fMLP Receptors

fMLP Receptors FrancËois Boulay*, Marie-JoseÁphe Rabiet and Marianne Tardif Department of Molecular and Structural Biology, DBMS/BBSI UMR 314 CEA-CNRS CEA, 17 Rue des Martyrs, Grenoble, Cedex 9, F 38054, France * corresponding author tel: (33) 04-76-88-31-38, fax: (33) 04-76-88-51-85, e-mail: [email protected] DOI: 10.1006/rwcy.2000.23004.

SUMMARY The N-formylated peptide receptor (FPR) is a seven-transmembrane Gi2 protein-coupled receptor expressed in neutrophils, monocytes, macrophages, eosinophils, dendritic cells, hepatocytes, and astrocytes. Upon agonist binding, it promotes a complex array of signaling cascades ± calcium mobilization, phosphoinositide hydrolysis, and activation of protein kinases ± that ultimately result in chemotaxis, release of proteolytic enzymes, production of superoxide by the NADPH oxidase, and activation of transcription factors. Activation of FPR is thought to play a critical role in host defense against bacterial infection and in inflammation. Following fMLP binding, the FPR is rapidly phosphorylated and desensitized. The human FPR has 69% and 56% amino acid sequence similarities with two other human receptors designated FPRL1 and FPRL2, respectively. FPRL1 binds fMLP with low affinity and lipoxin A4 with high affinity and is consequently referred to as the lipoxin A4 receptor. The human FPR, FPRL1, and FPRL2 genes are clustered on chromosome 19 in the 19q13.3 band.

BACKGROUND

Discovery In the mid-1970s, leukocyte chemotaxis was found to be triggered by the bacterial N-formyl peptide Nformyl-L-methionyl-L-leucyl-L-phenylalanine (CHOMet-Leu-Phe-OH, known as fMLP) (Showell et al.,

1976; Freer et al., 1980). fMLP binds with high affinity to a specific receptor (FPR) that initiates complex signaling cascades. Initial attempts to characterize a formyl peptide receptor biochemically and pharmacologically made use of affinity chromatography, radioligands, and ligands derivatized with photoactivatable and fluorescent groups (Williams et al., 1977; Niedel et al., 1980; Sklar et al., 1984; for a review see Allen et al., 1988). In 1990, the amino acid sequence of the human Nformyl peptide receptor was elucidated by an expression cloning strategy in COS-7 cells (Boulay et al., 1990a,b). More information on the structure and function of leukocyte chemoattractant receptors can be found in the two reviews by Murphy (1994) and Ye and Boulay (1997). Two additional human genes designated FPRL1 and FPRL2 (L for like) have been isolated by lowstringency crosshybridization with the human FPR cDNA probe (Bao et al., 1992; Murphy et al., 1992; Ye et al., 1992). The gene product of FPRL1 is also known as FPR2 and FPRH1. FPRL1 binds fMLP with low affinity (Kd > 400 nM). When expressed in CHO cells, FPRL1 has been found to bind lipoxin A4 with high affinity (Kd=1.7 nM) and consequently it is referred to as the lipoxin A4 receptor. The gene product of FPRL2, also known as FPRH2, is expressed in monocytes. FPRH2 is not activated by fMLP and its ligand is still unknown (Bao et al., 1992; Durstin et al., 1994).

Alternative names The N-formyl peptide receptor, originally known as the fMLP receptor or N-formyl peptide receptor (fMLP-R), is now referred to as FPR.

2198 FrancËois Boulay, Marie-JoseÁphe Rabiet and Marianne Tardif

Structure The protein deduced from the cDNA comprises 350 amino acids. Its hydropathy profile reveals features common to all members of the G protein-coupled receptor superfamily, characterized by seven domains highly enriched in hydrophobic residues. These domains are thought to form a bundle of transmembrane  helices joined by hydrophilic segments on the extracellular and intracellular sides of the receptor. The C-terminal region is enriched in serine and threonine residues that become phosphorylated upon agonist binding (Ali et al., 1993; Tardif et al., 1993).

Main activities and pathophysiological roles FPR is expressed in neutrophils and other phagocytic cells of the mammalian immune system, including macrophages, dendritic cells, hepatocytes, and astrocytes. Binding of fMLP activates a complex array of signaling cascades that ultimately result in chemotaxis, release of proteolytic enzymes from secretory granules, production of superoxide by the NADPH oxidase, and activation of transcription factors. These cellular functions are thought to play a critical role in host defense against bacterial infections and in inflammation.

GENE

Accession numbers Two cDNAs coding for allelic forms of the FPR (R26 and R98) were isolated from a plasmid cDNA library

prepared from differentiated HL-60 cells. Both cDNA clones confer high-affinity binding sites for fMLP to COS-7 cells (Boulay et al., 1990a). Several cDNA clones that differ from each other by point mutations were further isolated from different sources (Table 1).

