The N-formyl peptide receptor: A model for the study of chemoattractant receptor structure and...

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Pharmacol. Then. Vol. 74, No. 1, pp. 73-102, 1997 Copyright 0 1997 Elsevier Science Inc.

ELSEVIER

Associute Editor: I’. K. Chiang

The N-Formyl Peptide Receptor: A Model for the Study of Chemoattractant Receptor Structure and Function

Eric R . Prossnitz and Richard D. Ye DEPARTMENT OF IMMUNOLOGY, THE SCRIPPS RESEARCH INSTITUTE, 10550 NORTH TORREY PINES RD., LA JOLLA, CA 92037, USA

ABSTRACT. N-formyl peptides, such as fMet-Leu-Phe, are one of the most potent chemoattractants for phagocytic leukocytes. The interaction of N-formyl peptides with their specific cell surface receptors has been studied extensively and used as a model system for the characterization of G-protein-coupled signal transduction in phagocytes. The cloning of the N-formyl peptide receptor cDNA from several species and the identification of homologous genes have allowed detailed studies of structural and functional aspects of the receptor. Recent findings that the receptor is expressed in nonhematopoietic cells and that nonformylated peptides can activate the receptor suggest potentially novel functions and the existence of additional ligands for this recentor. PHARMACOL. THER. 74(l): 73-102. 1997. 0 1997 Elsevier Science Inc.

KEY WORDS. Chemoattractants, G-protein-coupled receptor, inflammation, N-formyl peptide, N-formyl peptide receptor.

CONTENTS

1. IN~~DUCTION. . . . . . . . . . , . . . .

2. THE LIGANDSFORTHEN-FORMYL FEPTIDERECEPTOR. . . . . . . . . . . . . 2.1. SYNTHETIC ANDBACTERIALLY

DERIVEDN-FORMYLPEPTIDES . . . . 2.2. N-FORMYLPEPTIDESFROM

OTHERSOURCES. . . . . . . . . . . . 2.3. -FHESTRUCTUREOF

N-FORMYLPEPTIDES . . . . . . . . . 2.4. NONFORMYLATEDPEPTIDES . . . . .

3. N-FORMYLFEPTIDE-INDUCED LEUKOCYTEFUNCTIONS. . . . . . . . . . 3.1. ADHESION,EMIGRATION AND

CHEMOTAXIS . . . . . . . . . . . . . 3.2. MICROBICIDALACTIVITIES. . . . . .

3.2.1. RESPIRATORY BURST OXIDASE . . . . . . . . . . . .

3.2.2. DEGRANULATION . . . . . . . 3.2.3. PHAGOCYTOSIS . . . . . . . .

3.3. CYTOIGNEGENEEXPRESSION. . . . . 3.4. SIGNALTRANSDUCTION

MEDIATORS . . . . . . . . . . . . . . 3.4.1. PHOSPHOLIPASES . . . . . . . 3.4.2. PROTEIN PHOSPHORYLATION. 3.4.3. PHOSPHATIDYLINOSITOL

3.KINASE. . . . . . . . . . . . 4. THERECEPTORSFORN-FORMYL

PEPTIDES . . . . . . . . . . . . . . . . . . 4.1. BIOCHEMICAL CHARACTERIZATION

OFTHEN-FORMYLPEPTIDE RECEPTOR. . . . . . . . . . . . . . . 4.1.1. TISSUE CULTURECELL

LINEMODELS. . . . . . . . . . 4.1.2. PROTEASE AND

DEGLYCOSYLATIONSTUDIES . 4.1.3. N-FORMYL PEPTIDE RECEPTOR

SOLUBILIZATION AND BIOPHYSICALPROPERTIES. . .

4.1.4. N-FORMYCPEPTIDERECEPTOR

74 PURIFICATION AND RECONSTITUTION . . . . . . . 82

74 4.1.5. DETERMINATION OF THE N-FORMYLPEPTIDERECEPTOR

74 ASAG-PROTEIN-COUPLED RECEPTOR . . . . . . . . . . . 82

74 4.1.6. N-FORMYL PEPTIDE RECEPTOR INTERACTIONSWITHTHE

75 CYTOSKELETON . . . . . . . . 83 75 4.1.7. FLUORESCENTLIGANDS AS

PROBESFORN-FORMM. 76 PEPTIDERECEPTOR

DYNAMICS . . . . . . . . . . . 84 76 4.2. N-FORMYLPEPTIDERECEPTOR 78 EXPRESSIONPATTERNS . . . . . . . . 85

4.2.1. EXPRESSION OFTHEN-FORMYL 78 PEPTIDERECEPTORIN 78 NONHEMATOPOIETICCELLS. . 85 78 4.2.2. EXPRESSION ANDFUNCTION 79 OFTHEN-FORMYLPEPTIDE

RECEPTORINOTHERSPECIES . 85 79 4.3. CLONINGOFN-FORMYLPEPTIDE 79 RECEPTORCDNAS . . . . . . . . . . 85 80 4.4. STRUCTURE-FUNCTION

RELATIONSHIPSOFTHEN-FORMYL 81 PEPTIDERECEPTOR . . . . . . . . . . 87

4.4.1. LIGANDBINDINGDOMAINS . . 87 81 4.4.2. G-PROTEIN COUPLING

SPECIFICITIES. . . . . . . . . . 89 4.4.3. N-FORMYL PEPTIDE

81 RECEPTOR-G-PROTEIN INTERACTIONSITES. . . . . . 90

81 4.4.4. N-FORMYL PEPTIDE RECEPTOR PHOSPHORYLATIONAND

81 DESENSITIZATION . . . . . . . 92 5. CONCLUSIONS,~%RSPECTIVESAND

FUTUREDIRECTIONS. . . . . . . . . . . . 94 82 REFERENCES . . . . . . . . . . . . . . . . . . 94

74 E. R. Prossnitz and R. D. Ye

ABBREVIATIONS. C5,, activated fifth complement component; CAM, cell adhesion molecule; CAMP, cyclic AMP; CD, cluster designation; DG, diacylglycerol; DMSO, d imethyl sulfoxide; Dpg, dipropylglycine; endo F, endo-P-N-acetylglucosaminidase F; ERK, extracellular signal-regulated kinase; fMLF, N-formyl-methionyl- leucyl-phenylalanine; FPR, N-formyl peptide receptor; GPCR, G-protein-coupled receptor; GRK, G-protein- coupled receptor kinase; ICAM, intercellular cell adhesion molecule; Ig, immunoglobulin; IL, interleukin; IP,, 1,4,5-inositol &phosphate; LTB,, leukotriene 3,; LXA+ lipoxin A,; MAP, mitogen-activated protein; NF-KB, nuclear factor-KB; PAF, platelet-activating factor; PI, phosphatidylinositol; PUK, phosphatidylinositol3-kinase; PIP,, phosphatidyiinositol trisphosphate; PKC, protein kinase C; PL, phospholipase; PMA, phorbol myristate acetate; PPAD, phosphoramidon; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; VCAM, vascular cell adhesion molecule; WGA, wheat germ agglutinin.

1. INTRODUCTION

Phagocytic leukocytes respond to a large number of chemoattractants with directed cell movement, activation of integrins, generation of superoxide anions, and release of granule contents. These functions constitute the first-line host defense against invading microorganisms. However, in several pathological conditions, including ischemia-reperfu- sion injury (e.g., frostbite and stroke), and other inflammatory and autoimmune diseases, the inappropriate activation of phagocytes is often the major cause of tissue damage. Over the last 25 years, a large number of chemoattractants have been identified. These include the “classical” chemoattractants (N-formyl peptides, the activated complement fragment Csa, leukotriene B4 (LTB,), and the phospholipid platelet-activating factor (PAF), among others), and a class of 8-10 kDa chemotactic cytokines that have characteristic conserved cysteine residues. It is now clear that these chemoattractants function by binding to heptahelical G-protein-coupled receptors (GPCRs), with subsequent activation of multiple signal transduction cascades. The scope of chemoattractant receptor research has expanded recently following the discovery that several G-protein-coupled chemokine receptors also serve as co-factors for human im- munodeficiency virus-l entry into host cells.

Among all the chemoattractants so far discovered, the N-formyl peptide is unique in many aspects. It is one of the first identified leukocyte chemoattractants. The prototype N-formyl peptide, CHO-Met-Leu-Phe (fMLF), contains only 3 amino acids and is the smallest chemotactic peptide. Yet, fMLF is also one of the most potent peptide chemoattractants, being able to induce all major phagocyte functions. These properties, together with the relative ease of its synthesis, have allowed the extensive use of fMLF as a model chemoattractant for the study of phagocyte functions. The requirement for G-proteins in phagocyte activation was first described with studies of the N-formyl peptide receptor (FPR). In 1990, the receptor for fMLF became the first chemoattractant receptor to have its primary structure delineated by molecular cloning. Interestingly, the fMLF receptor is so far the only receptor that recognizes an exogenous chemoattractant, the bacterially derived N-formyl peptides.

In this review, we will attempt to give an overview of the structural and functional aspects of the FPR. The reader is also referred to several excellent reviews regarding other aspects of N-formyl peptide ligands and receptors (Allen et al., 1990; Snyderman and Uhing, 1992) and chemoattractant receptors in general (Murphy, 1994).

2. THE LIGANDS FOR THE IV-FORMYL PEPTIDE RECEPTOR 2.1. Synthetic and Bacteriaily Derived N-Fomyi Peptides

It has long been observed that viable bacteria in infected tissues attract polymorphonuclear leukocytes, presumably by releasing factors rhat serve as chemoattractants (Harris, 1954). In 1968, Ward and colleagues confirmed that fil- trates from several gram-positive and gram-negative bacteria contain chemotactic activities. Their study suggested that the generation of bacterial chemotactic activity is related to bacterial growth, and, based on gel filtration data, that the chemotactic factors are of low molecular weight. Furthermore, since all the experiments were performed in the absence of serum, the observed bacterial chemotactic activity was independent of serum factor activation, which had been reported (Ward et al., 1965). In landmark articles published in 1975, Schiffmann and co-workers (1975a,b) discovered that N-formylmethionyl peptides are chemoattractants for neutrophils and macrophages, whereas similar peptides with nonformylated N-termini lack chemotactic activity. This important finding is in complete agreement with the observation that newly synthesized bacterial proteins contain formylated methionine at the N-terminus, and together support the notion that the bacterially generated chemotactic factors could be N-formylated peptides. To correlate the chemotactic activities of the synthetic N-formyl peptides that Schiffmann characterized with those of the bacterially isolated chemotactic factors, Marasco and colleagues (1984) purified and identified N-formyl-Met-Leu- Phe as the major peptide from Escher&a cob cultures that attracts neutrophils. Subsequently, Rot and co-workers (1987) reported the isolation of a chemotactic peptide from culture fluids of StaI&&~occus aureus that consisted of equimolar quantities of methionine, leucine, phenylalanine and isoleucine. Of the synthetic tetrapeptides prepared based on this composition, fMet-Ile-Phe-Leu and fMet- Leu-Phe-Ile displayed the highest chemotactic potency for human monocytes. Taken together, these studies provided unequivocal evidence that bacterially generated N-formyl peptides can serve as potent chemoattractants for human phagocytes.

2.2. N-formrl Peptides from Other Sources

Apart from bacteria, mitochondria in mammalian cells also synthesize proteins with N-formyl methionine. Carp (1982) showed that in at least one case studied, mitochondrial pro-

N-Fonnyl Peptide Receptor Structure and Function 75

teins indeed possess chemotactic activity. It has also been reported that the N-terminus of a mitochondrially encoded NADH dehydrogenase subunit can potently induce elastase release from rabbit neutrophils (Shawar et al., 1995). It is conceivable that mitochondrial IV-formyl peptides may be released from disintegrated cells at sites of inflammation and tissue damage, thereby attracting and activating phagocytes for their scavenging and tissue repair functions.

2.3. The Structure of N-Formy Peptides

Following Schiffmann’s report, a series of studies were con- ducted to delineate the structural requirements for the biological activities of synthetic N-formyl peptides. Showell et al. (1976) reported that the chemotactic capability of synthetic N-formyl peptides corresponded with their ability to stimulate secretion of lysosomal enzymes from neutrophils. In this work, fMet-Leu-Phe was identified as the most potent chemotactic peptide from among 24 synthetic peptides studied (EDso for chemotaxis = 7.0 + 1.7 X 10-l’). Freer and co-workers (1980, 1982) examined a large number of synthetic peptides with and without N-formyl methionine, and compared their biological activities using lysosomal enzyme release and chemotaxis assays. Based on these extensive studies, a model for fMet-Leu-Phe interaction with rabbit neutrophils was proposed. The requirements for a potent IV-formyl peptide are: (1) A formyl group at the N-terminus. This may allow H-bonding to a H-bond acceptor in the binding pocket of the receptor. (2) The methionine side chain, which may occupy a hydrophobic pocket in the receptor. The relatively electron-rich sulfur atom of the methionine may interact with a positively charged area of the receptor. (3) The leucine and phenylalanine side chains, which may interact with hydrophobic areas of the receptor (Freer et al., 1982).

Several investigators have attempted to determine the structure of the prototypic chemotactic peptide Wet-Leu-Phe in relation to its ligand-binding pocket. However, the lack of sufficient quantities of receptor protein has hampered the effort. Taking advantage of the binding of fMLF to an immunoglobulin (Ig) light chain (Bence-Jones) dimer, Edmund- son and Ely (1985) studied the structural properties of the N-formyl peptide using X-ray crystallographic techniques. Data derived from this study suggest that the bound fMLF fills a wedge-shaped cavity, with its N-formyl group forming a hydrogen bond with the phenolic hydroxyl group of a tyrosine residue at the base of the cavity. The side chains of the peptide sweep back towards the entrance of the cavity to conform to the space available for binding. Although the interaction of fMLF with the Bence-Jones protein crystals by no means is identical to its interaction with the phagocyte receptor, there are some similar aspects. Di-, tri- and tetrapeptides with free a-amino groups did not show binding to the dimer, reflecting the requirement for the N-formyl group. The binding interactions in both cases are predominantly hydrophobic in character. However, fMet-Leu-Phe- Lys could not bind to the dimer due to spatial restrictions.

Becker and colleagues (1979) used NMR to determine the conformation of fMLF in dimethyl sulfoxide (DMSO), and found it to adopt a structure similar to that of an anti- parallel P-pleated sheet. Gavuzzo and co-workers (1989) studied the crystal structure and conformation of fMet-Leu- Phe-OMe, a more potent chemoattractant than fMet-Leu- Phe-OH for the rabbit neutrophils. The results suggest that the peptide backbone is folded at the Leu residue without intramolecular hydrogen bonds, and the Leu side chain is oriented on the same side as that of the Phe residue and on the opposite side to that of the Met residue, respectively. This structure differs from that of fMLF determined in solution, but the authors suggest that the folded conformation of fMLF-OMe found in the crystal should be relevant to its biological activity since the receptor is capable of recogniz- ing a folded conformer (Gavuzzo et al., 1989). Dentin0 et al. (1991) used a different approach to study the conformation of an fMLF analogue. They replaced the Leu with dipropylglycine (Dpg), which is stereochemically constrained. The resultant peptide was then studied in crystal form using X-ray diffraction and also in solution by NMR, infrared spectroscopy, and circular dichroism. In the solid state, the Dpg analogue adopts an extended P-sheet-like structure. There is an intramolecular hydrogen bond between the NH and CO groups of the Dpg residues, resulting in a fully extended conformation at position 2. Both NMR and infrared spectroscopy studies indicated the formation of an intramolecular hydrogen bond, and suggest an extended antiparal- lel p conformation, whereas circular dichroism spectra in three different solvents indicated more conformational flexibility of the Dpg analogue compared with an analogue with l-aminocyclohexanecarboxylic acid at position 2. In- terestingly, the Dpg analogue is also biologically more potent than both the I-aminocyclohexanecarboxylic acid analogue, as well as the parent peptide.

