Somatostatin Receptors

Somatostatin Receptors David E. Elliott* Division of Gastroenterology-Hepatology, Department of Medicine, University of Iowa, 4611 JCP, Iowa City, IA 52242, USA * corresponding author tel: 319-353-8574, fax: 319-353-6399, e-mail: [email protected] DOI: 10.1006/rwcy.2001.23012.

SUMMARY SSTR2 is a cell surface receptor for somatostatin. Somatostatin is a small cyclic peptide made by neuroendocrine cells and activated macrophages. It has immunoregulatory and anti-inflammatory properties. Murine and human inflammatory cells express receptors for somatostatin. Five different somatostatin receptors (SSTR) are known. Murine T cells, B cells, and macrophages express exclusively SSTR2 mRNA in vivo. Blocking SSTR2 prevents the immunoregulatory activity of somatostatin. Patients also express SSTR2 at sites of inflammation. The immunoregulatory and anti-inflammatory activities of somatostatin are mediated by SSTR2.

BACKGROUND Somatostatin was first described as a regulatory growth hormone released by pituitary cells but is now known to regulate many cell types. It helps to regulate the release of many different hormones throughout the body (Patel, 1999). Somatostatin regulates stomach acid secretion and intestinal motility. It also inhibits inflammation and regulates T lymphocyte function (Blum et al., 1992). Somatostatin is made by macrophages at the site of inflammation (Weinstock et al., 1990). Somatostatin production by splenic macrophages is regulated by cytokines and inflammatory mediators (Elliott et al., 1998; Weinstock and Elliott, 1998). Somatostatin affects cell function by binding to specific cell surface receptors. Five different mammalian somatostatin receptors have been identified. Murine and human inflammatory cells express

Cytokine Reference

somatostatin receptor subtype 2 (SSTR2), which is the subject of this review.

Discovery Somatostatin (abbreviated SST, SOM, SMS, SRIF) is a small cyclic immunoregulatory peptide. The first SST receptor was cloned by Yamada et al. (1992). Five different receptors for SST were rapidly identified. They are named SSTR1 through SSTR5. Each SSTR is a product of a distinct gene but they share significant homology (Figure 1). The five receptors fall into two groups as determined by their affinity for synthetic agonists (Table 1). SSTR2, 3, and 5 comprise one group while SSTR1 and 4 comprise a second group. SSTR2 is the only somatostatin receptor expressed at sites of inflammation in mice (Elliott et al., 1994, 1999). Blocking murine SSTR2 function prevents somatostatin-mediated attenuation of in vitro IFN production (Elliott et al., 1999). This suggests that SSTR2 is the receptor responsible for the immunoregulatory activity of somatostatin in mice. SSTR2 is also prominently displayed in human sites of inflammation (Reubi et al., 1994; ten Bokum et al., 1999a, 1999b) and may signal the anti-inflammatory activity of somatostatin in patients.

Alternative names Somatostatin receptor subtype 2, somatostatin receptor 2, SSTR2, SSTR2A, SSTR2B, Smstr2, SRIF-1 receptor (no longer used).

Copyright # 2001 Academic Press

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David E. Elliott Figure 1 Alignment of human SSTRs by the method of Corpet (1988).

Structure The five somatostatin receptors share considerable homology (Figure 1). All except SSTR2 lack known introns. SSTR2 has occult introns as described below. All SSTRs are seven-transmembrane G

protein-coupled receptors. Each contains a highly conserved amino acid sequence YANSCANP(I/V)LY in the seventh transmembrane spanning region that serves to identify this receptor family. A schematic representation of SSTR2 is shown in Figure 2. Somatostatin likely binds in the transmembrane regions that may form a pocket including residues

Somatostatin Receptors

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Table 1 Comparison of the five known somatostatin receptors SSTR1