Sequence See Figure 1.

Chromosome location and linkages The FPR gene is approximately 7.5 kb in length and organized into three exons and two large introns. It is present in the human genome as a single copy located in chromosome 19 in the 19q13.3 band adjacent to the 13.3-13.4 interface. The gene for FPR is clustered with that for FPRL1, FPRL2, and the C5aR in a 200 kb fragment (Bao et al., 1992; Murphy et al., 1992, 1993; Perez et al., 1992; Gerard et al., 1993; Haviland et al., 1993; Alvarez et al., 1994). As shown in Figure 2, a large open reading frame (ORF) of 1050 bp is entirely encoded by exon 3. The 50 untranslated region of cDNAs isolated from neutrophil-like HL-60 cells and monocytes appears to be formed by the 74bp-long exon 1 and 12 bp from exon 30 , probably resulting from an alternative splicing of exon 2. The promoter region is separated from the initiation ATG codon by 5.2 kb. Two Alu repeats are present in the intronic region and a third is found in the 30 untranslated region. The absence of intron in the coding region is very unusual for G protein-coupled receptors. However, it appears to be a general feature of the chemoattractant receptor subclass. The region upstream of exon 1 does not contain the classical CAAT and TATA boxes that are commonly

Table 1 GenBank accession numbers of N-formyl peptide receptor and homologs Species

FPR

FPRL1 cDNA

FPRL2 cDNA

Human

allelic R26 cDNA: M33598

M84562, M88107, X63819, M76672

L14061, M76673

allelic R98 cDNA: M33537 FPR gene: L10820 Gorilla

cDNA: X97736

X97738

X97742

Macaque

cDNA: X97734

X97737

X97740

Orangutan

cDNA: X97735

X97744

X97741

Chimpanzee

cDNA: X97745

X97739

X97743

Mouse

FPR gene: L22181

U78299

Rabbit

cDNA: M94549

fMLP Receptors 2199 Figure 1 FPR gene organization. Exons 1, 2, and 3 are in capital letters in green boxes. A sequence motif identical to the cytokine 2-specific sequence that binds the nuclear factor NF-GM (orange box, bp 287±293) and a consensus sequence that binds the nuclear transcription factor NFB (gray box, bp 390±399) are indicated. The putative nonconsensus TATA sequence 19 bp upstream of the initiation transcription start (bp 456±563) is indicated by a yellow box. The open reading frame (ORF) encoding the FPR protein is indicated by a pink box in exon 3. (Full colour figure may be viewed online.)

1 61 121 181 241 301 361 421

ttatggggtt aacttccccc ctcttccttt cttgaccctt agcactgaac ttctaaacag cttggggcca ttcagttcct

aatcttggtg actcccttac cattgcctcc ggagggagca ctctgcatcc gcccagccac tcaaaaatca ttacccctcc

gtgtgcatgg ctctctctgt ctctgattct ggggcccgga acagagactg tgtcctaatg gaagaagctc tcctgttcct

gtgtggacgc gtttctggtc tctcaccaca cacttggatt aggctgagaa ccattaaagc agacttccta tggtgtatgt

gctgtcctgc tccatccctc gtgcttgctg tcttggccct atacagtcag agacagtata tttcctgcta tttgctgcaa

caactgtctc atgacttctt ctttctttac tgttgttgag gtacatgagt ttggtgtatt cccagctggt tcattagaGC

EXON 1 481 541

CTGAGTCACT CTCCCCAGGA GACCCAGACC TAGAACTACC CAGAGCAAGA CCACAGCTGG TGAACAGTCC AGgtaagaaa ---//--- Alu repeat 1 --//--- tactcctaca

EXON 2 2101 2161

gCCTGTCTCC AGTTGGACTA GCCACAATTC AAGTGCTTGA AAACCACATG TGgtgagtga ---//-- Alu repeat 2 -------- aggaaatgac cacgactgca ctatttcagG

EXON 3 5351 6461 6521 6581 6641 6931

AGCAGACAAG TCCCAGCTCC TTAGGATTAC AATAAACAGA ggtcagggtg agaagatact

ORF AGCTTCGTCT CCACTCATCA TATGAGTTTA ggaaatgata ttatataggg

TGAGGAGGGA CACCTTGAGT TAGGCTGAGC GAAAAAAAAA AAAAGCCTTT ttattgactt cttttttgat -------//------- Alu caggagcggtg

found in the promoters of many proteins. A nonconsensus TATA box (TATGTT) is located 19 bp upstream of exon 1 and an inverted CCAAT box (ATTGG) is present at position ÿ129 from the initiation transcription site. Sequences similar to the consensus sequences for binding of the nuclear factors NF-GM (Shannon et al., 1988) and NFB (Baeuerle and Henkel, 1994) are present in this region and may be involved in the regulation of FPR expression (Figure 2). Although transcription of FPR in HL-60 cells can be activated by dibutyryl cyclic AMP, no cAMP-responsive element is contained in the promoter region (Perez et al., 1992; Haviland et al., 1993; Murphy et al., 1993). A systematic study of the

GCTGGGGGAC ACAGGCATTT GTGTCCCCTG tttggacctc

repeat 3

ACTTTCGAGC CCTGCTTATT ATTTGGGGAG agcctcgggt ----//----

promoter region with reporter genes has not yet been achieved.