2.4. Nonjbrmyk-ued Peptides

Although early studies indicated that the N-formyl group was essential for optimal agonist potency (Freer et al., 1982), more recent investigations suggest that nonformylated peptides may also bind the FPR and activate neutrophil functions. In fact, the synthetic pentapeptide Met-Nle- Leu-Phe-Phe-OH, either N-formylated or N-acetylated, is at least lo-fold more potent than the parent prototype fMLF in evoking transient alteration of the intracellular Ca2+ concentration in human neutrophils. Moreover, the unacylated form, H-MNleLFF, is also a good activator of neutrophil functions, but is lo-fold less active than fMLF (Gao et al., 1994). Employing the novel screening method of a tethered ligand library to identify receptor peptide agonists, Chen et al. (1995) selected a peptide with the sequence Met-Met-Trp-Leu-Leu as being the most active pentapep tide. This nonformylated peptide was comparable to fMLF in activity, but when synthesized in its N-formylated form, was 100-500 times more potent. These results confirm that the


N-formylated form of a peptide is consistently at least lOO- fold more potent than its nonformylated counterpart, but also demonstrate that nonformylated peptides of greater length (pentapeptides) can effectively bind and activate the FPR. In addition to formylated peptides, amino terminal urea-substituted and carbamate-modified peptides have also been shown to be potent agonists for the FPR (Derian et al., 1996; Higgins et al., 1996). Altering the amino acid composition of these substituted peptides can convert an agonist to an antagonist, further supporting the notion that the bioactivity of these chemotactic peptides is not strictly determined by the formyl group at the amino terminus.

These recent findings may have a profound effect on the traditional views of the FPR and its ligand. First, the interaction between the FPR and its ligands may display greater flexibility than previously appreciated. Results from recent studies suggest that the binding pocket may be able to ac- commodate an amino terminal group larger than a formyl group (Derian et al., 1996; Higgins et al., 1996). The roles of the side chains of these ligands may also be more important than was previously thought. Secondly, the finding that an N-terminal formylated group is not an absolute requirement for FPR binding and activation suggests the possible existence of additional types of ligands for the FPR. This notion is substantiated by the observation that the mouse FPR binds fMLF only weakly (Gao and Murphy, 1993). Thus, the FPR may have multiple physiologically relevant ligands similar to the mannose &phosphate receptor, which is also a receptor for the structurally distinct insulin-like growth factor II (Morgan et al., 1987).

3. N-FORMYL PEPTIDE-INDUCED LEUKOCYTE FUNCTIONS 3.1. Adhesion, Emigration and Chemotaxis

Neutrophils, which represent the major and most studied site of action of the FPR, are in constant motion through- out the body, both into and out of the circulation, exercis- ing their main functions of locating and responding to chemoattractants generated by tissue damage and the inva, sion of foreign organisms. The ability of neutrophils to con centrate at sites of inflammation is a combined result of diapedesis, defined as the movement of cells from the blood stream across capillary walls, followed by chemotaxis, defined as the directed movement of a cell up a biochemical gradient over time. These cellular responses are triggered by chemotactic agents, such as fMLF, Csa, PAF, and a multitude of chemokines, and involve numerous adhesion molecules, including those of the Ig family (cell adhesion molecules, CAM’s), integrins and selectins. The series of events that leads to the accumulation of neutrophils at inflammatory sites begins with interactions between the neutrophil and endothelial cells (Rosales and Juliano, 1995).

Unactivated endothehal cells demonstrate little ability to recognize circulating neutrophils. However, when stimulated, endothelial cells rapidly up-regulate surface expression of P-selectin (Bevilacqua, 1993). Binding of neutro-

phils to P-selectin does not require activation of the neutrophil (Geng et al., 1990), since the ligand for P-selectin is a carbohydrate (sialyl Lewis x), normally expressed on the neutrophil (Larsen et al., 1990; Siegelman, 1991; Mulli- gan et al., 1993). This interaction results in a low-affinity, transient interaction between the cells causing neutrophils to roll along the surface of the endothelium. L-selectin, which can be up-regulated on neutrophils in response to fMLF, has also been shown to mediate rolling (Jutila et al., 1990; Berg et al., 1993), as well as integrin activation (Si- mon et al., 1995). The next step leading to neutrophil accumulation at inflammatory sites involves neutrophil activation by chemoattractants generated at the site of inflammation (e.g., fMLF) or by activated endothelial cells (e.g., interleukin [IL]-8).

Neutrophil stimulation results in the activation of integrins, which are heterodimeric CXB cell surface molecules responsible for mediating cell-cell and cell-extracellular matrix interactions (Hynes, 1992). Integrins bind to such extracellular matrix components as laminin, fibronectin and vitronectin, as well as to the Ig family of CAMS, which includes intercellular CAM (ICAM) and vascular CAM (VCAM). Upon binding of a ligand, integrins are capable of transducing a signal to the interior of the cell, in part through interactions with the cytoskeleton (Burridge and Fath, 1989; Larson and Springer, 1990). The ability of the recombinant FPR to mediate fMLF-dependent cell adhesion to activated endothelium via integrins was demonstrated in transfected lymphocytes (Honda et al., 1994), where a role for the low molecular weight G-protein RhoA has been demonstrated (Laudanna et al., 1996). The fMLF- stimulated homotypic aggregation of neutrophils has also been shown to require integrins in addition to L-selectin (Simon et al., 1993). Integrins, themselves, can also be activated through signal transduction processes emanating from the cell interior, which manifest themselves as an increase in ligand affinity with no concomitant rise in integrin number (Faull et al., 1993; Williams et al., 1994). The major integrin species of leukocytes represent B2 chains in combination with one of three different (Y chains (Arnaout, 1990). The significance of these integrins in leukocyte function is indicated by the existence of a disease, leukocyte adhesion deficiency, in which afflicted individuals, with mutations of the BZ integrin subunit, exhibit recurring infections and elevated neutrophil counts (Kishimoto et al.,

1987). With the activation of integrins, and the subsequent in-

creased binding interaction with ICAMs and VCAM, neutrophils shed L-selectin, stop rolling and begin to spread along the endothelium, greatly increasing the surface area of contact (Jutila et al., 1990; Lawrence and Springer, 1991). Prolonged stimulation of endothelial cells results in the synthesis of E-selectin and ICAM- 1, which appear to be required for the transendothelial migration of neutrophils (Luscinskas et al., 1991). Furthermore, binding interactions between platelet endothelial cell adhesion molecule, local- ized in the intercellular junctions between endothelial

N-Formyl Peptide Receptor Structure and Function 77

cells, and neutrophil glycosylated aminoglycans also appear to represent a critical step in diapedesis (Muller et al., 1993).

At this point, when the neutrophil has crossed the endothelial barrier, it begins to migrate chemotactically towards the site of inflammation. Interactions between neutrophil integrins and the extracellular matrix play a vital role in this process. In addition, actin-mediated pseudopod formation, integrin-based de-adhesion mechanisms and cellular contraction combine to result in the directed movement of neutrophils. Upon the introduction of a neutrophil to a biochemical gradient of fMLF, the cell assumes a polar- ized morphology, with a broad pseudopod located at the leading edge of the cell and a small, knob-like protrusion, or uropod, at the rear of the cell. As the cell crawls, the pseudopod adheres to the matrix, while the uropod de- taches, the latter process possibly regulated by the calcium- dependent phosphatase, calcineurin (Hendey et al., 1992). Along with high rates of directed actin polymerization and depolymerization, these processes combine to result in the directed movement of the neutrophil.

Whether actin polymerization directly generates the force required for protrusion of the pseudopod is unclear. Alternative models involving osmotic forces resulting from the formation of the actin gel have been proposed to power pseudopod formation, with subsequent actin polymerization providing a stabilizing framework (Oster, 1988). Either way, actin polymerization and its regulation represent essential facets of neutrophil migration (Downey, 1994). That the pseudopod contains all of the necessary machin- ery for directed movement was demonstrated by the fact that pseudopods that have been detached from the rest of the cell retain the ability to migrate towards chemoattractant (Keller and Bessis, 1975). In the cell, actin exists in two states: unpolymerized, monomeric actin (G-actin) and double-helically filamentous, polymerized actin (F-actin). F-actin filaments display polarity both structurally and functionally (Stossel, 1989). The formation of actin nuclei, groups of 2-3 actin monomers, appears to be the rate-limiting step in overall actin polymerization. In unstimulated neutrophils, approximately two-thirds of the actin is in an unpolymerized state. Upon stimulation by fMLF, the amount of polymerized actin rapidly and transiently doubles (Howard and Meyer, 1984). However, in the migrating neutrophil, there is a highly controlled equilibrium between polymerization at the leading edge and depolymerization at the trailing edge of the cell. One aspect of this control involves the sequestering of G-actin by proteins such as thymosin p4 and profilin (Goldschmidt-Clermont et aI., 1992). Other mechanisms include capping of formed filaments to prevent monomer addition or loss (Weeds and Maciver, 1993), control of the adenine nucleotide bound to the actin (Car- lier et al., 1993), and most likely, the asymmetry of receptor-initiated signaling cascades. Multiple roles for fMLF- generated phosphatidylinositol (PI) phosphates have been suggested due to their ability to interact with actin-sequest- ing proteins (Lassing and Lindberg, 1985; Janmey and

Stossel, 1987; Eberle et al., 1990). A direct role for PI 3-kinase (PUK) in neutrophil motility is unlikely since specific inhibitors of this enzyme do not block actin polymerization or chemotaxis (Thelen et al., 1995; Vlahos et al., 1995).

Receptor recycling is believed to be required for sustained cellular responses such as chemotaxis. Perez and co- workers (1986) originally demonstrated that wheat germ agglutinin (WGA) specifically and irreversibly inhibits fMLF- induced neutrophil chemotaxis. Although WGA did not affect either the binding or internalization of formylated peptides, it did interfere with the re-expression of the FPR. Also unaffected were fMLF-induced superoxide anion generation and degranulation. The results suggested that WGA, perhaps by binding to the fMLP receptor, inhibits fMLF-induced chemotaxis by blocking the re-expression of receptors required for continuous migration. Subsequently, it was demonstrated that removal of cell surface sialic acid by neuraminidase also inhibits fMLF-induced chemotaxis (Perez et al., 1987). Analysis of the fate of the FPR demonstrated that the receptor internalized normally, but accumulated within the cell and was not re-expressed. In another study, Painter and Aiken (1995) utilized an inhibitor of surface membrane neutral endopeptidase, phosphoramidon (PPAD), to examine the relationship between ligand degradation and receptor recycling. After down-regulation of the FPR by fMLF, PPAD blocked the normal re-expression of surface receptor. Internalized fML[jH]F was hydro- lyzed by neutral endopeptidase at a rate proportional to receptor re-expression, suggesting that removal of ligand from the receptor and subsequent hydrolysis are rate-limiting. Since PPAD is known to inhibit neutrophil chemotaxis, but not other rapid neutrophil responses (Painter et al., 1988), removal of ligand from the receptor, along with the receptor’s subsequent recycling, appear to be required for chemotaxis.

A functional association between the FPR and the glyco- syl-PI-linked Fc receptor (Fey RIIIB) has been demonstrated to play a role in chemotaxis (Kew et al., 1992). Pre- treatment of human neutrophils with monoclonal antibodies to Fey RI11 specifically inhibited chemotaxis in response to fMLF. However, there was no effect on the neutrophil response to a wide range of other chemotactic factors, including C&, LTB,, IL-8, and PAF. Binding to the Fc binding site of Fey RIIIB appeared to be essential for altering the fMLF-induced response, since other anti-Fey RI11 antibodies that recognize the ligand binding site reproduced the inhibitory effect. However, a monoclonal antibody that does not recognize the Fc binding site of Fey RIIIB had no effect on fMLF-induced chemotaxis. That Fey RIIIB activation was not required for inhibition of fMLF-induced chemotaxis was suggested by removal of cell surface Fey RIIIB by PI-phospholipase (PL) C, which abolished the neutrophil chemotactic response to fMLF, but did not affect movement toward Csa, IL-8, or LTB,. Taken together, these results suggest that an interaction between the FPR and Fey RIIIB is required for FPR-mediated neutrophil chemotaxis. Additional complexity is suggested by a possible role for di-


rect interactions between the FPR (and possibly other chemoattractant receptors) and cytoskeletal actin, as described in Section 4.1.6 (Jesaitis et al., 1993; Jesaitis and Klotz, 1993).

3.2. Microbic&l Activities

Once the neutrophil has arrived at the site of inflammation, it ceases to move, likely due to the lack of a defined

chemoattractant gradient. At the higher concentrations of chemoattractants found here, the cell begins to carry out its remaining major functions: phagocytosis of foreign particles

(Brown, 1995), release of protease-containing granules (Tapper, 1996) and generation of superoxide anions (Cha-

neck et al., 1994).

3.2.1. Respiratory burst oxidase. Reactive oxygen species are generated by a membrane protein complex, the respiratory burst oxidase, which utilizes NADPH to catalyze the one-electron reduction of molecular oxygen to the su-

peroxide radical Or-. (Babior, 1992). This radical goes on to react with other molecules, generating such compounds

as hydrogen peroxides and hypochlorous acid. The oxidase is completely inactive in the unstimulated neutrophil, but

is rapidly activated upon stimulation with chemoattractants such as fMLF. Perhaps because of the extremely toxic nature of the products of the oxidase, the activity is highly

regulated, requiring the assembly of multiple components for enzymatic activity.

The respiratory burst oxidase consists of four primary components. The identification of these components was

greatly aided through studies of cells from patients with de- fective oxidases, which results in the condition known as chronic granulomatous disease (Smith and Cumutte, 1991). The complex consists of two membrane-bound components

gp91rhox and p22phox, which combine to form a flavohemo-

protein, cytochrome b55s. In unstimulated cells, the cyto-

chrome is found in intracellular vesicles, which fuse with the plasma membrane upon cell stimulation (Sengelov et

al., 1992). Two cytosolic components, p47rhox and p67rhox, become phosphorylated and subsequently translocate to the cell surface upon stimulation to generate an active complex. Two additional proteins of the ras superfamily are also involved in producing an active oxidase. The first, raplA, copurifies with the cytochrome bsss (Babior, 1992). A functional role for raplA has been suggested by the expression of mutant forms of this protein, which result in reduced activity of the oxidase (Maly et al., 1994). The second such protein, rac2, exists in an inactive complex with rhoGDI in

the cytoplasm (Abo et al., 1991; Knaus et al., 1991). Cell- free systems have demonstrated that rat is required for oxi-

dase activity (Abo et al., 1992; Heyworth et al., 1993). Cells treated with anti-sense rac2-based oligonucleotides demonstrated a decreased level of oxidase activity, attesting to its role in viva (Dorseuil et al., 1992). Rac2 recently has also been shown to activate p21-activated kinase, which is capable of phosphorylating portions of p47rhox, providing

additional clues to the activation mechanisms of the neutrophil oxidase (Knaus et al., 1995).