SSTR2

SSTR3

SSTR4

SSTR5

14q13

17q24

22q13.1

20p11.2

16p13.3

Chromosome Human Murine Rat

11 6

10

Size (amino acids) Human

391

369

418

388

363

Mouse

391

369

428

384

362

Human

M81829

M81830

M96738

L07061

D16827

Mouse

M81831

M81832

M91000

U26176

U82697

Somatostatin 14

<2

<1.5

<1.5

<2

<1

Somatostatin 28

<2

<4

<6

<8

<0.5

Octreotide

> 1000

<2

<35

> 1000

<35

CH275

<5

> 1000

> 1000

<1000

> 1000

Vapreotide

> 1000

<5

<30

<50

<1

NC8-12

> 1000

<0.5

<0.5

> 1000

> 1000

L-779,976

> 1000

<0.5

> 700

300

> 1000

L-803,087

<200

> 1000

> 1000

<1

> 1000

Accession number

Affinity (IC50, nM)

Asp125, Asn276, and Phe294. The actual threedimensional structure of SSTR2 has not been solved. SSTR2 has an occult intron within the coding region of the transcript. In humans, the primary transcript codes for SSTR2A, a 369 amino acid protein. Excision of the occult intron produces a splice variant that codes for SSTR2B, a 356 amino acid protein with a novel intracellular C-terminus. SSTR2A and SSTR2B share the N-terminal 331 amino acids that code for the extracellular Nterminus, the transmembrane-spanning regions, and the intra- and extracellular loops between the transmembrane-spanning regions. This shared region encompasses the ligand-binding complex and G protein-binding areas. Only the 25(B) to 38(A) residues at the C-terminus differ. Like the human protein, mouse SSTR2A is a 369 amino acid protein (Figure 2) with a unique 38 amino acid C-terminus. Murine SSTR2B is shorter than its human counterpart at 346 residues with a unique 15 amino acid Cterminus. Greater than 99% of murine inflammatory cell transcripts are for SSTR2A while 1% are for SSTR2B (Elliott et al., 1999). SSTR2A is by far the dominant SSTR expressed in vivo by murine T cells, B cells, and macrophages.

SSTR2 has three potential transcriptional start sites (Kraus et al., 1998). The entire gene may be up to 50 kb in length. The alternative start sites provide novel untranslated exons at the 5 0 end of the transcript. Splenocytes appear to use the transcriptional start site closest to the coding sequence (Kraus et al., 1998). Which promoter site is preferred by inflammatory cells is not known.

Main activities and pathophysiological roles Somatostatin is a cyclic peptide first discovered as the regulator of pituitary growth hormone release. It is now known to have a wide anatomic and phylogenetic distribution (Patel, 1999). Somatostatin regulates the function of many cell types through the five known somatostatin receptors. Somatostatin is antiinflammatory (see somatostatin) and inhibits murine T cell IFN release (Blum et al., 1992). T cells, B cells, and macrophages express mRNA for SSTR2. SSTR2 is the exclusive SSTR expressed in vivo by murine inflammatory cells as determined by RT-PCR (Elliott

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David E. Elliott Figure 2 Schematic of murine SSTR2A.

et al., 1994, 1999). Somatostatin likely regulates inflammation through SSTR2. Using the murine schistosomiasis model of granulomatous inflammation (schistosomiasis) (Elliott, 1996), our laboratory showed that murine inflammatory cells express receptors that specifically bind somatostatin with high affinity (Kd 4.28  10ÿ9 M) (Blum et al., 1992). Murine schistosome egg granuloma cells express mRNA only for SSTR2 (Elliott et al., 1994, 1999). Blocking SSTR2 with specific rabbit antiserum prevents somatostatinmediated inhibition of T cell IFN production (Elliott et al., 1999). B cells and macrophages also express SSTR2 mRNA (Elliott et al., 1999). Thus, somatostatin may regulate B cells and macrophages in addition to T cells. Somatostatin may influence IFN production by regulating antigen-presenting cells. However, it can directly regulate T cell IFN release. The murine TH1 cell line D1.1 (Boom et al., 1988) expresses SSTR2 mRNA (Elliott et al., 1994). Somatostatin and octreotide prevent IFN production by D1.1 T cells stimulated by crosslinking with anti-CD3 and