PROTEIN

Accession numbers SwissProt: Human FPR: P21462 Gorilla FPR: P79176 Macaque FPR: P79189 Orangutan FPR: P79235

2200 FrancËois Boulay, Marie-JoseÁphe Rabiet and Marianne Tardif Figure 2 Chromosomal localization, structural organization, and polymorphism of the human FPR gene. The third exon contains the coding sequence as a whole. Sequence analysis of cDNAs (pINF10, pINF12) and genomic clones (pINF14, G6) isolated from different sources revealed amino acid differences in positions 11, 101, 123, 192, 256, 293, and/or 346. Residues that differ from the sequence of clone R98 are listed. These modifications do not alter the functional and pharmacological properties of the receptor, suggesting that there is a polymorphism of the FPR gene. Chromosome localization and polymorphism of FPR gene

13.4

13.3

13.2

12 11 11 12

13.1

q

13.1

13.2

13.3

p

Chr 19

Exon 1

Exon 2

Exon 3 ORF

1 kb Clones R98

I

II

III

I

L

11

101

IV R

V A 192

VI P 256

Polymorphism

123 R26 G6 pINF 10 pINF 12 pINF 14

V V

VII N A 346 293 E

N

T

E D A H

K

Chimpanzee FPR: P79241 Rabbit FPR: Q05394 Mouse FPR: P33766

Sequence The amino acid sequence of FPR has been deduced from the cDNA (Figure 3).

Description of protein FPR is 350 amino acids long with a calculated molecular mass of 38,420 Da, but biochemical studies indicate that the receptor migrates as a broad band on SDS-PAGE with an apparent molecular weight of 50±70 kDa typical of a glycoprotein. The hydropathy profile reveals seven stretches of about 20±25 residues highly enriched in hydrophobic amino acids that are most likely folded in  helices.

The interhelical hydrophilic loops are relatively short, as seen in most chemoattractant receptors. By analogy with the visual-light receptor rhodopsin, and based on biophysical and biochemical studies, FPR is thought to span the plasma membrane seven times. The seven transmembrane  helices are joined by intra- and extracellular hydrophilic loops. Three N-glycosylation sites are present in the putative extracellular regions, two of them are located in the N-terminal region. The C-terminal region is cytoplasmic, with multiple serine and threonine residues that are phosphorylated in an agonist-dependent manner (Ali et al., 1993; Tardif et al., 1993). No palmitoylation site is found in the C-terminal domain (Figure 4). A direct determination of the three-dimensional structure has not yet been achieved, but a model for a probable structural organization has been proposed by Baldwin (1993) upon alignment of 204 sequences, including that of FPR. The proposed model fits relatively well with the projection map of bovine rhodopsin which was determined by electron microscopy of two-dimensional crystals. The folding of the hydrophilic loops is presently unknown but of particular importance because it conditions the interactions with the ligand at the extracellular surface, and with signaling and regulatory molecules at the cytoplasmic side. A structural constraint is probably imposed to the first and second extracellular loops by the presence of a disulfide bridge between two cysteine residues, Cys98 and Cys176, which have an invariant position in the G protein-coupled receptor family. The importance of this disulfide bridge to stabilize an active ternary structure of receptors has been established for rhodopsin (Karnik and Khorana, 1990) as well as for muscarinic and 2-adrenergic receptors (Dohlman et al., 1990; Kurtenbach et al., 1990).

Relevant homologies and species differences The human FPR has 34%, 69%, and 56% amino acid sequence similarities to human C5aR, FPRL1 and FPRL2, respectively. The overall degree of sequence similarity to murine and rabbit FPRs is 76% and 78%, respectively. The molecular evolution of FPR has recently been examined in nonhuman primates (chimpanzee, gorilla, orangutan, and macaque). The amino acid sequence similarities with the human counterpart range from 95% to 99%, with the highest similarity observed in chimpanzee and highest divergence observed in macaque (Alvarez et al., 1996). While the highest divergence is observed in extracellular loops, the transmembrane and the

fMLP Receptors 2201 Figure 3 FPR cDNA and deduced amino acid sequence (GenBank L10820). The putative transmembrane domains predicted from the hydropathy plot (Kyte and Doolittle, 1982) are indicated by blue boxes. Three putative N-glycosylation sites are shown by gray boxes. (Full colour figure may be viewed online.)

cytoplasmic domains are highly conserved. The divergence in the extracellular loops suggests that there are few structural constraints imposed by the ligand on these domains. Thus, despite a high degree of divergence of the N-terminal domain and the second extracellular loop of rabbit FPR with their counterpart in human FPR, both receptors bind fMLP with similar affinity (Ye et al., 1993), suggesting that amino acids that have undergone an evolutionary change are not essential for ligand

binding. The transmembrane domains and intracellular loops may have a higher restriction on divergence imposed by interaction with and activation of the heterotrimeric G protein(s).