3.2.2. Degranulation. In addition to the generation of superoxide radicals, neutrophils possess a battery of granules capable of inflicting significant damage upon foreign organ-

isms, as well as host tissues. Granule contents are released through regulated exocytosis to two possible locations: intracellular, through fusion with phagosomes, or extracellular, through fusion with the plasma membrane (Tapper,

1996). Neutrophils contain a minimum of four classes of granules that differ in both content and order (or ease) of

exocytosis. Primary or azurophil granules contain lysosomal hydrolases, myeloperoxidase and defensins. Granules lack- ing peroxidases include specific or secondary granules, ge-

latinase granules and secretory vesicles (Borregaard et al., 1987; Kjeldsen et al., 1992). A specific order of release has been observed for neutrophils in which cytoplasmic calcium has been artificially elevated. With increasing ease of exocytosis the order is: azurophil, specific, gelatinase granules and secretory vesicles. The latter required very low in-

creases in cytosolic calcium, or no increase when stimulated with fMLF (Sengelov et al., 1993). Although the mechanisms of degranulation are still largely unclear, roles for low

molecular weight G-proteins and lipid mediators have been

established (Bokoch, 1995).

3.2.3. Phagocytosis. As discussed in the previous sec-

tion, degranulation, and for that matter superoxide production, can occur at two distinct sites within the cell. Since both of these events involve the cell membrane, they can occur either at the cell surface or at the surface of internal

compartments resulting from phagocytosis. In the former case, toxic species are released from the cell, where their action becomes diluted and can result in host tissue damage. In the latter case, release into phagosomes results in the

concentration of these destructive processes away from host tissues (Ohno et al., 1982). Furthermore, because phagocytosis is stimulated by factors distinct from those involved in

superoxide generation and degranulation, an additional level of specificity is introduced into the system (Brown, 1995). Receptors mediating phagocytosis often recognize not the pathogen itself, but host proteins, such as IgG,

which coat the pathogen, a process referred to as opsoniza- tion. Engagement and crosslinking of phagocytic receptors leads to rearrangement of the actin cytoskeleton, which is essential in the process, since cytochalasins (Densen and Mandell, 1978) and botulin toxin C2 (Grimminger et al.,

1991) block phagocytosis. It has been shown that phagocytic receptor activation leads to spatially limited signal transduction and actin rearrangements in the vicinity of the activated receptors (Ohno et al., 1982; Brown, 1995). During the process of phagocytosis, the FPR has been demonstrated to accumulate at the site of target internalization (Kindzelskii et al., 1994). FPR accumulation was dependent on the activation of the receptor, as demonstrated by the lack of accumulation of antagonist-bound receptor. Fur-

N-Forrnyl Peptide Receptor Structure and Function 79

thermore, this accumulation was unique, in that accumulation of the IL-8 receptor, Fc yIIIB receptor, CD1 lb/CD18 or urokinase-type plasminogen activator receptor was not observed. The role of FPR activation and accumulation in phagocytosis remains to be determined.

3.3. Cytokine Qene Expressiun

Monocytes are known to be a major source of pro-inflammatory cytokines; however, neutrophils also are capable of cytokine production that may regulate the local cytokine network and facilitate the recruitment of additional leukocytes to the site of acute inflammation (Lloyd and Oppen- heim, 1992; Cassatella, 1995). Recent studies indicate that neutrophils are capable of RNA synthesis in response to a limited number of pro-inflammatory stimuli, including fMLF (Beaulieu et al., 1992). Cassatella and co-workers (1992) demonstrated that neutrophils responded to fMLF with IL-8 mRNA up-regulation and protein synthesis. That this response is sensitive to pertussis toxin suggests the involvement of the FPR. Browning et al. (1997) recently identified different regulatory mechanisms for fMLF-stimulated cytokine gene expression in neutrophils as compared with monocytes. They found that in monocytes, fMLF stimulates the activation of nuclear factor-KB (NF-KB), a transcription factor that is central to the regulation of the expression of pro-inflammatory immediate-early genes (Baeuerle and Henkel, 1994). In freshly prepared peripheral blood mononuclear cells, fMLF (l-100 nM) induced a KB binding activity that was receptor-dependent and involved the p50- and p65-subunits of the NF-KB/Rel family of proteins. However, the activation of NF-KB by fMLF appeared to be cell specific. Neutrophils that responded to fMLF with cytokine gene expression failed to activate NF-KB, whereas FPR-transfected HL-60 cells were responsive to tumor necrosis factor-a, but not fMLF, for NF-KB activation. Differentiation of FPR-transfected HL-60 cells with DMSO conferred the capability of the cells to activate NF-KB in response to fMLF, indicating the involvement of a factor(s) expressed in differentiated cells. These results provide the first evidence for chemotactic peptide stimulation of a transcription factor, and suggest that this function is highly reg ulated and requires signaling components likely to be specific for chemoattractant-induced NF-KB activation in myeloid cells. Additionally, it was found that neutrophils express very little of the NF-KB/Rel proteins, p50 and ~65, which may explain the lack of an NF-KB response in these cells (Browning et al., 1997). Since fMLF-stimulated IL-8 production in neutrophils is a transcription-dependent event, other transcription factors, such as NF-IL-6 and activating protein-l, are likely involved. The signal transduction pathway leading to fMLF-induced transcription activation remains to be characterized.

3.4. Signal Transduction Molecules 3.4.1. Phospholipases. Stimulation of neutrophils with fMLF leads to the rapid generation of numerous hydropho-

bic and hydrophilic phospholipid-derived cleavage products, which act as second messengers, and are essential to many of the functions described above. Three types of PLs (PLC, PLA2, and PLD) have been characterized as being involved in neutrophil activation.

3.4.1.1. Phospholipase C. The central PLC involved in neutrophil activation is a phosphoinositol-specific enzyme, PLCB,, which generates two second messengers: 1,4,5-inositol trisphosphate (IPJ and diacylglycerol (DG). Activa- tion of this PL, as well as the other two PLs described in Sections 3.4.1.2 and 3.4.1.3, is rapid and dependent upon G-protein activation, as treatment with pertussis toxin blocks activation by chemoattractants such as fMLF (Bokoch and Gilman, 1984; Reinhold et al., 1990; Cockcroft et al., 1991; Cockcroft, 1992). PLC,, is directly activated by the @y-subunits of G-proteins upon their activation by receptors (Camps et al., 1992; Katz et al., 1992). The IP, generated by PLC binds to specific Ca++ channels to release Ca++ from intracellular stores (Berridge, 1993), yielding the characteristic transient rise in cytosolic Ca++ observed upon stimulation with chemoattractants. DG, in conjunction with the released calcium, activates protein kinase C (PKC) (see Section 3.4.2.1).

3.4.1.2. Phospholipase A2. PLA, generates arachidonic acid from the phospholipids of the inner face of the plasma membrane. Neutrophil PLAZ-mediated signal transduction results from the activity of an 85 kDa cytoplasmic form of the enzyme (cPLAJ (Clark et al., 1991; Sharp et al., 1991). Secretory and membrane-bound forms of the enzyme have also been identified (van den Bosch et al., 1990). Arachi- donic acid generated by cPLA1 provides the precursors for inflammatory mediators, such as leukotrienes and prosta- glandins (Kramer, 1993). Activation of cPLAz appears to be independent of PLC activation (Cockcroft et al., 1991), rather requiring phosphorylation by mitogen-activated protein (MAP) kinase (Grinstein et al., 1993; Lin et al., 1993).

3.4.1.3. Phospholipase D. Activation of PLD by fMLF results in the generation of phosphatidic acid and choline from phosphatidylcholine. Phosphatidic acid, itself, may be involved in the activation of the respiratory burst oxidase (Billah, 1993), but is also dephosphorylated to produce DG, which activates PKC. DG production via PLD is slow, but prolonged, compared with that derived from PLC,, (Baggi- olini et al., 1993), and may be responsible for sustained PKC activity during phagocytosis (Fallman et al., 1992). PLD activation requires Ca++ (Kessels et al., 1991), since activation is possible by the Ca++ ionophore A23187 (Re- inhold et al., 1990). In Ca++-depleted cells, fMLF stimulation cannot lead to PLD activation as it does when Ca++ is present (Kessels et al., 1991). The low molecular weight G-protein Arf has been identified as a regulator of PLD in HL-60 cells (Brown et al., 1993; Cockcroft et al., 1994), leading to speculation that membrane traffic regulation by Arf might be a result of PLD activity (Kahn et al., 1993). In addition to Arf, the low molecular weight G-protein Rho


has also been demonstrated to act directly on PLD (Singer et al., 1995).

3.4.2. Protein phosphorylation. Protein phosphorylation plays a key role in intracellular signaling. In neutrophils, fMLF stimulation induces protein phosphorylation of serine, threonine and tyrosine residues, thereby altering the charge or conformation of substrate proteins, as well as the binding properties of substrates. fMLF-stimulated phosphorylation correlates closely with many important cellular functions in phagocytes.

3.4.2.1. Protein kinase C. Stimulation of phagccytes by chemoattractants, including fMLF, leads to G-protein-coupled PLC activation. The resultant generation of IP3 and DG are responsible for elevated intracellular calcium and PKC activation, respectively. PKC represents a large family of enzymes acting on serine and threonine residues (Nishizuka, 1992; Hug and Sarre, 1993). The activities of some PKCs (01, p, and y forms) are sensitive to intracellular free calcium levels, whereas others (6, E, and 5 isoforms) are not dependent on calcium for their activities (Hug and Sarre, 1993). Various forms of PKCs are also regulated by their specific tissue and subcellular distributions (Mochly-Rosen, 1995). Neutrophils contain the CY, p, and 5 isotypes of PKC, but do not have the y isotype of PKC (Pontremoli et al.,

1990; Stasis et al., 1990; Majumdar et al., 1991; Obel et al., 1991; Smallwood and Malawista, 1992). Activators of PKC, such as phorbol esters and cell-permeant DGs, activate the plasma membrane-associated respiratory burst oxidase to generate superoxide anions. A calcium and DG-dependent PKC (PKC-P) phosphorylates a number of cytosolic proteins, including the 47 kDa components of the respiratory burst oxidase system (Kramer et al., 1988). PKC-P is the most abundant PKC isozyme in neutrophils, and responds to fMLF and phorbol ester stimulation with translocation from the cytosol to the membrane (Majumdar et al., 1991). fMLF stimulation also activates calcium-independent, phosphatidyl serine and DG-dependent PKC isotypes; but their function in neutrophil activation is not clear (Stasis et al., 1990; Majumdar et al., 1991). In addition, activation of PKC leads to rapid and transient phosphorylation of a specific target, the myristoylated alanine-rich C kinase substrate (Thelen et al., 1990, 1991). This function may be important for the regulation of the reversible attachment of actin filaments to the plasma membrane (Thelen et al., 1991; Hartwig et al., 1992).

3.4.2.2. Mitogen-a&a&protein kinase. In neutrophils, stimulation with fMLF induces tyrosine phosphorylation of several polypeptides, as detected by in vitro kinase renaturation assays (Grinstein and Furuya, 1992). One of the peptides has been identified to be the 41 kDa extracellular signal- regulated kinase (ERK)- 1, which is tyrosine phosphorylated within seconds after fMLF stimulation. Torres et al. (1993) confirmed this finding and reported the identification of two tyrosine phosphorylated proteins with apparent molecular masses of 40 and 42 kDa, which were recognized by an

antibody against MAP kinase. fMLF-stimulated MAP kinases were able to phosphorylate the substrate’s myelin basic protein and T-669, a peptide derived from the epidermal growth factor receptor. The tyrosine kinase inhibitor genistein attenuated fMLF-stimulated phosphorylation of the 40 and 42 kDa proteins in a dose-dependent manner, which was accompanied by a decreased production of superoxide anion in fMLF-stimulated neutrophils (Torres et al., 1993). These initial studies suggest a link between fMLF induction of MAP kinase activation and the stimulation of the respiratory burst oxidase system. However, it was reported later that MAP kinase activation can be dissoci- ated from the oxidative burst in neutrophils and differentiated HL-60 cells, based on the finding that cyclic AMP (CAMP) treatment reduced the fMLF-stimulated oxidative burst while it had no inhibitory effect on MAP kinase activation, possibly acting at a site distal to MAP kinase (Yu et al., 1995).

Worthen and co-workers (1994) investigated the mechanisms by which the FPR mediates the activation of MAP kinase. They immunoprecipitated Raf-1 activity and detected Ras activation in fMLF-stimulated neutrophils (Worthen et al., 1994). It was further demonstrated that this function of fMLF was sensitive to pertussis toxin treatment of the cells, but was not dependent upon PKC activity. Thus, fMLF stimulation of MAP kinase activity in neutrophils appears to involve G,-mediated receptor regulation of the Ras/Raf/ MAP kinase pathway. MAP kinase activation by the P-y- subunits of G-proteins has been shown by others (Crespo et al., 1994). In addition to fMLF, Csa and IL-8 also stimulate this kinase pathway (Buhl et al., 1995; Knall et al., 1996).

Recently, it has been reported that receptor tyrosine kinases and GPCRs activate MAP kinase through a common signaling pathway (van Biesen et al., 1995). Gi-protein- coupled receptors activate MAP kinase by Gp,-mediated tyrosine phosphorylation of the adaptor protein She, leading to a functional association between She, Grb2 and SOS. The subsequent MAP kinase activation presumably is Ras- dependent. In neutrophils, fMLF stimulates a PKC-independent pathway through a Src-related tyrosine kinase, lyn, which binds and phosphorylates She on tyrosine residues (Ptasznik et al., 1995). Lyn interacts with She via its SH2 domain, which leads to the formation of a complex between lyn, She and PI3K. It was proposed that regulation of She by GPCRs such as FPR may be important for the modulation of the MAP kinase cascade. However, additional pathways may exist for FPR-mediated MAP kinase activation. The mouse L-cell fibroblast cell line lacks the kinase lyn; yet, when stably transfected to express the FPR, the cells responded to fMLF with tyrosine phosphorylation of the ERK-1 and ERK-2 proteins (Torres and Ye, 1996). fMLF-stimulated MAP kinase activation in L-cells occurred in the absence of She phosphorylation and was blocked by pertussis toxin, suggesting G,-mediated signaling without the involvement of lyn and She. It will be interesting to determine whether the alternative pathway for MAP kinase activation found in the L-cells also exists in neutrophils.


3.4.3. Phosphatidylinositol3kinase. Stimulation of neutrophils with fMLF results in an increase in the levels of PI

trisphosphate ( PIP3), a response that can be blocked by treatment of the cells with pertussis toxin prior to stimulation (Traynor-Kaplan et al., 1988, 1989). That the elevated PIP3 level may contribute to the activation of the oxidase system was originally suggested based on the observation that LTB,, which induces minimal oxidant production, stimulates significantly less PIP3 production (Traynor- Kaplan et al., 1989). PIP, production correlates temporally with actin polymerization in fMLF-stimulated neutrophils (Eberle et al., 1990). A number of laboratories subsequently have demonstrated activation of PI3K in fMLF-stimulated neutrophils.