anti-CD4 in the absence of antigen-presenting cells (Elliott et al., 1999). Human inflammatory cells also bind somatostatin. Somatostatin binds specifically to the human Isk-B cell line and to the Jurkat, U266, and MT-2 T cell lines (Nakamura et al., 1987; Sreedharan et al., 1989). Scintigraphy with radiolabeled agonists of somatostatin demarcates areas of granulomatous inflammation in patients with diseases such as Wegener's granulomatosis, tuberculosis, sarcoidosis, and aspergillosis (Ozturk et al., 1994; van Hagen et al., 1994a, 1994b, 1994c; Postema et al., 1996). This demonstrates that cells within an inflammatory reaction express receptors for somatostatin (John et al., 1996). Studies on patients showed that SSTR2 is expressed by cells at sites of inflammation in rheumatoid arthritis and sarcoidosis (ten Bokum et al., 1999a, 1999b). Lymphoid germinal centers in human intestine contain cells bearing somatostatin receptors (Reubi et al., 1992) that label with antibody specific for SSTR2 (Reubi et al., 1999). The submucosal veins of patents with Crohn's disease and ulcerative colitis

Somatostatin Receptors exhibited greatly increased specific somatostatin binding in areas of inflamed intestine. Veins in uninvolved areas did not have increased binding (Reubi et al., 1994). The increased binding of somatostatin by veins in inflamed intestine is probably due to SSTR2 (Reubi et al., 1994). Two isoforms of SSTR2 exist (SSTR2A, SSTR2B) due to alternate splicing. Murine granuloma cells, splenocytes, and thymocytes express predominantly SSTR2A which accounts for 99% of the SSTR2 transcripts as measured by quantitative RT-PCR (Elliott et al., 1999). SSTR2 couples to protein phosphotyrosine phosphatases (PTPase) and inhibitory G proteins (Buscail et al., 1994; Dent et al., 1997). It is not known if SSTR2 regulates inflammatory cell function by these pathways. Mouse models demonstrate that somatostatin and SSTR2 regulates immune inflammatory reactions. However the role of SSTR2 in human disease is unknown. There are no known diseases directly attributed to loss or overexpression of SSTR2.

GENE

Accession numbers Human SSTR1: M81829 Human SSTR2: M81830, L13033, L34689, AF184174 Human SSTR3: M96738 Human SSTR4: L07061 Human SSTR5: D16827 Mouse SSTR1: M81831 Mouse SSTR2: M81832, X68951, AF008914 Mouse SSTR3: M91000 Mouse SSTR4: U26176 Mouse SSTR5: U82697 Rat (Rattus norvegicus): M96817 (SRIF-1),

M93273, X98234 Cow (Bos taurus): L06613 Pig (Sus scrofa): D21338 Caenorhabditis elegans: AF016434 (with introns)

Sequence For the human SSTR2 gene on chromosome 17q24 and mRNA sequences, see Figure 3 compiled from accession numbers M81830, L13033, L34689 and AF184174. In mice, the SSTR2 gene encompasses nearly 50 kb because two distant transcriptional start sites exist (Kraus et al., 1998). It is not known if these

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distant start sites are also active in humans. The human sequence encompasses the 5 0 -flank for the transcription start closest to the coding sequence. Features of the human gene are: 5 0 flank (promoter): 1±3722 Transcriptional start site: 3723 CDS (SSTR2A): 3815±4924 Occult intron: 4808±5148 CDS (SSTRB): 3815±4808, joined to, 5149±5226 For the mouse SSTR2 gene sequence from chromosome 11, see Figure 4 compiled from accession numbers: M81832, X68951, and AF008914. The murine SSTR2 gene encompasses nearly 50 kb because two distant transcriptional start sites exist (Kraus et al., 1998). Splenocytes appear to use only the promoter region and transcription start site closest to the protein-coding sequence. This mouse sequence encompasses the 5 0 flank for the transcription start closest to the coding sequence. Features of the mouse gene are: 5 0 flank (promoter): 1±2000 Transcriptional start site: 2001 CDS (SSTR2A): 2091±3200 Occult intron: 3084±3422 CDS (SSTRB): 2091±3083, joined to, 3423±3470

Chromosome location and linkages Human SSTR2 gene is on chromosome 17q24; mouse SSTR2 gene is on chromosome 11.