Affinity for ligand(s) The ligand-binding properties of FPR were originally characterized on plasma membrane preparations from

2202 FrancËois Boulay, Marie-JoseÁphe Rabiet and Marianne Tardif Figure 4 Schematic representation of the structure of human FPR in the plasma membrane. The folding of the polypeptide chain and the position of the transmembrane  helices were predicted by hydropathy plot (Kyte and Doolittle, 1982). Light blue circles indicate residues that result in a significant reduction in fMLP binding when replaced by an alanine residue (Miettinen et al., 1997). Residues with white letters on a dark blue background indicate the region (V83-R-K-A-Met87) identified as the site of crosslinking with CHO-Met-p-benzoyl-Lphenylalanine-Phe-Tyr-N "-(fluorescein)-Lys-OH (Mills et al., 1998). Regions of the model shown in green point to the peptide sequences which may be involved in FPR-Gi2 interactions (Bommakanti et al., 1995). Residues with white letters on a red background correspond to residues that are phosphorylated by GRK2 in vitro (Prossnitz et al., 1995a). (Full colour figure may be viewed online.) Human FPR structural model

T P A

N T E M G G S I 10 T P L S S N

190

S

Y S 20 A G

90

H W P F

G L

F L D

L

Y F T

F

G

A V

V

Y

L

G N

T

L

D

C F

G

A

I

L

Y

W

V N

V F

S

L

T 60

A

M

P

G

I

V

I

V

K

P

G A

V

S

G

K

I

A

A Y

A T

T H

K I H 230 K

Q

Y

K E I 280 G

R

I

I

A

R

V D

V V

A

T S L L A A V A F V Y N F Q S P L S C N M W C P L L F F A Y V A Y A F F S M L G V Q R D F L 240

M

L

G M

270 E

T

S S

I

Q

L

I

L

S 140 V R T C R V H C 130 N V L H P V W T Q

Y T

A

W

I

V

F

V A M L I 200 V R G

I

I

D

T

M

F

L

V

L

L

L 120

A

R

L

L

F

L

I

R

V P

I A I

F

G

F

T

I

I

I

L

S

V 50

G

V

D L

I

T T

T

N

A

G

P V

R F

I

T

S

C A V

G

K

T T

F

V

F

F

P

L

Y

L

V

M

L C K

Y A

170

F T

W F

K R

I

I

T

30 Y

G

M A

Out

F

W T N D P K E R I K

F N

G

Y

In

N-terminal

P R G L I K S S

R E

310

R L

C-terminal K A Q L A V E A S P L T 350

S N T

A T

340

polymorphonuclear neutrophils, monocytes, and macrophages, and cell lines (HL-60 and U937) differentiated into neutrophil-like cells with dibutyryl cAMP. Binding was measured using CHO-Met-Leu[3H]Phe-OH and 125I-labeled CHO-Nle-Leu-PheNle-Tyr-Lys-OH peptides, and N-formyl peptides derivatized with photoactivatable and fluorescent groups. The affinity of FPR for the prototypic tripeptide CHO-Met-Leu-(3H)Phe-OH is relatively high (Kd=1 nM). As for other G protein-coupled receptors, the incubation of membrane preparations from cells expressing native or recombinant FPR with nonhydrolyzable GTP analog, which dissociates the G protein from the receptor, results in a severe decrease in receptor affinity for N-formyl peptides (Kd=10±20 nM) (Koo et al., 1982; Prossnitz et al.,

S

D T

Q 330

T

S D E

T

L

A

R E L H S A P L A

I

320

1995b; Wenzel-Seifert et al., 1998). The ionic environment and pH have also been found to affect receptor affinity. Removal of Na‡ results in a rapid and reversible increase of affinity for fMLP (Zigmond et al., 1985). This phenomenon has recently been explained by the fact that Na‡ stabilizes the FPR in an inactive state with a reduced ability to interact with Gi protein (Wenzel-Seifert et al., 1998). Moreover, the use of fluorescein-derivatized peptides has indicated that the ligand-binding pocket is able to accommodate no more than six residues, and contains at least two microenvironments, one hydrophobic and another charged and supporting protonation. It has been suggested that the role of protonated residues is to stabilize ligand binding at neutral pH (Fay et al., 1993).

