, which may reverse the specificity of the PTH1- and PTH2-Rcs for their respective ligands, P T H / P T H r P and PTH (229). An interesting study reported by Shimizu and coworkers has incorporated data generated by the ligandand receptor-centered approaches in a very innovative way to design a constitutive active ligand-tethered hPTH1-Rc (Fig. 14) (246). Four concepts were established: (1) A peptide as small as PTH(1-14) can stimulate weak cAMP formation with both wild-type and N-ECD-truncated rPTH1-Rc, rANt (18). (2) Residues 1-9 in PTH (1-14) are critical for interacting with the rANt (18). (3) Position 13 in PTH photocross-links in the proximity of R ]86 in PTH1-Rc (26,27). (4) The hydrophobic residues F TM and L 187 in PTH1-Rc are functionally important for the interaction with the 3-14 portion of PTH(1-34) (172). In this ligand-tethered hPTH1-Rc, the N-ECD was truncated from E 182, juxtaposed to the TM1 (AN-ECD-hPTH1-Rc), and was extended by a Gly4 spacer (Ga-AN-ECD-hPTH1-Rc) linked to PTH(1-9) (246). Transient expression of this construct in COS-7 cells resulted in 10-fold higher basal cAMP levels compared to the control, wild-type

PTH/PTHrP/REcEPTOR INTERACTIONS /

~NH2

[A1-V-S-E-I-Q-L-M-H9 ]

PTH[ 1-9]

[AI-V-S-E-I-Q-L-M-H-NI° I

PTH[ 1-10]

[AI-V-S-E-I-Q-L-M-H-N-L 11]

PTH[1-11]

[A'-V-S-E-I-Q-L-M-H-N-R 1']

Arg11PTH[1-11]

y

H2N~77-/'~G_G_G_G

HOOC ~

81

H2N

HOOC hPTH1-Rc

HOOC -

Tether-G4-AN-ECD-

AN-ECD-hPTH1-Rc

hPTH1-Rc FIG. 14 Schematics include the wild-type hPTH1-Rc, the AN-ECD-hPTH1-Rc, and the Tether-G4-AN-ECDhPTH1-Rc. Also listed are the different N-terminal sequences derived from PTH, which are tethered to E 182 (solid diamond) via a tetraglycine (G4) spacer. All the receptor constructs retain the 23-amino acid native hPTH1-Rc signal sequence. Therefore, the putative N-terminal residue in all the receptors is y23 generated on signal peptidase cleavage. Modified from Shimizu et aL (246).

hPTH1-Rc in the same expression system. T e t h e r i n g the extended and more potent [Arg11]PTH(1-11) resulted in 50-fold higher basal c-AMP levels than those seen with the wild-type hPTH1-Rc. Interestingly, similar to the PTH(1-14) (18) in which Val 2, Ile 5, and Met 8 were the most critical residues for activation they were also the most critical ones for the constitutive activity of the [Arg11]PTH(1-11)-G4-AN-ECD-hPTH1-Rc (246). The elegance of this study is in devising a unique way to specifically "immobilize" the principal activation domain of the ligand in the proximity to the contact sites critical for receptor activation. The correspondence between the substitutions in the PTH (1-11) that increase the efficacy of the free and the tethered peptide supports the notion that both exercise the same contact points responsible for receptor activation. The high effective molarity of the tethered ligand minimizes the role of binding affinity as it is known for the free ligand, thus allowing the identification of residues within the tethered ligand essential for induction of activity. However, the accessibility to the tethered ligand-receptor system is limited to the recombinant technology and therefore to coded amino acids, and the stringent requirements for efficient expression may turn out to be major obstacles in practicing and extending this approach in the future. It

remains to be shown whether the tethered ligand-receptor system may be a source for identifying structural constraints that can contribute to the refinem e n t of the experimentally based ligand-receptor model and to rational drug design. The elimination of most of the entropic c o m p o n e n t from the ligand-receptor interaction may generate contact interactions and produce activation mechanisms that differ from those involved in the interaction with a diffusable ligand. The quality of any model, namely, its capacity to represent ligand-receptor interactions realistically and predict the nature of the interface, is based primarily on the data and procedures used in construction of the model. A model can become highly speculative and thus only remotely relevant to biology if overloaded with data derived from indirect and circumstantial conclusions. It is important to avoid overinterpretation of model and r e m e m b e r the assumptions and approximations used in its construction. Last, any extrapolation derived from the model must be tested in order to validate its predictive potential. Therefore, the evaluation of any models for complexes of PTH and PTHrP with PTH1-Rc and PTH with PTH2-Rc should follow the above-mentioned principles.