The fungal metabolite wortmannin and the synthetic PI3K inhibitor LY294002 selectively block PI3K activation, and have been used extensively to study the effects of PI3K on cellular functions. Neutrophil PI3K activation can be blocked by these inhibitors, resulting in diminished oxidant production following fMLF stimulation (Okada et al., 1994; Ding et al., 1995; Vlahos et al., 1995). The effects of these inhibitors appear to be quite specific, as fMLF-induced calcium flux, CD1 lb-dependent adhesion and actin polymerization were not affected (Vlahos et al., 1995). This latter finding suggests the selective involvement of PI3K in some, but not all, neutrophil functions.

Unlike the activation of PUK by receptor tyrosine kinases such as platelet-derived growth factor, fMLF-induced PUK activation appeared not to involve tyrosine phosphorylation of the ~85 protein (Vlahos and Matter, 1992). This is consistent with the finding that a newly cloned PUK isotype, ~1107, can be activated by the py-subunits of G-proteins without interaction with the p85-subunits of the PUK (Stephens et al., 1994; Stoyanov et al., 1995). However, immunoprecipitation studies indicate that neutrophils contain the p85/pllO heterodimer PUK that can be activated following fMLF stimulation (Okada et al., 1994; Ding et al., 1995). That this p85/pllO form of PUK is responsible for the majority of fMLF-stimulated PIP, formation via the lyn tyrosine kinase has been shown recently in neutrophils through the use of multiple tyrosine kinase inhibitors (Ptasznik et al., 1996).

4. THE RECEPTORS FOR N-FORMYL PEPTIDES

4.1. Biochemical Characterization of the N-Formyl Peptide Receptor 4.1.1. Tissue culture cell line models. Tissue culture cell lines have proven to be very useful tools over the years for the study of chemoattractant receptors, in particular the FPR. Two of the first and still most widely used lines are the HL-60 promyelocytic and U937 promonocytic blast cell lines, which exist in an arrested state of maturation, yet can be stimulated to differentiate further into granulocytic- and monocytic-like cells (Collins et al., 1978; Harris and Ralph, 1985; Collins, 1987). HL-60 cells originally were characterized in 1977 from a patient with acute promyelocytic leukemia (Collins et al., 1977; Gallagher et al., 1979), whereas U937 cells were first cultured in 1976 from a patient with

diffuse histiocytic lymphoma (Sundstrom and Nilsson, 1976). Numerous additional human myeloid cell lines, arrested at different stages of differentiation, have also been described (Lubbert et al., 1991). Niedel and co-workers (1980b) demonstrated that a subpopulation of HL-60 cells were capable of binding (and internalizing) various formylated peptides with a similar rank order of potencies to that displayed by neutrophils. The subpopulation of cells was distinct from the rest of the culture, displaying morphological characteristics typical of well-differentiated promyelocytes. The fraction of cells capable of binding formyl peptides could be greatly increased through treatment of the culture with polar compounds, which similarly induced morphological cell differentiation. Formyl peptide binding activity could also be demonstrated on U937 cells only when they had been induced to differentiate with dibutyryl CAMP or DMSO (Kay et al., 1983). Photoaffinity labelling of proteins with molecular masses of 54-91 kDa could be demonstrated as for the neutrophil. Furthermore, both cell types, when differentiated, are capable of reproducing many of the functions of the neutrophil and macrophage, including superoxide generation, degranulation and chemotaxis, and thus, have served as excellent model systems for a multitude of studies (Harris and Ralph, 1985). Prossnitz and co-workers have also demonstrated that the recombinant FPR is capable of mediating an actin polymerization response, when expressed in undifferentiated HL-60 cells (Prossnitz et al., 1993b), as well as a chemotactic response, similar to that observed in neutrophils, when expressed in undifferentiated U937 cells (R. R. Kew and E. R. Prossnitz, unpublished observations).

4.1.2. Protease and deglycosylation studies. Initial studies to characterize the receptor responsible for binding N-formy lated peptides were carried out by Niedel and co-workers (1980a). The molecular mass of the N-formyl peptide chemotactic receptor on human neutrophil membranes was determined using covalently affinity-labelled probes. N-formyl- Nle-Leu-Phe-NleW-Tyr-Lys was cross-linked to the receptor with dimethyl suberimidate, N-formyl-Nle-Leu-Phe-Nle- ~*5I-Tyr-Lys-Nepsilon-4-azido-2-nitrophenyl was cross-linked by photoactivation, and N-formyl-Nle-Leu-Phe-Nle-1251-Tyr Lys-Nepsilon-bromoacetyl reacted spontaneously with the receptor. In each case, a broad band with a molecular mass between 55 and 70 kDa was specifically labelled. Pro- teolytic papain treatment of whole cells resulted in a product with a molecular mass of 35 kDa (Dolmatch and Niedel, 1983). However, in membrane preparations, 28 and 23 kDa fragments were generated, suggesting that the receptor spans the membrane with sites accessible on both faces. This conclusion was substantiated by digestion with pronase, which generated no FPR fragments when whole cells were treated, but multiple fragments when membranes were treated. The 35 kDa papain fragment was functionally active, being capable of binding ligand with a & identical to that of the FPR from untreated cells, internalizing ligand and stimulating granule P-glucuronidase release (Dolmatch and Niedel, 1983).


The breadth of the radiolabelled FPR band on sodium

dodecyl sulphate-polyacrylamide gel electrophoresis (SDS- PAGE) suggested the protein might be glycosylated. This was definitively demonstrated by virtue of the receptor’s binding to and specific elution from WGA (Painter et al., 1982; Dolmatch and Niedel, 1983). It was further demonstrated that the FPR from neutrophils, monocytes and differ-

entiated HL-60 cells, when treated with endo-P-N-acetylglucosaminidase F (endo F) produced two fragments with

masses of 40-50 kDa and 33 kDa, both of which retained the ability to bind formylated peptides, suggesting that the

FPR contains two N-linked glycosylation sites (Malech et

al., 1985). The fact that the 33 kDa papain generated FPR fragment could not be further degraded by endo F indicated that the oligosaccharide chains are located at one or the

other terminus of the receptor. In fact, since the endo F and papain generated products were of essentially identical size (33 kDa), the membrane-associated papain cleavage product must have represented virtually the entire peptide back-

bone of the receptor, which provided both ligand binding and signal transducing functions.

4.1.3. N-formyl peptide receptor solubilization and

biophysical properties. The ability to radiolabel the FPR with specific photoaffinity probes allowed the physiochemical

properties of the receptor to be determined even before it could be purified. The FPR was shown to be tightly associated with the membrane, as conditions used to extract pe-

ripheral membrane proteins were ineffective at extracting the FPR (Baldwin et al., 1983). Efficient solubilization of

the FPR required detergents capable of disrupting the membrane bilayer. These included Triton X-100, octylglucoside, CHAPS, deoxycholate and digitonin, with only digitonin

yielding solubilized receptor with significant binding activity. Utilizing Triton X-100 solubilized, photoaffinity-labelled

FPR, Allen and co-workers (1986) demonstrated by gel filtration that the receptor eluted as a single molecular spe-

cies, with a Stokes radius of 40 A. Sedimentation equilibrium analysis in D20-HZ0 mixtures provided a molecular mass for the Triton X-100- and digitonin-solubilized FPR of 63 kDa. This correlated well with the molecular mass determined from SDS-PAGE (SO-70 kDa), indicating that the detergent-extracted FPR was monomeric in nature. Deter-

mination of the partial specific volume yielded values between 0.829 and 0.880 cc/g, indicating either the presence of tightly bound endogenous lipid or that the protein was hydrophobic. A frictional ratio of 1.1-1.3 was determined and was consistent with an elongated globular protein with

an axial ratio of approximately 3:l. With an average radius of 40 A, but an elongated structure, the FPR would be more than able to span a plasma membrane lipid bilayer.

4.1.4. N-formyl peptide receptor purification and reconstitution. The fact that the FPR could be solubilized effectively in a number of detergents made possible at- tempts at purifying the receptor. One of the earliest ap proaches was taken by Goetzl et al. (1981), who solubilized

the FPR with NP-40 and subjected the extract to affinity chromatography on fMLF-Sepharose, followed by gel filtration. Four proteins were thus isolated, with the major band representing a sharp 68 kDa species. This was in contrast to the diffuse 50-70 kDa band reported by others. Further- more, this 68 kDa protein was never specifically demonstrated to bind fML[jH]F. A subsequent report by Huang (1987) described a purification of the digitonin-solubilized

rabbit FPR from peritoneal neutrophils employing fMLF- Sepharose in conjunction with WGA-agarose affinity chromatography. Upon SDS-PAGE analysis of the final mate-

rial, the majority of the protein was present in a diffuse band between 50 and 70 kDa. Alternative purification ap-

proaches, not utilizing ligand affinity steps, were developed by Allen and co-workers (1990). Prepared by utilizing human neutrophil membranes extracted with octyl glucoside, the solubilized proteins were sequentially chromatographed on multiple materials, followed by SDS-PAGE, excision of

the 50-70 kDa region, and finally, deglycosylation of the eluted proteins. This procedure yielded a discrete 33-35

kDa protein upon silver staining that colocalized with the photoaffinity radiolabelled, deglycosylated receptor. De-

spite these numerous approaches by multiple investigators, insufficient material was obtained to generate reliable

amino acid sequence data or specific antibodies. Reconstitution of the FPR binding activity utilizing par-

tially purified receptor preparations was performed initially by Hoyle and Freer (1984). The receptor was solubilized from differentiated HLF60 cells with NP-40 and partially purified with fMLF-Sepharose and gel filtration, as de-

scribed above. When reconstituted into phosphatidylcholine vesicles, the FPR displayed unaltered affinity and ligand selectivity, as compared with the unextracted recep-

tor. Subsequently, Cupo et al. (1989) developed a unilamel- lar phospholipid vesicle system containing the FPR and associated G-proteins. Of the several detergents investigated,

only octyl glucoside and deoxycholate were capable of extracting the FPR in a form that remained functionally active upon reconstitution. The reconstituted receptor bound

fML[3H]F, whereas endogenous Gi,-protein, detected in the vesicle preparation, bound [%]GTPyS. Furthermore, addition of the nonhydrolyzable guanine nucleotide GTPyS shifted the FPR from a high- to a low-affinity binding state for ligand. With the current methods available for the over-

expression of the recombinant FPR in Sf9 insect cells

(Quehenberger et al., 1992) and mammalian cells (Dids- bury et al., 1992; Prossnitz et al., 1993b), reconstitution of

the FPR should provide exciting new insights into FPR function.

4.1.5. Determination of the N-formyl peptide receptor as a G-protein-coupled receptor. The involvement of heterotrimeric G-proteins in FPR-mediated signal transduction was first indicated by the studies of Koo et al. (1983), which demonstrated that nonhydrolyzable guanine nucleotide analogs caused a reduction in the binding affinity of the FPR towards ligand. At about the same time, it was


demonstrated that N-formylated peptides stimulated GTPase activity in neutrophil homogenates (Hyslop et al.,

1984). Further support for the role of G-proteins of the Gi class in FPR function came from studies showing that treatment of neutrophils with pertussis toxin, which ADP-ribo- sylates the a-subunit of members of the G, class of G-proteins, blocked the ability of formyl peptides to activate neutrophil functions, including arachidonic acid release, degranulation and superoxide production (Bokoch and Gil- man, 1984; Lad et al., 1985; Okajima et al., 1985). GTP sensitivity of ligand binding, as well as GTPase activity in membrane preparations, could be restored by the addition of purified Gi protein (Okajima et al., 1985). PLC activity in isolated neutrophil membranes was also shown to require GTP and to be pertussis toxin-sensitive (Smith et al., 1985). In differentiated HL-60 cells, the FPR was capable of stimulating the cholera toxin-catalyzed ADP-ribosylation of both G,z and Gi3 a-subunits, an effect that could be blocked by prior treatment with pertussis toxin (Gierschik et al., 1989). Furthermore, the FPR was shown to act catalytically, as it was demonstrated to be capable of activating up to 20 G-protein molecules/liganded receptor (Gierschik et al., 1991). A subsequent study utilizing digitonin-permeabilized, differentiated HL-60 cells produced a ratio of about three activated G-proteins/fMLF-activated receptor (Wieland et al., 1995). The FPR appears to interact with a G-protein pool that is common to other chemoattractant receptors, including the LTB, and C5a receptors, in HL-60 cells, suggesting that multiple receptors may couple to the same pool of G-proteins (McLeish et al., 198913; Jacobs et al., 1995). The observed correlation between the rate of guanine nucleotide exchange following HL-60 cell stimulation with various chemoattractants (fMLF, C& and LTB,) and the rate of effector activation has led to the hypothesis that differential functional responses are the direct result of receptor-specific differences in G-protein affinity for GTP (Jacobs et al., 1995). Following activation, desensitization of FPR-mediated responses by extended fMLF stimulation has been shown to uncouple cellular responsiveness, at least in part, at the site of the receptor-G-protein interaction (Wilde et al., 1989; McLeish et al., 1989a).

That the FPR interacts directly with G-proteins at the physical level was first demonstrated by Polakis et al. (1988). Cholate extracts of affinity-labelled differentiated HL-60 cells were purified through multiple columns, yielding a final receptor preparation containing a complex with almost equimolar amounts of a pertussis toxin-sensitive G-protein. Peptide mapping studies of this G-protein corresponded with the then recently cloned Gm2 from U937 cells (Dids- bury et al., 1987). Recent studies using G-protein antisense mRNA in differentiated HL-60 cells to reduce G-protein levels have suggested that G, and not Gi is responsible for coupling to the FPR (Goetzl et al., 1994). The significance of these results remains unclear, as the major neutrophil GTP binding protein has been shown to be G,* (Bokoch et al., 1988). Using a different approach, the existence of an FPR-G-protein complex in octyl glucoside extracts of pho-

toaffinity-radiolabelled neutrophil membranes was demonstrated (Jesaitis et al., 1989). Using velocity sucrose gradient sedimentation, the sedimentation rate of the radiolabelled FPR was determined following extraction, and shown to consist of two forms: one with a sedimentation coefficient of 4S and another with a coefficient of 7s. By the addition of GTPyS, the 7S form could be converted to the 4S form. Conversely, by the addition of purified G-protein, the 4S form could be converted into the 7S form, although this could be prevented by prior pertussis toxin treatment of the purified G-protein preparation (Bommakanti et al., 1992). No complex formation was measurable when the bovine retinal G-protein transducin was used at concentrations 30 times the EC,, for Gi. The EC50 for Gi was the same for receptors obtained from formyl peptide-stimulated (desensitized) or unstimulated cells (Bommakanti et al., 1992). This result suggests that perhaps accessory proteins involved in desensitization and retained in membrane preparations (Wilde et al., 1989; McLeish et al., 1989a) might be lost in the extraction process.