fMLP Receptors 2203 To delineate the amino acid residues of FPR that are responsible for high-affinity fMLP binding, receptor chimeras were constructed by sequential replacement of FPR segments with the corresponding regions in FPRL1 or murine FPR which bind the prototypical peptide fMLP with low affinity (Gao and Murphy, 1993; Quehenberger et al., 1993). Based on amino acid differences between the transmembrane domains of FPR and FPRL1, mutants of FPR were generated to pinpoint residues responsible for high-affinity ligand binding (Miettinen et al., 1997). This has led to the identification of 10 amino acid residues, located in  helices 2±7, that may participate in binding of fMLP. An alternative approach used a `gain-of-function' strategy by selectively replacing the nonconserved region of FPRL1 with those of FPR (Quehenberger et al., 1997). However, although a number of mutants or chimeric receptors display reduced or improved ligand-binding capabilities, none of these approaches has enabled the ligand binding site to be localized conclusively. By an elegant strategy that combines the photolabeling properties of CHO-Met-p-benzoyl-L-phenylalanine-Phe-Tyr-N "-(fluorescein)-Lys-OH, the ability of an antifluorescein antibody to immunoprecipitate crosslinked fluoresceinated peptides, and the use of matrix-assisted laser desorption ionization mass spectroscopy, a major photolabeled cyanogen bromide peptide, Val-Arg-Lys-Ala-Hse, corresponding to residues 83±87 of FPR has recently been identified (Mills et al., 1998). In the current three-dimensional model, this peptide lies at the interface between the second transmembrane domain and the first extracellular loop (Figure 4).

Cell types and tissues expressing the receptor The general assumption was that FPR is expressed on cells of myeloid origins, such as neutrophils, monocytes, macrophages, eosinophils, and differentiated myeloid cell lines HL-60, U937, and NB4. However, several recent studies have indicated that FPR has a much broader expression pattern than previously thought. Dendritic cells derived from human blood most likely expressed FPR since fMLP elicits their migration and intracellular calcium mobilization (Sozzani et al., 1995). Expression of FPR has been demonstrated in the human hepatocarcinoma cell line HepG2 by in situ hybridization analysis and radioligand-binding studies (McCoy et al., 1995). This is consistent with a recent study indicating that permeabilized hepatocytes and Kupffer cells stain positively with an antibody directed against the

C-terminal domain of FPR (Becker et al., 1998). The presence of FPR on cells of the central nervous system, such as human astrocytes, microglia, and the immortalized human astrocyte cell line HSC2 has been demonstrated by flow cytometry, indirect immunofluorescence, and RT-PCR analysis (Lacy et al., 1995). An immunocytochemical study on normal human tissues suggests that the expression of FPR can be expanded to other organs and tissues (Becker et al., 1998). Although the results of this study need to be correlated with in situ hybridization analysis, FPR or an antigenically related receptor appears to be present on some epithelial cells with secretory functions, and some endocrine cells such as follicular cells of the thyroid. Smooth muscle cells of the muscularis mucosa and muscularis propria of ileum, and arterioles, as well as endothelial cells and some neurons of the motor, sensory, and cerebellar systems are also positively stained. Radioligand-binding studies indicate that FPR maps to the lamina propria of the gastrointestinal mucosa but not to epithelial cells (Anton et al., 1998). In the retina, the pigmented retinal epithelial cells as well as the retinal rods and the cones are positively stained (Becker et al., 1998). Thus, the broad tissue and cell localization of FPR, summarized in Table 2, suggests that FPR may have unanticipated physiological roles.

Regulation of receptor expression In nondifferentiated myeloid cells such as HL-60, NB4, and U937 cells, FPR mRNA is not detectable. A number of chemical agents including dimethylsulfoxide, dibutyryl cAMP, and all-trans retinoic acid confer to these cells a granulocyte-like phenotype with a concomitant expression of FPR on the cell surface. IFN and TNF have been shown to enhance expression of FPR in HL-60 granulocytes but the underlying mechanism remains unclear (Klein et al., 1992). In neutrophils, FPR is present on the cell surface and in intracellular compartments. The surface recruitment of FPR is triggered by inflammatory stimuli including fMLP, PAF, IL-8, leukotriene B4, GM-CSF, and TNF. Upregulation of FPR appears to result from the mobilization of secretory vesicles (Sengelov et al., 1994) but a pool of receptor is also present in the membrane of gelatinase granules and possibly of specific granules (English and Graves, 1992). In differentiated HL-60 cells, FPR is not upregulated, most likely because these cells are deficient in specific granules. There is presently no evidence as to whether FPR expression is modulated by inflammatory stimuli in cells that are not of myeloid origin.