PROTEIN

Sequence See Figure 5.

Relevant homologies and species differences SSTR2 is highly conserved between species at the protein level (Table 2).

Affinity for ligand(s) See Table 1.

Figure 3 Nucleotide sequences for human SSTR2 gene and mRNA. Lower case is not transcribed. Uppercase is transcribed. Protein-coding sequence shown in bold. Difference in sequence between SSTR2A and SSTR2B shown in italics.

Somatostatin Receptors

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Figure 4 Nucleotide sequences for mouse SSTR2 gene and mRNA. Lower case is not transcribed. Uppercase is transcribed. Protein-coding sequence shown in bold. Difference in sequence between SSTR2A and SSTR2B shown in italics.

Cell types and tissues expressing the receptor SSTR2 is widely expressed. Murine T cells, B cells, and macrophages express SSTR2 mRNA (Elliott et al., 1999). Other cell types also express SSTR2 (Patel, 1999).

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David E. Elliott

Figure 5 Amino acid sequences for SSTR2 protein deduced from mRNA sequences.

Table 2 Percentage identity of SSTR2A between species Human

Mouse

Rat

Human

100%

93%

92%

Mouse

93%

100%

98%

Rat

92%

98%

100%

Cow

94%

94%

94%

Pig

96%

95%

94%

inflammatory cell SSTR2 expression. Other agents that did not appear to alter steady state splenocyte SSTR2 mRNA levels included TNF (30 ng/mL), somatostatin (10ÿ6 M), octreotide (10ÿ6 M), anti-CD3 (145±2C11, 1 (g/mL), IL-4 (200 U/mL), and TGF 1 (250 pg/mL) (Elliott et al., 1999). Murine pituitary tumor cell (AtT-20) SSTR2 mRNA expression is regulated by forskolin (which increases cAMP) (Patel et al., 1993). AtT-20 cells initiate SSTR2 transcription at all three potential alternate sites, suggesting that they make use of all three promoters (Kraus et al., 1998). Splenocytes appear to utilize only the transcription initiation site closest to the protein-coding region. Alternative promoter usage may explain why cAMP or other factors appear to regulate SSTR2 mRNA in some cell types but not others. SSTR2 has several possible phosphorylation sites (Figure 2). The receptor can desensitize and this is likely due to alteration in phosphorylation. Dynamic regulation of SSTR2 may occur by protein phosphorylation rather than alteration in transcription.

Release of soluble receptors No soluble receptor has been identified.

SIGNAL TRANSDUCTION

Regulation of receptor expression SSTR2A mRNA appears to be expressed constitutively by murine T cells, B cells, and macrophages (Elliott et al., 1999). Cytokines and inflammatory mediators that regulate somatostatin expression, such as IFN (200 U/mL), IL-10 (30 ng/mL), LPS (30 g/ ml), PGE2 (110ÿ6 M), or dibutyryl cAMP (110ÿ4 M) do not appear to alter steady state

Associated or intrinsic kinases SSTR2 appears to couple with protein tyrosine phosphatases. Dent et al. (1997) using a CD4/CD8 double positive early murine T lymphoma cell line (S49) showed that engagement of SSTR2 increased cell membrane PTPase activity and inhibited constitutive Raf (MAP kinase pathway) activity. The specific PTPase activated was not identified. SSTR2 also increases PTPase activity in transfected COS-7,

Somatostatin Receptors and 3T3 fibroblast cells (Buscail et al., 1994). The PTPase activity may be due to SHP-1. SHP-1 precipitated with SSTR2 in cotransfected CHO cells (Lopez et al., 1997). Stimulation of PTPase was pertusis toxin sensitive in these models, implying a requirement for inhibitory G protein involvement.

THERAPEUTIC UTILITY

Effect of treatment with soluble receptor domain For SSTR2 agonists, see somatostatin chapter.