4.1.6. IV-formyl peptide receptor interactions with the cytoskeleton. In addition to forming a complex with G-proteins, the FPR has also been shown to interact with the cytoskeleton (Jesaitis et al., 1984). When neutrophils were exposed to N-formyl-Met-Leu-[3H]Phe at 37”C, the FPR formed a high-affinity complex with the ligand, which was insensitive to GTP. Cells incubated at 4°C did not form this complex, and exhibited a dissociation rate 50-100 times greater than the cells incubated at 37°C. Further- more, the high-affinity complexes were stable to detergent solubilization of the cells with Triton X-100, co-isolating with the detergent-insoluble cytoskeleton. This association was transient, as it was effectively “chased” with unlabelled ligand. The possible involvement of the cytoskeleton in FPR-mediated signaling was investigated by treating neutrophils with dihydrocytochalasin, a cytoskeleton disrupting agent (Jesaitis et al., 1986). When dihydrocytochalasin was added either prior to or after fMLF, the rate and dura- tion of superoxide production in neutrophils was aug- mented. In the presence of dihydrocytochalasin, the formation of slowly dissociating surface complexes of occupied receptor and cytoskeleton was inhibited. If cells were first preincubated with fMLF at 15”C, where the majority of the surface receptors became irreversibly occupied and contin- ued to co-isolate with the cytoskeleton, subsequent expo- sure of the cells to fMLF at 37°C resulted in no superoxide production. This procedure, which effectively desensitized the cells, was blocked by dihydrocytochalasin, which similarly inhibited co-isolation of the FPR with the cytoskeleton. These results led to the hypothesis that conversion of the FPR to a slowly dissociating, cytoskeleton-associated form may provide a role for receptor-cytoskeleton interactions in the termination of neutrophil activation.

Cell fractionation studies carried out on neutrophils exposed to photoaffinity ligand at 4”C, which does not result in cellular activation, and at 15”C, which results in FPR de-


sensitization, revealed that FPR complexes could be isolated from two distinct plasma membrane domains (Jesaitis et al., 1988). When the cells had not been activated (at 4”C), the FPR was found in a light plasma membrane domain enriched in heterotrimeric G-proteins, existing in the FPR-G-protein 7s complex described in Section 4.1.5. However, when the cells were effectively desensitized (at 15”C), the FPR was found in the 4s form in a heavy plasma membrane domain depleted in G-proteins, but enriched in the cytoskeletal proteins actin and fodrin. More recent studies have indicated that the FPR may directly bind to actin. Using a nitrocellulose overlay technique of SDS- PAGE-separated cytosol and plasma membrane fractions of neutrophils, Jesaitis et $. (1993) demonstrated that the solubilized, photoaffinity-labelled FPR specifically bound to a 41-43 kDa protein band. This binding could be blocked by incubating the FPR with free neutrophil cytosolic actin, which has a molecular mass of approximately 42 kDa. Fur- ther studies demonstrated that addition of free actin to solubilized FPR could produce an increase in the sedimentation rate in sucrose density gradients, similar to that produced by the addition of G-proteins. A functional effect of actin was demonstrated in that the addition of actin to solubilized FPR increased the amount of photoaffinity label bound by the FPR. Unfortunately, it was not determined whether this was due to an increase in affinity for the ligand or to an increase in the number of receptors capable of binding the ligand. Providing further evidence of an FPR- actin interaction in ho, Klotz et al. (1994) demonstrated a parallel release of the FPR and actin from membranes by cytoskeleton-disrupting agents and a role for ATP in the association of the FPR with the cytoskeleton, as indicated by energy-depletion studies, suggesting that either receptor phosphorylation or another energy-consuming process was required (Klotz and Jesaitis, 1994). Sarndahl and co-workers (1996) recently have shown that the association of the FPR with the cytoskeleton can be induced by neutrophil stimulation with Csa or by direct activation of G-proteins. The mechanisms involved in this association remain to be

determined.

4.1.7. Fluorescent ligands as probes for Ndformyl peptide receptor dynamics. As is apparent from the previous dis- cussions, the ability to easily modify the FPR ligand by iodi- nation and crosslinking reagents has been of great benefit in studies of the receptor and neutrophil activation. An alternative approach used to visualize the FPR and determine the fate of the receptor utilized fluorescently labelled ligands. Niedel et al. (1979) originally utilized N-formyl- Nle-Leu-Phe-Nle-Tyr-Lys derivatized with rhodamine on the E amino group of the terminal lysine to demonstrate that the ligand and, therefore, the receptor became clustered and was internalized. Sklar and co-workers (Sklar, 1987; Posner et al., 1994) subsequently have developed ele- gant spectrofluorometric and flow cytometric techniques that have greatly increased our knowledge of the dynamics of the FPR. These approaches have provided mechanistic

details based on the kinetics and spectroscopic properties of ligandsreceptor interactions. Early studies with cell suspen- sions in cuvettes utilized N-formyl-hexapeptides derivatized with fluorescein in conjunction with a high-affinity antibody directed against the fluorescein moiety (Sklar et al., 1981). This antibody possessed two important properties: first, it was capable of rapidly binding to free fluoresceinated ligand in solution, with the ligand-antibody complex binding poorly to the receptor; second, the antibody significantly quenched the fluorescence of the ligand upon binding, leaving only cell-associated ligand capable of fluorescence. Following the establishment of these techniques, flow cytometric techniques were evaluated for their suit- ability in discriminating between free and bound ligand (Sklar and Finney, 1982). It was demonstrated that with ligand concentrations of about 1 nM, flow cytometry in- trinsically discriminated between cell-accumulated ligand and the dilute ligand in the surrounding solution (Sklar et al., 1984). Thus, it was not even necessary to wash the cells free of unbound ligand prior to analysis.

Early studies utilizing these techniques examined the kinetics of FPR internalization, taking advantage of the fact that acidification of the medium quenched fluorescein, emission intensity, whereas internalized ligand remained unaffected for a number of minutes. Thus, in neutrophils, it was shown that the FPR began to be internalized about 30 set after ligand addition, with a half-time of 3-5 min (Sklar et al., 1982; Finney and Sklar, 1983). Using the antifluorescein antibody described above, investigations into the dissociation rate of ligands from whole cells revealed the presence of two classes of binding sites (Sklar et al., 1985a). Studies of receptor occupancy-response relationships demonstrated that whereas almost 100% of the receptors needed to be occupied to achieve maximal superoxide production, less than 1% receptor occupancy was required to generate maximal actin polymerization (Sklar et al., 1985a,b). The next advance in these studies was the ability to permeabilize cells with digitonin, which, again in conjunction with the antifluorescein antibody, allowed detailed studies of the ternary ligand-receptor-G-protein (LRG) complex to be made (Sklar et al., 1987). Simple permeabiliza- tion led to the production of a slowly dissociating complex (referred to as LRG), whereas the addition of guanine nucleotides, or pretreatment with pertussis toxin, led to the production of a rapidly dissociating complex (referred to as LR), with a half-time of dissociation differing by two orders of magnitude (Sklar et al., 1987). Association rate constants were indistinguishable for LR and LRG (Fay et al., 1991). Such studies also revealed that once the ternary LRG complex forms, activation of the G-protein occurs rapidly within approximately 0.1 sec. Analyses of the ex- perimentally derived rate constants with respect to the ternary complex model were only consistent with a model in which approximately 50% of the receptor existed in a preformed or very rapidly forming complex with G-protein, with the remaining receptor associating with free G-protein over a period of minutes (Fay et al., 1991).


In addition to the two receptor complexes described (LR and LRG), an additional form was identified on intact cells. The properties of this state were that it formed slowly over time, demonstrated slow ligand dissociation, like LRG, but was insensitive to the addition of GTP nucleotides, unlike LRG. It was believed that this form of the FPR, termed LRX, may represent a desensitized state of the receptor. Since the LR and LRG forms of the receptor are transient, an approach using energy depletion was employed, allowing the various receptor states in whole cells to be detected (Sklar et al., 1989). Under normal circumstances, the major form of the FPR detected after cell activation, but prior to internalization, is LRX. Under conditions of energy (ATP and GTP) depletion, the LRG form of the receptor was observed. This indicated that the formation of the desensitized LRX form required energy, possibly through phosphorylation of the receptor. The simultaneous depletion of GTP was also required to observe LRG, since this state is normally destabilized by the presence of GTP in the cell. Confirmation that the slowly dissociating form of the FPR observed under conditions of energy depletion was in fact LRG was provided by inhibiting its formation by pretreatment with pertussis toxin. This is in contrast to the slowly dissociating LRX form, the formation of which is unaffected by pertussis toxin. Kinetic studies revealed that receptor desensitization occurs with a half-time of about 10 set, whereas a receptor uncoupled from G-proteins may require minutes to encounter a G-protein and be activated. It is, therefore, more likely that once uncoupled, the receptor is more likely to become desensitized than to activate G-protein. Thus, it is the 50% of receptors that are apparently pre- coupled (or are maintained in a rapidly coupling state) that are responsible for initiating the signal transduction cascade.

Fluorescent techniques have also been used to study the properties of the ligand binding pocket within the FPR. Sklar and co-workers (1990) have used a family of fluorescent peptides containing four, five or six amino acids, with fluorescein conjugated to the C-terminal lysine, to determine the topography of the binding pocket. Peptides containing either four or five amino acids displayed quenching of the terminal fluorescein upon binding to the receptor. However, the ligand containing six amino acids displayed no receptor-dependent quenching. The fluorescein of this ligand was accessible to high concentrations of the antifluorescein antibody while still bound to the receptor. These results suggested that the C-termini of the tetra- and pentapeptides were buried within the receptor, while the C-terminus of the hexapeptide was exposed at the surface of the receptor. Spectroscopic analyses of the quenching of fluoresceinated peptides indicated that protonation of the pentapeptide’s fluorophore by the receptor was likely occurring upon binding, whereas quenching of the tetrapeptide was most likely due to interactions with hydrophobic regions of the receptor (Fay et al., 1993). Utilizing the method of fluorescence recovery after photobleaching, which examines protein mobility within the membrane, Johansson and co- workers (1993) used fluorescent agonist and antagonist to

demonstrate that only the agonist-occupied FPR undergoes aggregation and immobilization in the plane of the mem-

brane prior to internalization.

4.2. N-fir-my1 Peptide Receptor Expression Patterns 4.2.1. Expression of the N-formyl peptide receptor in nonhematopoietic cells. The cellular distribution of FPR is not restricted to neutrophils and monocytes, as previously thought. A partial cDNA sequence for the FPR has been isolated from human brain (Adams et al., 1992). Expression of FPR recently was demonstrated in hepatocytes, dendritic cells, and astrocytes (Lacy et al., 1995; Sozzani et al., 1995). Since chemotaxis and degranulation are not the primary functions of hepatocytes and astrocytes, the role of the FPR in these cells remains to be defined. Study of the FPR and Csa receptor in HepG2 cells suggests that it may be able to mediate the expression of acute phase genes (Haviland et al., 1995). Indeed, fMLF recently has been shown to be able to activate NF-KB (Browning et al., 1997), a transcription factor that modulates the expression of pro-inflammatory cytokines and growth factor genes. FPR-stimulated migration of dendritic cells may be important for the modulation of T-cell activation.

4.2.2. Expression and function of the N-formyl peptide receptor in other species. The FPR has been found in the phagocytes of other species. Early studies of the N-formyl peptides employed rabbit neutrophils, as well as human neutrophils (Kermode and Becker, 1986). The rabbit FPR has very similar binding and functional properties compared with the human FPR. In a few instances, a given peptide may be slightly more potent for the rabbit FPR than the human receptor (Sukumar et al., 1985). Recently, cloning of the rabbit FPR indicated that it is 78% identical to the human FPR (Ye et al., 1993). Interestingly, the mouse FPR, which is 76% identical to the human FPR, binds fMLF with -2OO-fold lower affinity and is equally poor at calcium mobilization when stimulated with fMLF in Xeno- pus oocytes (Gao and Murphy, 1993). Surprisingly, not all the lower species share an unresponsive FPR. The receptors from rat, as well as guinea pig, apparently respond well to fMLF (Ohta et al., 1985; Remes et al., 1991). On the other hand, the mouse is not the only species whose neutrophils do not express a high-affinity FPR. Porcine neutrophils have been shown to be unresponsive to fMLF stimulation (El-Awar et al., 1990). A more interesting finding is that equine neutrophils responded to fMLF with potent degranulation, but the same ligand failed to induce chemotaxis (Sny- derman and Pike, 1980). The mechanisms underlying these species differences are not clear at present, but support the idea that other ligands for the FPR may exist in other species. Delineation of FPR sequences from various species may provide a structural basis for these functional differences.

4.3. Cloning of N-fownyl Peptide Receptor cDNAs

A number of laboratories attempted to clone the FPR cDNA using approaches based on receptor protein purifica-

86

tion (Allen et al., 1990), functional expression in Xenopus oocytes (Murphy et al., 1990), and functional expression with fluorescence-activated cell sorting (R. D. Ye et al., unpublished data). In 1990, Boulay and co-workers (1990a,b) first reported the isolation of a cDNA that encodes a high- affinity FPR. They used a COS-7 cell-based transient expression system and a highly specific, iodinated N-formyl

peptide derivative as a probe. This approach resulted in the cloning of two isolates from a differentiated HL-60 cDNA library, which is known to contain the messenger RNA for

the FPR (Sullivan et al., 1987). Except for a small allelic

difference (see below), the two cDNA isolates are nearly

identical and encode a polypeptide of 350 amino acids. Transfection of the cDNAs into COS-7 cells conferred a high-affinity binding site for the N-formyl peptide deriva-

tive (Kd = 0.5-l nM) and a low-affinity binding site (Kd = 5-10 nM). The authenticity of the cloned FPR cDNA was further confirmed by functional analysis in transfected

mouse and human cell lines ( Prossnitz et al., 199 1; Didsbury et al., 1992) and in injected Xenopus oocytes (Murphy and

McDermott, 1991). The FPR is representative of a subgroup of GPCRs that

are mainly expressed in leukocytes and serve as chemoattractant receptors. Structurally, the FPR contains seven hy-

drophobic segments connected by hydrophilic sequences typically seen with GPCRs of the rhodopsin family (Mur-

phy, 1994). The FPR, h owever, does not contain large loop structures found in several GPCRs for catecholamines. A model of the structure of the FPR is shown in Fig. 1. In particular, the first and the third intracellular loops of the FPR

are small, with 5 and 16 amino acids, respectively. The car- boxy1 terminal portion of the receptor contains 47 amino acids and is of medium size compared with other GPCRs

(Prossnitz et al., 1993a). The relevance of the sizes of these intracellular domains with receptor-G-protein interaction

subsequently has been studied and will be discussed in Sec- tion 4.4.3. The DRY sequence, located at the amino termi-

nal portion of the second intracellular loop of most GPCRs,

E. R. Prossnitz and R. D. Ye

becomes DRC in the FPR. A total of 11 serine and threonine residues are found in the carboxyl terminus of the FPR that may be potential sites for phosphorylation. No consen- sus sequence for PKC phosphorylation is found in the FPR. On the extracellular surface, a pair of cysteine residues are conserved between extracellular loops 1 and 2. The amino terminus of the receptor is relatively small (26 amino acids) and does not contain predominantly charged residues often seen in chemokine receptors. There are two N-glycosylation sites at the beginning of the amino terminus and one

site in the second extracellular loop.

Allelic variations exist in the FPR gene from different individuals. The two cDNA clones initially isolated by Bou-

lay et al. (1990a) contain a Leu + Val substitution at position 101 and a Ala + Glu switch at position 346. Perez and co-workers (1992) b q su se uently reported amino acid substitutions at positions 11 (Ile 3 Thr) and 256 (Pro + Glu). The above substitutions apparently cause no alteration in the functional properties of the FPR. Additional variants

include a Asn + Lys substitution at position 192 and an Arg + His switch at position 123 (Boulay et al., 1990a; Perez et al., 1992). The latter is a highly conserved residue

among all rhodopsin-like GPCRs. Whether these variants are functional remains to be examined.