2204 FrancËois Boulay, Marie-JoseÁphe Rabiet and Marianne Tardif Table 2 Cell type and tissue expression of FPR, FPRL1, and FPRL2 Receptor

Cells and tissues

Detected by

FPR

Neutrophils, macrophages, U937, HL-60, NB4

CHO-Met-Leu-(3H)Phe-OH binding, northern and western blotting

Astrocytes, microglia, HSC2, HepG2

Flow cytometry, indirect immunofluorescence, and RT-PCR

Hepatocytes, Kupffer cells, endothelial cells, epithelial enzyme-secreting cells, smooth muscle cells of muscularis propria and mucosa of ileum, arterioles, retina, some neurons

Immunohistochemistry

FPRL1

Neutrophils, monocytes, HL-60, colonic epithelial cells (T84, HT29, Caco-2, CL.19A) (Gronert et al., 1998), medial tissues of coronary arteries (Keitoku et al., 1997)

Northern blotting or RT-PCR

FPRL2

Monocytes, lung, medial tissue of coronary arteries (Keitoku et al., 1997)

Northern blotting or RT-PCR

SIGNAL TRANSDUCTION

Associated or intrinsic kinases FPR has no associated or intrinsic kinases. Activation of kinase cascades involves heterotrimeric G proteins.

Cytoplasmic signaling cascades The majority of fMLP-mediated leukocyte responses are largely inhibited by pertussis toxin (PTX), a bacterial toxin that ADP-ribosylates and, thereby, inactivates the  subunit of heterotrimeric G proteins of the Gi class but not of the Gq class (for review see Snyderman and Uhing, 1992). A large body of evidence indicates that fMLP-induced responses require the physical association of FPR to Gi2 protein (Bommakanti et al., 1992, 1995; Gierschik et al., 1989). Although agonist-occupied FPR does not couple to the PTX-insensitive Gq/11 subfamily (Amatruda et al., 1993), a small PTX-resistant activity is nevertheless observed in neutrophils, monocytes, and differentiated HL-60 cells. This may result from the coupling of FPR to residual molecules of G16 (Amatruda et al., 1995), a promiscuous G subunit restricted to a subset of myeloid cells in the early steps of differentiation (Amatruda et al., 1991). More details on the coupling of FPR to G proteins can be found in a recent review by Ye and Boulay (1997). The triggering and control of neutrophil functions by fMLP clearly require a coordinated activation of several pathways (Figure 5). In neutrophils, fMLP modulates the activity of adenylylcyclase, causing a

small increase in cellular cAMP by a mechanism that is not well understood (Spisani et al., 1996). The most remarkable effect of fMLP is the induction of a robust activation of tyrosine kinases, phosphatidylinositol-specific phospholipase C (PI-PLC), phospholipase D (PLD), phospholipase A2 (PLA2), phosphatidylinositol 3-kinase (PI3K), and of the mitogen-activated protein kinase (MAPK) cascades. The interaction of FPR with Gi2 leads to the exchange of GDP bound to G for GTP. This causes the dissociation of G from GTP-bound G. Free G interacts with second messenger-generating enzymes such as the PI-PLC 2 isoform (Camps et al., 1992; Katz et al., 1992) and the PI3K isoform (Stoyanov et al., 1995). PI-PLC 2 is a PLC isoform restricted to certain myeloid cells, which breaks phosphatidyl 4,5-bisphosphate (PIP2) into inositol trisphosphate (IP3) acting to mobilize calcium from intracellular stores, and diacylglycerol (DAG). A more sustained production of DAG is generated by phosphohydrolysis of phosphatidic acid resulting from phosphatidylcholine breakdown by phospholipase D (PLD) (Exton, 1994). The interplay of Ca2‡ and DAG leads to the activation of protein kinase C isoforms which are thought to contribute to the triggering of various neutrophil functions. The pathway leading to increased PLD activity is not yet completely characterized. A growing body of evidence indicates that PLD activation is dependent on the small GTPases, RhoA and the ADP-ribosylation factor (ARF). PLD activation requires the PTX-sensitive Gi2 protein but is not obligatory dependent on PI3K activation (Fensome et al., 1998). The mechanism by which ARF and RhoA are activated is still uncertain. In leukocytes,

fMLP Receptors 2205 Figure 5 Model for the distinctive signaling pathways induced by FPR activation in neutrophils. Phospholipases are shown in yellow, second messengers in green, small G proteins in blue. PI3 kinase, protein kinase C, and Src-related kinases are indicated by violet boxes. The MAP kinase cascades are shown in red. Abbreviations: PLA2, phospholipase A2; AA, arachidonic acid; PLC, phospholipase C; PIP2, phosphatidylinositol 4,5 bisphosphate; PIP3, phosphatidylinositol 3,4,5 trisphosphate; IP3, inositol trisphosphate; DAG, diacylglycerol; PLD, phospholipase D; PA, phosphatidic acid; PKC, protein kinase C; MAPK, MAP kinase. For details see text and the review by Bokoch (1995). (Full colour figure may be viewed online.)