References Blum, A. M., Metwali, A., Mathew, R. C., Cook, G., Elliott, D., and Weinstock, J. V. (1992). Granuloma T lymphocytes in murine schistosomiasis mansoni have somatostatin receptors and respond to somatostatin with decreased IFN-gamma secretion. J. Immunol. 149, 3621±3626. Boom, W. H., Liano, D., and Abbas, A. K. (1988). Heterogeneity of helper/inducer T lymphocytes. II. Effects of interleukin 4and interleukin 2-producing T cell clones on resting B lymphocytes. J. Exp. Med. 167, 1350±1363. Buscail, L., Delesque, N., Esteve, J. P., Saint-Laurent, N., Prats, H., Clerc, P., Robberecht, P., Bell, G. I., Liebow, C., and Schally, A. V. (1994). Stimulation of tyrosine phosphatase and inhibition of cell proliferation by somatostatin analogues: mediation by human somatostatin receptor subtypes SSTR1 and SSTR2. Proc. Natl Acad. Sci. USA 91, 2315. Corpet, F. (1988). Multiple sequence alignment with hierarchical clustering. Nucl. Acids Res. 16, 10881±10890. Dent, P., Wang, Y., Gu, Y. Z., Wood, S. L., Reardon, D. B., Mangues, R., Pellicer, A., Schonbrunn, A., and Sturgill, T. W. (1997). S49 cells endogenously express subtype 2 somatostatin receptors which couple to increase protein tyrosine phosphatase activity in membranes and down-regulate Raf-1 activity in situ. Cell. Signal. 9, 539±549. Elliott, D. E. (1996). Methods used to study immunoregulation of schistosome egg granulomas. Methods: A Companion to Methods in Enzymology 9, 255±267. Elliott, D. E., Metwali, A., Blum, A. M., Sandor, M., Lynch, R., and Weinstock, J. V. (1994). T lymphocytes isolated from the hepatic granulomas of schistosome-infected mice express somatostatin receptor subtype II (SSTR2) messenger RNA. J. Immunol. 153, 1180±1186. Elliott, D. E., Blum, A. M., Li, J., Metwali, A., and Weinstock, J. V. (1998). Preprosomatostatin messenger RNA is expressed by inflammatory cells and induced by inflammatory mediators and cytokines. J. Immunol. 160, 3997±4003. Elliott, D. E., Li, J., Blum, A. M., Metwali, A., Patel, Y. C., and Weinstock, J. V. (1999). SSTR2A is the dominant somatostatin receptor subtype expressed by inflammatory cells, is widely expressed and directly regulates T cell IFN-gamma release. Eur. J. Immunol. 29, 2454±2463. John, M., Meyerhof, W., Richter, D., Waser, B., Schaer, J. C., Scherubl, H., Boese-Landgraf, J., Neuhaus, P., Ziske, C.,