The cDNA for the rabbit FPR was isolated using the human cDNA as a probe (Ye et al., 1993). The deduced se-

quence of the rabbit FPR cDNA encodes a peptide of 352 amino acids that is 78% identical to the sequence of the human FPR. When expressed in transfected fibroblasts, the rabbit FPR displayed high- and low-affinity binding to the prototypic formyl peptide fMLF, with dissociation constants of 0.31 nM and 7.5 nM, respectively. The high-affinity site can be converted to a low-affinity site by the inclusion of

GTPyS in membrane-binding assays. Homologous and heterologous desensitizations were also observed in cells trans-

fected with the rabbit FPR cDNA (Ye et al., 1993). Gao

and Murphy cloned the mouse equivalent of the human FPR, along with 4 other genes that bear significant se-

cno CHO Extracellular Surface

Surface

FIGURE 1. Schematic repre- sentation of the structure of the FPR. Individual amino acids are indicated by filled circles; the membrane region is represented by the box. CHO, potential iv-linked glycosylation sites.

N-Fotmyl Peptide Receptor Structure and Function 87

quence homology to the FPR (Gao and Murphy, 1993; Murphy, 1994). Th e mouse FPR is 76% identical in sequence with the human FPR; yet, it is at least loo-fold less sensitive to the prototypic agonist fMLF when assayed in injected Xenopus oocytes. This functional difference has raised the possibility that fMLF may not be the physiological agonist for the mouse FPR. A comparison of the mouse FPR sequence with those of the human and rabbit revealed major differences in the three extracellular loops and at the membrane boundaries of the first extracellular loop. It is proposed that these differences may account for the significantly lowered affinity of the mouse FPR for the ligand fMLF.

GPCRs share high levels of sequence homology in certain domains, a feature utilized extensively for the cloning of structurally related receptors. Northern blot analysis of RNA samples from differentiated HL-60 cells identified two species that could be detected by the FPR cDNA probe in addition to the prominent FPR message (Boulay et al.,

1990a). Several groups subsequently have isolated the cDNAs corresponding to these species using low stringency hybridization approaches (Bao et al., 1992; Murphy et al., 1992; Ye et al., 1992). The first cDNA encodes a peptide of 351 amino acids that shares 69% sequence identity with the human FPR. When expressed in a fibroblast cell line, the protein encoded by this cDNA responded to micromolar concentrations of fMLF with calcium mobilization (Ye et al., 1992). In membrane-binding assays with fML[3H]F, the receptor displayed a Kd of 430 nM compared with a Kd of 1 nM for the human FPR in similarly transfected cells (Que- henberger et al., 1993). This receptor was given the name FPR2 (for its low-affinity binding to fMLF; Ye et al., 1992), FPRLl (FPR-like receptor 1; Murphy et al., 1992) and FPRHl (FPR homologue 1; Bao et al., 1992). More recently, Fiore et al. (1994) reported that FPR2 binds to lipoxin A4 (LXA,) with high affinity and responds to LXA, with GTPase activity in transfected Chinese hamster ovary cell membranes. This is an interesting finding since LXA, was not able to stimulate calcium mobilization in transfected fibroblast cells that responded to high concentrations of fMLF (R. D. Ye et al., unpublished data), despite being capable of stimulating calcium mobilization in peripheral blood monocytes (Roman0 et al., 1996). One possible explanation is that LXA4-induced signaling is different from that induced by fMLF, and that LXA4-stimulated calcium mobilization is a cell-specific event. These findings warrant additional studies of the receptor and its function.

A second gene, FPRU, was originally cloned from a genomic DNA library (Bao et al., 1992). It encodes a protein with 56% sequence identity to the human FPR and 83% sequence identity to the FPRU gene product. It subsequently was shown that the FPRLZ gene indeed is expressed in monocytes, but not in neutrophils (Durstin et al., 1994). This is different from the expression pattern of the FPRU gene, which is found in neutrophils, as well as in monocytes. FPRL2 expression is also induced during myeloid differentiation of HL-60 cells. Expression of the FPRLZ gene

product in Xenopus oocytes did not result in any functional responses to fMLF (Durstin et al., 1994). A comparison of the FPR sequence to those of its homologues is shown in

Fig. 2. That the FPR and its homologues may be derived from

the same ancestral gene through gene duplication events is suggested by the finding that these genes, together with the C5a receptor gene, are clustered in a relatively small region on human chromosome 19 (Bao et al., 1992). Further studies by others have indicated that the human FPR is encoded by a single-copy gene of -6 kb, with a genomic organization similar to several other GPCRs in that the coding sequence is contained in a single exon (Perez et al., 1992; Haviland et al., 1993; Murphy et al., 1993). The 5’ untranslated region contains two exons separated by Alu sequences. Two mRNAs are produced by alternative splicing of exon 2 in differentiated HL-60 cells and normal blood monocytes (Murphy et al., 1993). The 3’ untranslated sequence contains a third Alu sequence downstream of the polyadenylation site, thereby indicating that the different cDNA clones originally isolated may be derived by altema- tive polyadenylation (Haviland et al., 1993). Although CAMP regulates FPR gene expression in HL-60 cells in a time- and concentration-dependent manner, no CAMP-responsive ele- ment has been found in the 5’ untranslated region (Perez et al., 1992).

4.4. Structure-bnctiun Relationships of tk N-Formyl Peptide Receptor 4.4.1. Ligand binding domains. Given the incredible di- versity of ligands that stimulate GPCRs, from biogenic amines to lipids to oligopeptides to proteins to retinol and odorants, it is clear that there are probably almost as many mechanisms for the recognition of ligand by receptor. Al- though larger ligands, almost by necessity, must interact with the extracellular loop domains of receptors, in addition to any possible interactions with transmembrane domains, small ligands, such as retinol, biogenic amines and the chemoattractants fMLF and PAF, are believed to occupy ligand binding pockets deep within the transmembrane regions. Sklar and co-workers (1990) have demonstrated that formylated peptides up to five amino acids long are effectively not exposed to the extracellular mileau. With the cloning of the FPR, it became possible to investigate the molecular determinants involved in ligand recognition. Radel et al. (1991) utilized peptides corresponding to extracellular domains in an attempt to block fMLF binding to neutrophil membranes. A peptide representing the first extracellular loop (amino acids 84-100) was able to inhibit ligand binding to the FPR and, furthermore, was suggested to interact directly with the ligand.

Chimeric receptor approaches were used by a number of groups to investigate the FPR regions important in ligand binding. Perez et al. (1993) constructed chimeric receptors between the C5a receptor and the FPR (which show 34% amino acid identity), progressively substituting one receptor for the other and replacing specific domains of one re-


ceptor with the corresponding domain of the other. The results of ligand-binding studies on transfected COS cells indicated that essentially all of the extracellular domains and/or their adjacent transmembrane domains were involved in the formation of a ligand binding pocket. Addi-

tionally, both the extracellular amino terminus and intracellular carboxy terminus were observed to influence ligand

binding. A subsequent report by the same authors further studied the role of the amino terminus in fMLF binding (Perez et al., 1994). Amino acids 2-9 of the FPR were found to have a role in FPR function, as replacement of these res-

idues with the FLAG sequence (DYKDDDDK) caused a large decrease in receptor expression, as well as a moderate reduction in binding affinity. In addition, a synthetic pep-

tide corresponding to this region of the FPR specifically competed binding of ligand to the FPR. Alanine screening

mutagenesis of the second extracellular loop identified ad-

ditional residues involved in ligand binding (trp91 and

HUFPR

RbFPR

MuFPR

FPRLl

FPRLZ

HuFPR

RbFPR

MuFPR

FPRLl

FPRLZ

HuFPR

RbFPR

MuFPR

FPRLl

FPRLZ

HuFPR

RbFPR

MuFPR

FPRLl

FPRLZ

10 20 30 * * *

phe96, and to a lesser extent, his90, phe93, gly94 and 1~~99). The relevance of the mutagenesis studies on the amino terminus are brought into question by the pioneer- ing studies of Malech, who demonstrated that papain cleav,

age of the FPR, which removes the entire amino terminus, has no effect on the affinity of fMLF binding to the FPR (Malech et al., 1985). Additionally, Mery and Boulay

(1994) demonstrated high-affinity fMLF binding in a chi- merit FPR receptor containing the amino terminus of the C5a receptor. Thus, it is likely that certain alterations of the “disposable” amino terminus simply impede normal function of the FPR, and that this region is neither essential for, nor plays a role in, ligand binding.

A different chimeric receptor study was carried out by Quehenberger et al. (1993), who characterized 10 chimeric

receptors between 2 more closely related receptors, the FPR

and FPR2, an FPR homologue, which is approximately 70% identical to the FPR, yet binds fMLF with an affinity 400-

40 50 60 70 80 90 l l l * * l

METNSSLPTNIS--------GOTPAVSAGYLFLDIITYLVFAVTFVLOVLONGLVIWVAGFRMTHTVTTISYLNLP ::::::::::::::: ::: :::::::: : :: ::::: ~DS~~~~~g________~~~*~Tp:L~~VFS~~IL~~~~~~~~~~~*~sy

LNLALADFSFTSTLPFFIVTKALGGHWP ::::: :: : :: :: ::::::::: :::::::::::::::: :::: :::::::::::: ::: ::::::: : ::::: MDTNMSLLMNKSAVNLMNVSGSTQSVSAGYIVLDVFSYLIFA~~~NGLVI~~~~~TISY~~I~FCFTSTLPFYI~~~P

. . . . . . . . . . . . . . . ::: * :: ~E~F~TP~EY________EEVSYEg~~~~~RILPLWLG~~~~~~~~~~~~~AGF~TR~~~C~~~~sFTA~~~L~v~~~*~~

:::::: :::: ::: : :: ::: : : : ::::: :::::::::::::::::::: . . . . . . . . . . . . . . . . . . : ::: :: :: ::::

METNFSIPLNET--------EEVLPEPAGHTVLWIFSLLVHGVTFVFGVLONGLVIWAGFRMTRTVNTICY LNLALADFSFSAILPFRMVSVAMREKWP <__________~_~___________, <__________~_2_________>

100 110 120 130 140 150 160 170 180 * t l l * t l t t

FGWFLCKFGFTIVDINLFGSVFLIALIALDRCVCVLHPWJIIGPWVMALLLTLPVIIRVTTV--PGKT---G~ACTFNFSPWTNDP :::::::: ::::::::::::::::::::::: ::::::: :::: :::::::: ::: :::::::::::::: FGWFLCKFVFTIMINLFGSVFLIALIALDRCICVLHPVWAQNS~K~IVGPWI~LLTLPV~IRVTTLSHbRAP---~~~$DWg~~N~~ :::: ::: : :::::::::::::::::::::::::::::::: :::::::: ::::: ::::::::: :: FGWFMCKFIYTVIDINLFGSVFLIALIALDRCICVLHPVW~IVPWICAFLLTLPVIIRLTTV--~NSRLG~~~~~Fg~~~~ :::: :: : : ::::::::::: :::::::::::::::::::::::: ::: ::: : ::::: :::: ::: : FGWFLCKLIHIWDINLFOSVFLIGFIALDRCICVLHPVWAQNHRTVSLAMKVIVOPWILALVLTLPVFLFLT~--TIPN---~D~YCTFNFAS~~Tk

::: :: :: . . . . . . . . . . . . . . . . . . . . . . ~~~~r~~~I~iNLFVSVYLITIIALDRCI_HPA~~~~K~M~~~T~L~IFTI~~~~N~I~I__S~___~~~trI~~~~D~A

<_________M_3________> <__________~_4________>

190 200 210 220 230 240 250 260 270 280

KE~INVAVAML~GIIRFIIG~SAPMSIVAV~YGLIATKIH~QGLIKSSRP~RVLSFVAAA~FLCWSPYQ~~IATVRIR~-LM-G"YKEI~IAVDV

:: ::: : ::::::::::.... . . . . . . . . . ::::: ::::::::::::::::::::::: : ::: : : ::: : :: : VEKR~A~LTVRGIIRFIIGFSTPMSIVAICYGLITTKIHRaOLIKSSRPLRVLSFWAAFFLCWCPFQWALISTIQVRE-RLK-~TPGIVT~KI

::: :::: :::::: :::: :::::::::::: . :::::::::: E$RLKVAITMLTARGIIRFVIGFSLPMSIVAICYGLIAAh~I;KK~MIKSSRPLRVLTA~~$BI~~F~$bL~~LG~LK~-MCFYGKYKIiDILVNP ::: : :::

<________T&5_______> <__________~_6_________>

290 300 310 320 330 340 350

TS~FFNSCWPM~YVmDFR~RL~-P~~~~TS--DST;LTSDTATNST~PSA---EVELQA~ :: ::::::::::::::::::.... . . . . . . . ::::::::: : :: ::::: ::::

TSFVAFFNSCLNPMLYVFMGQDFRERLIHSLPASLERALS---~~~~I :: ::::::::::::::::::::::::::: :::::::: : ::::::::::: TSPLAFFNSCLNPMLYVFMGQDFRERLIHSLPASLERALTE--DSAQTSDTGTNLOTNSTSLSHNT~~ :: ::::::: :::::::: ::::::::::::: :::::: : ::: : :: : TSSLAFFNSCLNPMLWFVOODFRERLIHSLPTSLERALSE--DSAPTNDTAANCASPPA---~TE~Q~ ::::::::::::: :::: : : :::: :::::::::: : ::: : : :::: ::::::: TSSLAFFNSCWPILYVFMGRFQERLIRSLPTSLERALTEPPE---ETELQP(M <________T&,_______>

FIGURE 2. Alignment of the sequences of FPR and its homologues. The protein sequences of the human FPR (HuFPR), rabbit FPR (RbFPR), mouse FPR (MuFPR) and the homologues FPRLl and FPRL2 were aligned using the ALIGN program (Scientific & Educa- tional Software, PA). Gaps were introduced to allow for the best match of the sequences. Putative transmembrane domains (TM) are marked with arrows and numbered. The sequences are derived from GenBank with the accession numbers M33537 (HuFPR), M94549 (RbFPR), L22181 (MuFPR), MB8107 (FPRLl), and P25090 (FPRL2).

N-Formyl Pepcide Receptor Structure and Function 89

fold lower than that of the FPR (Kd = 1 nM). Seven chi- merit FPR/FPRZ receptors were generated by sequential replacement of FPR segments with the corresponding regions from FPRZ, as well as three reverse chimerae. Replacement of the first and the third extracellular loops of the FPR with those of FPR2 resulted in 275 and %-fold decreases in ligand-binding affinity, respectively. Introduction of both these domains into the FPR2 protein significantly increased ligand-binding affinity (Kd = 18 nM), whereas substitution of either the first or third extracellular loop alone improved ligand binding to a lesser degree (Kd = 90 and 372 nM, respectively). In all cases, alterations in ligand-binding affinity were closely correlated with functional changes in calcium mobilization. Substitution of either the amino or the carboxyl-terminal regions with those of the FPR2 had little effect on ligand-binding affinity. Gao and Murphy (1993) came to similar conclusions based on studies of FPR/FPRZ chimerae and a structure/function comparison of the high- affinity human and rabbit FPRs and low-affinity human FPR2 and murine FPR. In a follow-up to their original study, Quehenberger and co-workers (in preparation) have located three clusters of amino acids that are able to restore high-affinity fMLF binding to two distinct chimeric receptors exhibiting low-affinity ligand binding. Conversion of Ser84 to Arg and Met85 to Lys at the beginning of the first extracellular loop restored normal binding of fMLF to one of the chimeric receptors. Restoration of either of two pairs of amino acids at the end of the first extracellular loop (Glu89 to Gly and Lys90 to His or His102 to Phe and Ile103 to Thr) was able to restore high-affinity binding to the other chimera. Again, all increases in binding affinity were paralleled by increases in signal transduction, indicating a direct cause and effect relationship. A role for residues Arg84 and Lys 85 has also been suggested in ligand-binding experiments of synthetic FPR peptides (Radel et al., 1994). How- ever, a peptide of the same composition, but random sequence, was equally effective, but only when containing an arg and lys residue. Thus, simple charge-charge interactions may explain the effect of this peptide.