FMLP FMLP ?

Rac PAK

Rac

αi 2

Grb2 Sos Shc

γ

γ

β

β

PIP 2

IP 3 Src kinases

?

AG

Pl3Kγ Calcium stores

MEKK/Raf

Rho

PIP β 2

PIP3

?

MEK CA++ p38

SAPK/JNK

Rho/Art

MAPK PA AA

??

PKCs PLA2

DAG

PLD

SECRETORY RESPONSES superoxide proteases cytokines CHEMOTAXIS adhesion, locomotion cytoskeleton reorganisation

RhoA is thought to be involved in FPR-mediated triggering of rapid adhesion through integrins (Laudana et al., 1996). For more information on the role of GTPases of the Rho family (Rho, Rac, and Cdc42) in actin cytoskeletal organization the reader is referred to several recent reviews (Tapon and Hall, 1997; Zohn et al., 1998). Two protein kinase cascades, the extracellular signal-regulated kinases (p42/44 MAPK) and the stress-activated p38 MAP kinases are activated by fMLP in neutrophils (Worthen et al., 1994; Avdi et al., 1996; Krump et al., 1997; Nick et al., 1997; Rane et al., 1997). The activated MAP kinases in turn regulate the activity of downstream targets by phosphorylation. These two signaling pathways are thought to participate at different degree in adherence, superoxide production, and chemotaxis. The pathway leading to p38 activation is not completely deciphered and the role played by p38 in neutrophil responses remains to be clarified. In vivo, p38 activates the MAP kinaseactivated protein kinase 2 which may be involved in the superoxide production (Zu et al., 1996). In neutrophils, p42/44 MAPK activation is concomitant with the activation of the Src-like tyrosine kinase Lyn,

the phosphorylation of the adapter protein Shc, and the activation of the small GTPase p21Ras (Worthen et al., 1994; Ptasznik et al., 1995). The pathway for p42/44 MAPK activation has been recently reconstituted in COS-7 cells by cotransfection of cDNAs encoding PI3K , G , and proteins involved in the Ras pathway (Lopez-Ilasaca et al., 1997). The emerging picture is that free G recruits PI3K to the plasma membrane, thereby enhancing the activity of Src-like tyrosine kinase(s), which in turn phosphorylate(s) the adapter protein Shc. The Shc/Grb2/Sos/ Ras complex is formed, leading to activation of the downstream kinases Raf/MEKK-1 which activate MEK. This latter phosphorylates and, thereby, activates p42/44 MAPK. One important role for activated MAP kinases is to phosphorylate and activate cytoplasmic PLA2, which is translocated to the plasma membrane in a Ca2‡-dependent manner (Lin et al., 1993). The reduction of PLA2 activity by an antisense strategy has recently revealed that PLA2 is essential for activation of the superoxide production by the NADPH-oxidase (Dana et al., 1998). In other FPR-expressing cells, such as hepatocytes, MAP kinases most likely participate in signaling to the

2206 FrancËois Boulay, Marie-JoseÁphe Rabiet and Marianne Tardif nucleus for activation of early genes of the acute phase of inflammation. There are a number of lines of evidence from inhibitor studies that several neutrophil functions are dependent on the activation of PI3K. fMLP-induced superoxide production and chemotaxis are, for instance, completely inhibited by wortmannin, a selective inhibitor of PI3K. Reconstitution experiments in transfected COS-7 cells indicate that FPR induces cytoskeletal reorganization through G heterodimers, PI3K , the small GTPase Rac, and a guanosine exchange factor for Rac (Ma et al., 1998). However, the role of the different enzymes activated upon fMLP binding can vary from one cellular function to another. For instance, knockout experiments indicate that PI-PLC 2 is critical for Ca2‡ mobilization, superoxide production, and upregulation of MAC-1 (CD11b/CD18) but is dispensable for chemotaxis (Jiang et al., 1997). The observation that fMLPmediated chemotaxis of leukocytes from PLC 2deficient mice is enhanced suggests that PLC 2 is part of a negative feedback loop that attenuates chemotaxis (Jiang et al., 1997). A more extensive description of the different signaling cascades activated by chemoattractants can be found in recent reviews (Bokoch, 1995; Downey et al., 1995). The cellular responses elicited by fMLP are rapidly attenuated and cells become refractory to repeated stimulation with the same agonist. This process, termed homologous desensitization, is in part due to the agonist-dependent phosphorylation of FPR on its C-terminal domain (Ali et al., 1993; Tardif et al., 1993; Prossnitz et al., 1995a). Phosphorylation of FPR is an essential step in the functional desensitization of FPR and is required for receptor internalization (Prossnitz, 1997). However, neither the phosphorylation of FPR nor its internalization is required for chemotaxis (Hsu et al., 1997).