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Molling, K., Riecken, E. O., Reubi, J. C., and Wiedenmann, B. (1996). Positive somatostatin receptor scintigraphy correlates with the presence of somatostatin receptor subtype 2. Gut 38, 33±39. Kraus, J., Woltje, M., Schonwetter, N., and Hollt, V. (1998). Alternative promoter usage and tissue specific expression of the mouse somatostatin receptor 2 gene. FEBS Lett. 428, 165±170. Lopez, F., Esteve, J. P., Buscail, L., Delesque, N., Saint-Laurent, N., Theveniau, Nahmias, C., Vaysse, N., and Susini, C. (1997). The tyrosine phosphatase SHP-1 associates with the sst2 somatostatin receptor and is an essential component of sst2-mediated inhibitory growth signaling. J. Biol. Chem. 272, 24448±24454. Nakamura, H., Koike, T., Hiruma, K., Sato, T., Tomioka, H., and Yoshida, S. (1987). Identification of lymphoid cell lines bearing receptors for somatostatin. Immunology 62, 655±658. Ozturk, E., Gunalp, B., Ozguven, M., Ozkan, S., Sipit, T., Narin, Y., and Bayhan, H. (1994). The visualization of granulomatous disease with somatostatin receptor scintigraphy. Clin. Nucl. Med. 19, 129±132. Patel, Y. C. (1999). Somatostatin and its receptor family. Front. Neuroendocrinol. 20, 157±198. Patel, Y. C., Greenwood, M., Kent, G., Panetta, R., and Srikant, C. B. (1993). Multiple gene transcripts of the somatostatin receptor SSTR2: tissue selective distribution and cAMP regulation. Biochem. Biophys. Res. Commun. 192, 288±294. Patel, Y. C., Panetta, R., Escher, E., Greenwood, M., and Srikant, C. B. (1994). Expression of multiple somatostatin receptor genes in AtT-20 cells. Evidence for a novel somatostatin-28 selective receptor subtype. J. Biol. Chem. 269, 1506±1509. Postema, P. T., Kwekkeboom, D. J., van Hagen, P. M., and Krenning, E. P. (1996). Somatostatin-receptor scintigraphy in Graves' orbitopathy. Eur. J. Nucl. Med. 23, 615±617. Reubi, J. C., Horisberger, U., Waser, B., Gebbers, J. O., and Laissue, J. (1992). Preferential location of somatostatin receptors in germinal centers of human gut lymphoid tissue. Gastroenterology 103, 1207. Reubi, J. C., Mazzucchelli, L., and Laissue, J. A. (1994). Intestinal vessels express a high density of somatostatin receptors in human inflammatory bowel disease. Gastroenterology 106, 951±1214. Reubi, J. C., Laissue, J. A., Waser, B., Steffen, D. L., Hipkin, R. W., and Schonbrunn, A. (1999). Immunohistochemical detection of somatostatin sst2a receptors in the lymphatic, smooth muscular, and peripheral nervous systems of the human gastrointestinal tract: facts and artifacts. J. Clin. Endocrinol. Metab. 84, 2942±2950. Sreedharan, S. P., Kodama, K. T., Peterson, K. E., and Goetzl, E. J. (1989). Distinct subsets of somatostatin receptors on cultured human lymphocytes. J. Biol. Chem. 264, 949±952. ten Bokum, A. M., Hofland, L. J., de Jong, G., Bouma, J., Melief, M. J., Kwekkeboom, D. J., Schonbrunn, A., Mooy, C. M., Laman, J. D., Lamberts, S. W., and van Hagen, P. M. (1999a). Immunohistochemical localization of somatostatin receptor sst2A in sarcoid granulomas. Eur. J. Clin. Invest. 29, 630±636. ten Bokum, A. M., Melief, M. J., Schonbrunn, A., van der Ham, F., Lindeman, J., Hofland, L. J., Lamberts, S. W., and van Hagen, P. M. (1999b). Immunohistochemical localization of somatostatin receptor sst2A in human rheumatoid synovium. J. Rheumatol. 26, 532±535. van Hagen, P. M., Krenning, E. P., Kwekkeboom, D. J., Reubi, J. C., Anker, L., Lowenberg, P.J. B., and Lamberts, S. W. (1994a). Somatostatin and the immune and haematopoetic system; a review. Eur. J. Clin. Invest. 24, 91±99.

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van Hagen, P. M., Markusse, H. M., Lamberts, S. W., Kwekkeboom, D. J., Reubi, J. C., and Krenning, E. P. (1994b). Somatostatin receptor imaging. The presence of somatostatin receptors in rheumatoid arthritis. Arthritis Rheum. 37, 1521±1527. van Hagen, P. M., Krenning, E. P., Reubi, J. C., Kwekkeboom, D. J., Bakker, W. H., Mulder, A. H., Laissue, I., Hoogstede, H. C., and Lamberts, S. W. (1994c). Somatostatin analogue scintigraphy in granulomatous diseases. Eur. J. Nucl. Med. 21, 497±502. Weinstock, J. V., and Elliott, D. (1998). The substance P and somatostatin interferon- immunoregulatory circuit. Ann. N.Y. Acad. Sci. 840, 532±539. Weinstock, J. V., Blum, A. M., and Malloy, T. (1990). Macrophages within the granulomas of murine Schistosoma mansoni are a source of a somatostatin 1-14-like molecule. Cell. Immunol. 131, 381±390.

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ACKNOWLEDGEMENTS Grants from the National Institutes of Health (DK02428, DK25295, DK07663, DK38327), the Crohn's and Colitis Foundation of America, Inc. and the Veterans Administration supported this research.