4.4.2. G-Protein coupling specificities. With the cloning of the FPR, the door was opened for studies of structure- function relationships of the receptor. However, as the pa- per describing the cloning of the FPR demonstrated only binding of the ligand, the functional ability of the clone had not been established at that time (Boulay et al., 1990b). The first method used towards that end was that of mRNA injection into Xenopus oocytes. The advantages of this method are that a single oocyte can be injected with pure or crude mRNA preparations and assayed for receptor function 2-3 days later. That FPR function could be generated from crude mRNA was first demonstrated by Murphy et al. (1990) and Coats and Navarro (1990) prior to the cloning of the FPR by Boulay et al. (1990b). Both groups demonstrated that total mRNA from differentiated, but not undifferentiated, HL-60 cells was capable of generating an ion flux in response to formyl peptides. Furthermore, size

fractionation of the mRNA yielded a fraction of average

size 2 kb that possessed the maximal fMLF responsiveness. Navarro subsequently used this approach to identify a rabbit cDNA, isolated by hybridization cloning, as a receptor for fMLF (Thomas et al., 1990). This receptor later was definitively demonstrated to be a receptor for the chemokine IL-8 (Thomas et al., 1991; Lee et al., 1992). Following the cloning of the FPR by Boulay, it became possible to express the single recombinant FPR transcript in Xenopus oocytes. Despite expression of the receptor, as determined by ligand binding (Schultz et al., 1992), no functional responses could be detected (Murphy and McDermott, 1991; Schultz et al., 1992). However, co-injection of mRNA from undifferentiated HL-60 cells, which by itself did not confer responsiveness to fMLF, resulted in the generation of an ion flux in response to ligand. This result was in contrast to studies with all other nonchemoattractant GPCRs expressed in this system, which are capable of functional coupling with the injection of a single receptor mRNA transcript. Size fractionation of the undifferentiated HL-60 mRNA demonstrated that a fraction of 3-3.5 kb was responsible for yielding functional coupling upon co-injection. Since the FPR transcript was 1.4 kb, it was concluded that a complementary factor was required for FPR function (Murphy and McDermott, 1991; Schultz et al., 1992). The identity of this complementary factor has been suggested recently (see below; Burg et al., 1995).

The second approach utilized by Prossnitz et al. (1991) to investigate the functional capabilities of the newly isolated FPR clone involved stable transfection of the FPR cDNA into mouse L-cell fibroblasts with subsequent G418 selec- tion. Transfected cells demonstrated ligand binding both by flow cytometry and fML[jH]F binding. The ability to ana- lyze the cell population by flow cytometry would prove to be of great value because the distribution of FPR-expressing cells could be determined as a function of the relative or absolute number of receptors expressed per cell. A frequent result of the generation of stably transfected cell lines is a wide variation in the expression levels of individual clonally derived cells due, presumably, to positional effects from the recombination event. It is even possible to obtain G418- resistant cells that express no receptor at all. One solution to this problem is to isolate individual clones and determine receptor expression levels for each clone. The other approach is to sort the cells by flow cytometry, selecting only the cells that express high (or any other desired) levels of receptor. The advantage of the latter is that the selected population still represents the combined properties of many clones, largely eliminating any other possible clone-to- clone variations resulting from recombination.

Stably FPR-transfected L-cells expressed an average of 70,000-80,000 receptors/cell with a dissociation constant of 3 nM (Prossnitz et al., 1991). In contrast to Xenopus oocytes, when FPR-transfected L-cells were stimulated with fMLF, they responded with a rapid, yet transient, increase in the intracellular concentration of Ca++ with an ECsO of approximately l-3 nM fMLF. Cytoplasmic Ca++ concen-


trations were determined by loading the cells with the

Ca++-sensitive fluorophore indo-l and monitoring ligand- dependent fluorescence changes. The response could be completely inhibited by pretreatment of the cells with pertussis toxin, indicating that FPR-G-protein coupling in L-cells was similar to that observed in the neutrophil. This report established that expression of the single cDNA for the FPR

was necessary, and, more importantly, sufficient, in the absence of any additional neutrophil-specific cofactor, to me-

diate transmembrane signaling in response to fMLF. That

this coupling was not unique to L-cells was demonstrated by functional G-protein-coupling in additional cell lines, including human 293 kidney (Didsbury et al., 1992) and

Chinese hamster ovary cells (E. R. Prossnitz and R. D. Ye, unpublished observations). A physical association between the recombinant FPR expressed in L-cells and G,-protein was demonstrated both by co-immunoprecipitation and by

sucrose density sedimentation, where an affinity for G-protein similar to that of neutrophil-derived FPR was observed

(Schreiber et al., 1993).

Perhaps the most commonly used heterologous expression system involves transient transfection of monkey

COS-7 kidney cells, which, in fact, was used in the cloning of the FPR and Csa receptor (Boulay et al., 1990b, 1991; Gerard and Gerard, 1991). That these initial cloning reports

did not demonstrate functional properties of the cloned receptors was explained subsequently by demonstrations that the CSa receptor is unable to elicit PLC activity in transfected COS-7 cells (Amatruda et al., 1993). A similar result was also reported for 293 cells (Buhl et al., 1993), although

reports also exist describing functional calcium mobilization upon introduction of the FPR and Csa receptor (Dids- bury et al., 1992). Both the Amatruda et al. and Buhl et al.

studies showed that cotransfection with a recently discov-

ered myeloid-specific G-protein, Gu16 (Amatruda et al., 1991), could restore function of the transfected receptor. Furthermore, cotransfection with either G,, or Giz was in-

effective (Buhl et al., 1993). The relevance of this interaction remains unclear, however, since, although Gut6 is selectively expressed in certain myeloid cells, it is not

sensitive to pertussis toxin, and all known responses of chemoattractant receptors are sensitive. A possible explanation for the observed coupling of chemoattractant recep-

tors to Gct6 in heterologous cell types was provided by the observation that Guls appears to couple promiscuously a wide variety of receptors, including adrenergic, serotonin, opioid and muscarinic receptors, to PLC (Gffermanns and Simon, 1995). Recently, co-injection of Gut6 cRNA with either the transcripts for the FPR or the C5a receptor into Xenopus oocytes was shown to be sufficient to yield ligand- stimulated ion fluxes, suggesting Gal6 is most likely the complementing factor present in the U937 and HL-60 mRNA fractions described above (Burg et al., 1995). In- consistent with this conclusion is the fact that the mRNA for Gals was shown to be 2.2 kb in size (Amatruda et al., 1991), whereas the complementary factor was shown to be 3.5 kb in size (Murphy and McDermott, 1991). Since the

source of both RNAs was HL-60 cells, the reason for the discrepancy in size remains unclear. It does suggest, however, that the 3.5 kb message may code for an as of yet unknown protein.

To complicate the matter further, HL-60 cells have been shown to express significant levels of Gut6 (Amatruda et al.,

1991), which are down-regulated upon differentiation, sug gesting that chemoattractant receptors expressed in undif-

ferentiated or minimally differentiated HL-60 cells should functionally couple to Gal+ However, transfection of the FPR into HL-60 cells yielded calcium mobilization and actin polymerization responses that were entirely blocked by pertussis toxin, indicating that coupling through Gal6 was

not occurring to promote these responses (Prossnitz et al., 1993b). A solution to this dilemma may exist in the speci-

ficity of activation of PLC subtypes by G-proteins. Whereas PLC,,, which is largely absent in myeloid cells, is activated by the o-subunits of certain G-proteins (Kozasa et al.,

1993), such as G,, and Go1t6, PLCsz, which is expressed predominantly in myeloid cells and absent in COS and other

cell lines, is activated by the By-subunits of G-proteins (Wu et al., 1993a), such as Gil. However, additional com-

plexity of the system was suggested by the demonstration that co-expression of Gut6 and Gplvl, but not Gal+, in COS-7 cells was able to synergistically activate recombi-

nant PLCaz. It was suggested that Gu16 may act together with free GPlvl to activate PLC,,, while Gal6 may form heterotrimeric complexes with GPlvs and be stabilized in an inactive form (Wu et al., 1993a). When PLC,, was cotrans- fected with IL-8 receptors into COS cells, IL-8 receptors interacted with endogenous pertussis toxin-sensitive G-pro-

teins to release free By-subunits that could then specifically activate the B2 isoform of PLC (Wu et al., 1993b). This has

been confirmed recently for the FPR and Csa receptor, which were also shown not to couple to PLC,, (Jiang et al.,

1996). Thus, the spectrum of G-protein By-subunits expressed in myeloid cells is likely to be pivotal in determining the interactions of heterotrimeric G-proteins with both receptors and effecters. In support of this idea, the G,z-subunit recently was demonstrated to play an important role in fMLF-dependent HL-60 cell activation. Retinoic acid

treatment of DMSO-differentiated HL-60 cells resulted in de nova expression of Gy, with commensurate stimulation of FPR-G,z coupling and activation of PLC (Iiri et al.,

1995). Whether other, as of yet unknown, myeloid cell functions are mediated by Gal6 or other G-protein By-subunits remains unexplored.

4.4.3. N-Formyl peptide receptor-G-protein interaction sites. With the cloning and functional expression of the FPR, it became possible to investigate the structure-function relationship of the receptor with respect to G-protein coupling. The first approach to determining FPR domains involved in G-protein interactions involved the use of synthetic receptor-derived peptides to interfere with the FPR-G-protein interaction. Bommakanti et al. (1993) investigated the ability of numerous FPR-derived peptides to inhibit formation


of a GTP-sensitive, rapidly sedimenting physical complex between the FPR and purified G-proteins as analyzed by sedimentation on sucrose density gradients (as described in Section 4.1.5). The results demonstrated that a peptide of 15 amino acids in length, spanning residues 322-336, in the central portion of the carboxy-terminal tail, was capable of disrupting the FPR-G-protein interaction with an IC50 of approximately 0.5 mM. A peptide with the reverse sequence had no discernible effect at concentrations up to 3 mM. Although peptides from other regions of the FPR were also tested, namely from the second and third intracellular loops, they were ineffective. Prossnitz et al. (1993a) performed extensive site-directed mutagenesis of the charged and polar residues of the third intracellular loop of the FPR and analyzed both ligand-binding and calcium mobilization capabilities of the resulting receptors. Of the 10 mutants generated, all were observed to bind ligand normally, and 8 of the 10 displayed close to normal calcium mobilization. The two mutants, Lys227 + Glu and Arg238 + Gly, however, displayed moderate increases in the EC50 for fMLF- stimulated calcium mobilization, as compared with the wild-type receptor. Overall, the results suggested that the third intracellular loop of the FPR was not an important de- terminant in G-protein interaction and activation.

Schreiber and co-workers (1994) also utilized synthetic peptides in addition to a fusion protein containing the entire carboxy-terminus (C-terminal 47 amino acids) of the FPR, but analyzed numerous physical, as well as functional, assays of the FPR-G-protein interaction. Utilizing a competitive ELISA procedure, which measures the ability of Gi protein in solution to compete with the binding of a G,-derived peptide coating microtiter wells to an anti-Gi-derived peptide antibody, the FPR carboxy-terminal fusion protein was shown to be highly effective in binding G-protein with an ICsO of approximately 20 PM. Of three synthetic peptides spanning the carboxy-terminal tail, only one, corresponding to the first third of the tail (amino acid residues 308-322), demonstrated an ability to bind to G-protein, albeit at concentrations loo-fold higher than that of the fusion protein. This result suggested that either multiple regions of the FPR tail were required for binding to G-protein or that higher order structure, absent in the short synthetic pep tides, was required. Using this assay, a peptide from the second intracellular loop (amino acids 126-137) was also shown to be effective (I&, ~200 FM) in binding to G-protein. Other methods used to assess FPR-G-protein interaction included: (1) inhibition of pertussis toxin-catalyzed ADP- ribosylation of Gi-protein, which demonstrated, in addition to the two peptides already described, a peptide representing the central third of the carboxy terminus (amino acids 3 19- 340) to be effective; (2) inhibition of high-affinity G-protein- dependent fML[jH]F binding; and (3) inhibition of the FPR-G-protein physical complex, as analyzed by sedimentation on sucrose density gradients. Both of these latter methods indicated the peptide representing a portion of the second intracellular loop to be the most effective in disrupting the FPR-G-protein interaction. Taken together, these results

indicated the second intracellular loop and the carboxy ter-

minus, but not the third intracellular loop, to be the major sites of contact between the FPR and G-proteins.

Recently, Bommakanti et al. (1995) carried out a more extensive study of receptor-derived synthetic peptides blocking the physical association of the FPR with G-proteins. A total of 20 FPR-derived peptides representing all of the possible cytoplasmic surfaces of the FPR were studied for their ability to disrupt the FPR-G-protein complex. The most effective of these was a 23-mer peptide representing the entire second intracellular loop (amino acids 122-144, I(&, = 20 PM). Interestingly, peptides 5 amino acids longer (amino acids 119-146) and 8 amino acids shorter (amino acids 119-133), or 6 amino acids shorter (amino acids 134-150) were 25-, 50- and 5-fold less effective, respectively. Two additional peptides that displayed moderate ef- fectiveness were derived from the first intracellular loop (amino acids 43-61, IC,, = 100 (IM) and the central portion of the carboxy terminus loop (amino acids 322-336, I& = 400 PM). The former of these two peptides contained amino acids at the amino terminus that are very hydrophobic and most likely represent part of the first transmembrane domain. Further peptides representing the majority of the third intracellular loop extending into transmembrane domain 6 (amino acids 230-246, I&, = 1.4 mM) and the amino terminal portion of the carboxy terminus were only slightly effective (amino acids 304-3 19 and 3 15-329, IC& = 3 mM). That even these low-affinity peptides were relevant was demonstrated with a peptide of the reverse sequence of the third intracellular loop peptide, which was shown to be completely ineffective (IC+,o >lO mM). Overall, these results are consistent with numerous other studies, indicating the importance of the second intracellular loop, the amino terminal half of the carboxy terminal tail (Schreiber et al., 1994; Prossnitz et al., 199513) and a minor role for the carboxy terminal region of the third intracellular loop, particu- larly R238 (Prossnitz et d., 1993a). The suggestion that the first intracellular loop is involved in G-protein binding is novel and recently has been supported by mutagenesis studies of the FPR-GQ16 interaction (Amatruda et al., 1995; see below).