DOWNSTREAM GENE ACTIVATION

Transcription factors activated The regulation of gene expression in neutrophils, monocytes, and macrophages during the early inflammatory response is governed by the activities of transcriptional activators. Several studies have examined the effects of chemoattractants on the modulation of transcription factor gene expression in myeloid cells. fMLP was found to induce a transient increase of c-fos mRNA in human monocytes and peripheral granulocytes (Ho et al., 1987; Itami et al.,

1987). The fMLP-mediated signaling cascade leading to c-fos induction has not been studied in detail but, by analogy with other systems, one can anticipate the involvement of the p38 MAPK (Hazzalin et al., 1996). The nuclear factor kappa B (NFB) plays a critical role in immune cell function due to its unique ability to turn on the transcription of genes encoding signaling and host defense proteins as well as many cytokines. In the past few years, it has been reported that fMLP is able to stimulate the activation of NFB in eosinophils (Miyamasu et al., 1995), peripheral blood mononuclear cells (PBMCs) and dimethylsulfoxide-differentiated HL-60 cells but not in neutrophils (Browning et al., 1997). Compared with PBMCs and differentiated HL-60 cells, neutrophils contain a much lower level of NFB subunits, which could explain their lack of responsiveness. Even though NFB can be activated by TNF in FPR-transfected HL-60 cells, these latter are unresponsive to fMLP stimulation, suggesting that the downstream signaling pathway becomes functional only when cells are terminally differentiated (Browning et al., 1997).

Genes induced Several studies have examined the ability of chemoattractants to induce secretion of cytokines and chemokines. fMLP has been found to induce the release of IL-8 from eosinophils (Miyamasu et al., 1995) and neutrophils (Cassatella et al., 1992). This secretion is completely inhibited by actinomycin D in both cell types, indicating that fMLP modulates IL-8 production at the transcriptional level. Both fMLP and C5a were found to induce the release of GM-CSF from eosinophils (Miyamasu et al., 1995). However, although both chemoattractant receptors use the same downstream transduction pathway, fMLP is less active than C5a. In addition, it has been shown that fMLP turns on the expression of proinflammatory cytokines such as IL-1, IL-1 , and IL-6 in human PMBCs (Arbour et al., 1996), and that it stimulates the synthesis of proteins of the hepatic acute phase response, including complement C3 and 1-antichymotrypsin, from HepG2 cells (McCoy et al., 1995).

Promoter regions involved The promoter regions of GM-CSF and cytokine/ chemokine genes (IL-1, IL-6, and IL-8) possess NFB-binding sites (Baeuerle and Henkel, 1994; Baggiolini et al., 1994). The involvement of this promoter region in the transcriptional activation of these genes is supported by the observation that

fMLP Receptors 2207 fMLP-mediated secretion of IL-8 and GM-CSF by eosinophils is inhibited by the specific NFB inhibitor pyrolidine dithiocarbamate (Miyamasu et al., 1995). The role of NFB in the fMLP-mediated secretion of IL-8 by neutrophils is, however, less clear since fMLP does not appear to activate NFB in these cells (Browning et al., 1997). Regulatory regions that bind other transcriptional factors such as AP-1 and NFIL6 are likely to be involved. The involvement of AP1 would be consistent with the observation that fMLP induces a transient increase in c-fos mRNA. In the case of liver cells, the promoter regions involved in the induction of complement C3 and 1antichymotrypsin genes by fMLP have not been determined. These genes may be indirectly activated by secreted cytokines. Their induction could involve IL-1- and IL-6-responsive elements (Wilson et al., 1990).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors The most spectacular effects resulting from the activation of FPR are observed in polymorphonuclear neutrophils. Binding of fMLP to FPR induces the change of morphology, chemotaxis, neutrophil adhesion, microbicidal functions, and cytotoxic effects. Leukocytes release proteolytic enzymes from their granules and produce highly reactive oxygenderived free radicals in response to fMLP binding. These substances are cytotoxic and constitute the first reaction of the host against invading pathogens. There is presently no evidence that a dysfunctioning of FPR could result in an inappropriate release of cytotoxic mediators and, thereby, to inflammatory states.

Phenotypes of receptor knockouts and receptor overexpression mice The consequences of FPR knockout or receptor overexpression in mice have not been examined yet.

Human abnormalities Very few studies have examined the possibility that a defective FPR could result in human abnormalities or

pathogenesis. Perez et al. (1991) have reported the case of a patient with juvenile periodontitis in whom leukocytes show an abnormal responsiveness to fMLP. However, this study has not provided evidence for any mutation in the FPR gene.

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