82

/

CHAPTER4

FUTURE DIRECTIONS T h e most powerful insights into the nature of h o r m o n e - r e c e p t o r interactions are emerging from direct PAS studies of h o r m o n e - r e c e p t o r photoconjugates. Future experimentally based models of the bimolecular interface will be m o r e refined and better validated. As additional contact sites are demonstrated, other constraints are generated for the model, which further refines the entire model. W h e n sufficiently advanced, this experimentally based model of the PTH-PTH1-Rc interface will b e c o m e a powerful tool for u n d e r s t a n d i n g structure-based mechanisms responsible for differences in biologic activities of h o r m o n e agonists, signaling-selective agonists, antagonists, partial agonists, a n d inverse agonists. It will also provide the means to u n d e r s t a n d aberrant mechanisms underlying pathologic mutations of PTH1-Rc leading to the clinical disorders of Jansen's metaphyseal chondrodysplasia and Blomstrand's osteochondrodysplasia. Finally, the detailed and validated model of the h o r m o n e - r e c e p t o r complex will serve as a molecular template for design of therapeutically advantageous analogs of PTH and PTHrP. O n e area of research that has grown steadily in interest over the past decade is the potential utility of PTH- or PTHrP-derived agonists for the t r e a t m e n t of osteoporosis. It is very well established that low-dose intermittent administration of several forms of P T H stimulates b o n e formation, leading to an overall anabolic effect on b o n e (247,248). Further observations have b e e n m a d e in vivo in animals and in h u m a n studies (249-255). This beneficial effect on b o n e occurs despite the well-documented action of P T H in stimulating b o n e resorption via increased osteoclast n u m b e r and activity. Nevertheless, instigating osteoblasfic bone formation without concomitant activation of osteoclasts a n d resultant b o n e resorption remains an ultimate goal for t r e a t m e n t of osteoporosis. To this end, future focus on the developm e n t of signaling-selective PTH or PTHrP analogs is one of the m o r e promising directions for analog design. Unfortunately, the chronic n a t u r e of osteoporosis implies that long-term or even life-long t r e a t m e n t is required. This poses serious c o m p l i a n c e issues due to the fact that the administration of a peptide-based d r u g such as P T H is generally limited to p a r e n t e r a l routes. In the short a n d i n t e r m e d i a t e term, we anticipate the d e v e l o p m e n t of i m p r o v e d d r u g delivery systems that will greatly e n h a n c e the t h e r a p e u t i c potential of PTHa n d PTHrP-derived agonists a n d antagonists. However, in the long term, d e v e l o p m e n t of small n o n p e p t i d e PTH-mimetic drugs is of major interest. In the past few years a growing n u m b e r of small n o n p e p t i d e peptide-mimetic agonists for GPCRs have b e e n r e p o r t e d (256-262). This raises our expectations that either

through rational d r u g design (based on the P T H - P T H 1 - R c e x p e r i m e n t a l model), or high-throughp u t screening of collections of synthetic c o m p o u n d s , natural products, a n d culture broths, new lead molecules will be discovered. These "leads" can then be optimized chemically into n o n p e p t i d e PTH-rnimetic anabolic agents. To this end, studying the s t r u c t u r e activity relations a n d the degrees of structural tolerance of P T H ( 1 - 1 4 ) is a p r o m i s i n g initiative (18-20). Given the probability of substantial progress in developing a m o d e l for the h o r m o n e - r e c e p t o r complex, in analog design, in elucidating h o r m o n a l m e c h a n i s m s of action, a n d in peptide delivery systems, P T H or PTHrP agonists are likely to find clinical utility in t r e a t m e n t of disorders of calcium a n d b o n e metabolism.

REFERENCES

1. Chorev M, Rosenblatt M. Structure-function analysis of parathyroid hormone and parathyroid hormone-related protein. In: Bilezikian JP, Levine MA, Marcus R, eds. The parathyroids. New York: Raven, 1994:139-156. 2. Chorev M, Rosenblatt M. Parathyroid hormone: Structurefunction relations and analog design. In: BilezikianJP, Raisz LG, Rodan GA, eds. Principles of bone biology. San Diego, Academic Press, 1996:305-323. 3. PottsJT, Jr, Gardella TJ, Jfippner H, Kronenberg HM. Structure based design of parathyroid hormone analogs. J Endocrinol 1997;S15-$21. 4. Whitfield JF, Morley E Small bone-building fragments of parathyroid hormone: New therapeutic agents for osteoporosis. Trends Pharmacol Sci 1995;16:382-386. 5. Dempster DW, Cosman E Parisien M, Shen V, Lindsay R. Anabolic actions of parathyroid hormone on bone. Endocr Rev 1993;14:690-709. 6. Rosenblatt M. Peptide hormone antagonists that are effective in vivo: Lessons from parathyroid hormone. N Engl J Med 1986;315:1004-1013. 7. Rosenblatt M, Callahan EN, Mahaffey JE, Pont A, Potts JT, Jr. Parathyroid hormone inhibitors: Design, synthesis, and biologic evaluation of hormone analogues. J Biol Chem 1977;252:5847-5851. 8. Rosenblatt M, Chorev M, Nutt RF, Caulfield ME Horiuchi N, Clemens TL, Goldman ME, McKee RL, Caporale LH, FisherJE, LevyJJ, Reagan JE, Gay T, DeHaven E New directions for the design of parathyroid hormone antagonists. In: Massry SG, Fujita T, eds. New actions of parathyroid hormone. New York: Plenum, 1993:61-67. 9. Nussbaum SR, Rosenblatt M, Potts JT, Jr. Parathyroid hormone renal receptor interactions: Demonstration of two receptorbinding domains. J Biol Chem 1980;255:10183-10187. 10. Caulfield ME McKee RL, Goldman ME, Duong LT, Fisher JE, Gay CT, DeHaven PA, LevyJJ, Roubini E, Nutt RF, Chorev M, Rosenblatt M. The bovine renal parathyroid hormone (PTH) receptor has equal affinity for two different amino acid sequences: The receptor binding domains of PTH and PTH-related protein are located within the 14-34 region. Endocrinology 1990;127:83-87. 11. Gardella TJ, Wilson AK, Keutmann HT, Oberstein R, PottsJT,Jr. Kronenberg HM, Nussbaum SR. Analysis of parathyroid hor-

P T H / P T H r P / R E c F ~ V T O R INTERACTIONS

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

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