Site-directed mutagenesis studies have yielded still more detailed information on the mechanisms of coupling between the FPR and G-proteins. Prossnitz et al. (1995b) studied the function of numerous amino acids conserved amongst the superfamily of GPCRs. These included Asp71 (located in the second transmembrane helix), Arg123 (part of the Asp-Arg-Tyr sequence found at the beginning of almost all GPCRs) and Arg309-Glu310-Arg311 (part of a loosely conserved sequence located at the beginning of the carboxy teminus). Substitution of either Asp71 or Arg123 resulted in mutant receptors that bound ligand with only low affinity (Kd = 30-50 nM) independent of GTPyS. This is in contrast to the wild-type FPR, exhibiting a & for fMLF of l-3 nM, which is reduced to approximately 40 nM in the presence of GTPyS. Replacement of residues Arg309-Glu3 lo-Arg3 11 resulted in a receptor with


intermediate ligand-binding characteristics. Functional analyses revealed that substitution of either Asp71 or Arg123 re-

sulted in a mutant receptor that was unable to mediate fMLF-dependent calcium mobilization. Replacement of

residues Arg309-Glu3 IO-Arg3 11 yielded an EC50 for fMLF of 50 nM, compared with 0.5 nM for the wild-type FPR.

Utilizing velocity sedimentation on sucrose density gradients, it was demonstrated that the defect introduced in the Asp71 and Arg123 mutants was manifested as an inability of the liganded FPR to bind to G-protein. This is in contrast to

a defect in the ability of the liganded FPR to activate G-protein, despite the presence of a physical interaction between the proteins. It was speculated that these mutants acted by preferentially stabilizing the inactive, ligand-free conforma-

tion (or conversely destabilizing the active conformation)

of the FPR, which is unable to bind G-protein. Support for

this interpretation was provided by the Arg309-Glu310- Arg3 11 mutant, which demonstrated a 50-fold reduction in the binding affinity for G-protein, as determined by physi-

cal reconstitution, which paralleled almost exactly the reduction in the ability of the mutant to promote G-protein activation, as measured by calcium mobilization. Recent results, which demonstrate that the Arg123 mutant is capable

of supporting ligand-induced receptor internalization, suggest that, in fact, this mutant receptor is capable of undergoing a

ligand-dependent conformational change and that Arg123 is strictly involved in G-protein recognition (E. R. Pross-

nitz, unpublished observation). The observation that the C5a receptor could couple to

Gal6 in nonmyeloid cell lines (Amatruda et al., 1993), was used as a basis to examine regions of the FPR and C5a receptor responsible for this coupling. Amatruda et al. (1995)

confirmed that the FPR was also capable of coupling to G a16 in COS cells, but not to endogenous G-proteins or G,. Using a set of FPR/CS, receptor chimerae, it was shown that a chimera of the FPR containing the first intracellular loop of the C5a receptor was constitutively activated by a factor

of two. Subsequent site-directed mutagenesis of the first intracellular domain of the FPR (amino acids 54-62) demon-

strated this region to be a domain necessary for ligand- dependent activation of Ga16. Whether this region is also important in the stimulation of Gil,:, remains to be deter-

mined, although it is suggested by peptide inhibition of reconstitution studies of the FPR with G, (Bommakanti et al., 1995). It should be noted that the increase in receptor ac-

tivity described for the FPR/C,, receptor chimera here and the decrease in activity described by Prossnitz et al. (1995b) above represent only some of the many or possibly infinite end points for receptor activity. Where total receptor activity is the result of an equilibrium between active and inactive conformations (in a simplistic view), the number of factors affecting this equilibrium is large, with the current wild-type amino acid sequence representing a receptor that has simply resulted from an evolutionarily determined com- promise between sensitivity and unresponsiveness. For a thorough discussion on this topic as it relates to the &-adrenergic receptor system, see Samama et al. (1993).

4.4.4. N-Fomyl peptide receptor phosphorylation and desensitization. Following the stimulation of neutrophils

with chemoattractants such as fMLF, cells rapidly become refractory to further or subsequent stimulation with the same or other chemoattractants. Multiple mechanisms may contribute to desensitization, including receptor phosphorylation, internalization and receptor down-regulation, as

has been extensively studied for the &-adrenergic receptor (Hausdorff et al., 1990). Studies with membranes prepared from ligand-stimulated neutrophils have indicated that desensitization of chemoattractant receptors occurs, at least in part, by an uncoupling of the receptor-G-protein interaction. The ability of endogenous G-protein to bind and hy drolyze GTP was significantly reduced in membranes pre-

pared from cells treated with chemoattractant (Wilde et al.,

1989). Interestingly, Mg*+ was able to restore G-protein interactions with the receptor (McLeish et al., 1989a), sug

gesting the possibility that the neutralization of a negative charge (e.g., a phosphate moiety) on either the receptor or G-protein can restore function to the system. Two conven- tional types of desensitization have been described. Homol- ogous desensitization refers to the decrease in responsive-

ness of the liganded receptor, often through phosphorylation of the liganded receptor (Dohlman et al., 1991; Premont et

al., 1995). Heterologous desensitization refers to the inacti-

vation of one or often multiple unliganded receptor types

by another liganded receptor type. This form of desensitization may result from the phosphorylation of unliganded receptors by second messenger-activated kinases, such as pro-

tein kinase A or PKC. Didsbury et al. ( 1991) have demonstrated that the cal-

cium rises induced by chemoattractant receptors undergo a type of heterologous desensitization in which the FPR and C,, receptor can desensitize each other, but cannot desensitize or be desensitized by unrelated receptors such as the aI- adrenergic receptors. This form of desensitization has been termed receptor class desensitization because its properties

are intermediate between classic homologous and heterologous desensitization (Didsbury et al., 1991). This work was

extended to compare the desensitizing abilities of IL-8 and PAF with those of fMLF and C5a (Tomhave et al., 1994). All receptors underwent homologous desensitization of the

calcium mobilization response as expected. However, peptide chemoattractants (fMLF, Csa, and IL-8) desensitized each other’s calcium-mobilizing responses, but had no effect on calcium mobilization by purinergic receptors. PAF and LTB, were also desensitized by peptide chemoattractants, but not vice versa. Whereas all peptide receptors underwent homologous desensitization of agonist-stimulated GTPyS binding to leukocyte membranes, Csa and IL-8, but not fMLF, receptors were cross-desensitized by other pep tide chemoattractants. Furthermore, phorbol myristate acetate (PMA) treatment prior to membrane preparation inhibited Csa- and IL-g-, but not fMLF-, stimulated GTPyS binding. The results indicated that in addition to homologous receptor desensitization, peptide chemoattractant

(fMLF, C5a, and IL-8) receptors cross-desensitize each an-


other by at least two distinct processes. The first occurs at the level of receptor-G-protein interaction, and possibly involves receptor phosphorylation by PKC. The FPR is not susceptible to this mechanism due to a lack of PKC sites in its intracellular domains. The second occurs downstream of receptor-G-protein coupling, and results in desensitization of the Ca++ mobilization response to all peptide chemoattractants, including the FPR.

The first demonstration that chemoattractant receptors were phosphorylated upon ligand stimulation was provided by Tardif et al. (1993). Immunoprecipitation of the endoge- nously expressed phosphorylated FPR and Csa receptor from differentiated HL-60 cells was shown to be dose- and time-dependent. PMA, which stimulates PKC, was capable of inducing phosphorylation of the Csa receptor, but not the FPR, in the absence of agonist. Staurosporine, a PKC inhibitor, had no effect on fMLF-induced FPR phosphory lation, but partially inhibited C5,-induced Csa receptor phosphorylation. Furthermore, neither ligand was able to stimulate phosphorylation of the other ligand’s receptor. It was concluded that a kinase other than PKC was involved in the phosphorylation of these chemoattractant receptors. Receptor phosphorylation was also demonstrated by Ali and co-workers (1993), who utilized epitope-tagged FPR and C5a receptors stably expressed in rat basophilic leukemia, RBL-2H3 cells. Phosphorylated FPR migrated as a single broad band, whereas two differently migrating species of phosphorylated Csa receptor were observed on SDS-PAGE. PKC was shown to be largely responsible for the generation of the fast migrating C5a receptor species, whereas CTa induced the slow species. Homologous desensitization was shown to correlate with ligand-induced phosphorylation of both the FPR and Csa receptors. However, only the C5a receptor underwent heterologous desensitization induced by antigen complexes, PMA or thrombin, which correlated with receptor phosphorylation. These results confirmed that PKC plays a role in C5a receptor phosphorylation and desensitization, but not in that of the FPR. On the other hand, both receptors are homologously desensitized by a staurosporine-resistant kinase.

Further studies of the relationship between receptor phosphorylation and desensitization were carried out utilizing stable co-expression of the FPR, Csa receptor and IL-8 receptor A in RBL cells (Richardson et al., 1995). When co-expressed, C& and IL-8 receptors were cross-phosphory lated by activation of the other receptor. Stimulation of the FPR phosphorylated C,R and IL-8RA, whereas the FPR could not be phosphorylated by Cfia or IL-8 stimulation. Furthermore, C5a and IL-8 stimulation of GTPyS binding, as well as the Ca++ mobilization response, were desensitized by each other and also by fMLF. Stimulation of GTPyS binding by fMLF was not desensitized by either C5a or IL-8, although the Ca++ mobilization response and IP, synthesis were desensitized by both. These results indicate that receptor phosphorylation may provide a mechanism for desensitization at the level of G-protein activation. However, desensitization of the downstream FPR response (IP, generation

and intracellular Ca++ mobilization) occurs in the absence of FPR phosphorylation. These results suggest a novel form of desensitization at a site downstream of the receptor-G-

protein coupling event, but before activation of PLC. In an effort to identify and characterize the kinase in-

volved in the phosphorylation of the FPR (and possibly other chemoattractant receptors), Prossnitz and co-workers (1995a) utilized a fusion protein containing the carboxy terminal 47 amino acids of the FPR. This same fusion protein previously was shown to bind to G-proteins, blocking interaction with the FPR (Schreiber et al., 1994). Incuba- tion of the fusion protein with neutrophil cytosolic extracts resulted in the co-isolation of a kinase activity that could specifically phosphorylate the fusion protein. This activity was insensitive to PKC, protein kinase A or tyrosine kinase inhibitors, but was blocked by heparin, at concentrations known to inhibit GPCR kinase (GRK) 2 (Benovic et al., 1989). Utilizing purified GRKZ, phosphorylation of the FPR fusion protein was also demonstrated. Other GRKs were either significantly less effective (GRK3), or not effective at all (GRKS and GRK6). Phosphoamino acid analysis revealed that both serine and threonine (but not tyrosine) residues were phosphorylated. Mutagenesis of the FPR carboxy terminus contained in the fusion protein revealed that (1) acidic residues within the carboxy terminus (Glu326, Asp327 and Asp333) are essential for activity of GRK2 towards the fusion protein; (2) simultaneous substitution of Thr334, Thr336, Ser338, and Thr339 resulted in an approximately 50% reduction in phosphorylation, whereas simultaneous substitution of the upstream Ser328, Thr329, Thr331, and Ser332, or merely the Ser328 and Thr329, residues resulted in an approximately 80% reduction in phosphorylation; and (3) the introduction of negatively charged glutamate residues for Ser328 and Thr329 or Thr331 and Ser332 resulted in marked stimulation of phosphorylation (Prossnitz et al., 1995a). Expression of the FPR containing either the Thr334, Thr336, Ser338, and Thr339 substitution or the Ser328, Thr329, Thr331, and Ser332 substitution in U937 cells resulted in significant decreases in the level of receptor phosphorylation upon ligand stimulation, as well as a partial, in the case of the former mutant, or complete, in the case of the latter mutant, loss of desensitization (E. R. Prossnitz, unpublished data). These results

** ** * * ** FPR SI_~F.ALT.~STQTSQTAl’NSTLP

Pz= KgnrLLC~~Pc~~~~HQc;~~n~~n~Q~~~~

Rhodopsin GKNPIG~ST&~&VAPA

FIGURE3. Sequence alignment of the phosphorylated region of the carboxyl-terminus of FPR, &-adrenergic receptor and rhodopsin. Underlined amino acids represent acidic residues that occur in conserved sites in the three receptors. Phosphoq- lation sites are indicated by an asterisk. In the case of the JTPR, it is not known precisely which of the indicated sites are phosphorylated.


confirm that multiple phosphorylation sites are required for FPR desensitization in &o and that a hierarchy exists in the ability of the phosphorylated residues to induce desensitization.

Taken together, these results suggest a model for FPR phosphorylation in which kinase recognizes acidic residues in the FPR carboxy terminus and phosphorylates serine and threonine residues in a sequential manner, with phosphorylation of amino-terminal serine and threonine residues required for the subsequent phosphorylation of carboxyl-terminal residues. Recently, the amino acid residues of the PI-adrenergic receptor phosphorylated by GRK2 were determined (Freder- icks et al., 1996). This complemented previous results on rhodopsin kinase phosphorylation of rhodopsin (McDowell et al., 1993; Pullen and Akhtar, 1994), and together with the results of the FPR, begins to establish a pattern for GRK specificity. All three receptors possess a pair of acidic amino acids immediately upstream of the phosphorylation sites, followed by an additional one or two acidic amino acids at a position 5-9 amino acids downstream from the first pair and downstream of at least one phosphorylated residue (Fig. 3). In the case of the FPR, both sets of acidic residues ap pear necessary for phosphorylation (Prossnitz et al., 1995a). In addition, the FPR and the &-adrenergic receptor also contain an acidic residue 5-6 amino acids upstream of the first pair of acidic residues. The role of this amino acid cur- rently is unknown.

5. CONCLUSIONS, PERSPECTIVES AND FUTURE DIRECTIONS

Since the initial discovery of N-formylated chemotactic peptides nearly a quarter century ago, research in this field has expanded to multiple disciplines, including pharmaco- logical characterization of the peptide ligands and the formyl peptide receptor, biochemical studies of the signaling events mediated by the receptor, and more recently, molecular cloning of the receptor and its homologues. These studies have produced an enormous amount of data, making the FPR one of the best studied chemoattractant receptors. Despite all these efforts, however, a number of important issues remain to be explored. First, the identification of potent chemotactic peptides without N-formylation has prompted a re-examination of the structure and function relationship of the ligands. As can be expected, the interaction of these ligands with the FPR may require additional studies utilizing derivatized ligands and mutant receptors. Second, it is not clear why the receptors in several species, such as mouse, bind with only low affinity to N-formyl peptides. It is likety that there is an additional ligand for the FPR that can interact with the receptors across species. Cloning of the receptors from these species may help identify potentially new and possibly endogenous ligands. Third, the finding that FPR is expressed in nonleu- kocytic cells suggests novel functions that may be mediated by the receptor. Fourth, the structural changes of the FPR resulting from binding of the formyl peptides remain unde-

fined. As a small peptide ligand, fMLF binding may involve mechanisms shared by both small molecule ligands, as well as large peptide ligands for the GPCR superfamily. Under- standing the details of ligand binding, receptor signaling, desensitization and processing events may provide, therefore, insights into GPCR signal transduction in general. Fi- nally, the clinical relevance of the FPR will need to be investigated further. In particular, the role of FPR in such conditions as Crohn’s disease, ulcerative colitis and juve- nile periodontitis requires additional studies. The generation of knockout mice may be a useful approach if ap propriate protocols can be developed to evaluate the physiological and pathological consequences. The avail- ability of many powerful tools developed over the last de- cade undoubtedly will allow us to study this interesting receptor in more detail and to answer the many questions that still remain.

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