ing factor

receptor and signal ctranuction

mechanism SHWENDRAI: Department

SHUKLA of P irmacology,

School of MedtcusC Unwersitjsof Missouri

ABSTRACT Platelet-activating factor (PAF) is the most potent phospholipid agonist known to date. Radioligand binding studies using [3HJPAF and structurally different PAF antagonists have provided the characteristics of PAF receptor(s) and its heterogeneity. Although efforts have been made to isolate the receptor, it was not until the recent cloning of the PAF receptor that the molecular architecture of the receptor can be visualized. The receptor shows homology to the G protein-coupled receptors with seven transmembrane spanning segments. Several serine, threonine, and tyrosine residues are present at the cytoplasmic side, which could serve as sites for phosphorylation. PAF activates GTPase, causes phospholipid turnover via phospholipases C, D, and A2 pathways and also activates protein kinase C and tyrosine kinase. Further, PAF stimulates Ca2 + mobilization some of which may occur via receptor operated channel. Second messengers generated by these multiple signalling pathways play role (or roles) in PAF responses and in the PAF induced expression of primary response genes. These recent developments throw light on the PAF receptor and its signal transduction mechanisms. Shulda, S. D. Plateletactivating factor receptor and signal transduction mechanisms. FASEBJ. 6: 2296-2301; 1992. Key tion

Words: platelet-activating factor phospholipases . protein kinases




FACTOR (i-O-alkyl-2-acetyl-sn-glycerol3-phosphocholine) is a potent phospholipid mediator involved in a variety of pathophysiological events, e.g., inflammation, asthma, cardiovascular disease, reproduction, cerebral ischemia, etc. Historical developments on the discovery and identification of platelet-activating factor (PAF)2 have been described in several excellent review articles (1-4). PAF is produced endogenously in pathological conditions and also upon stimulation of cells/tissues (3, 4). Further, PAF also exerts a wide variety of effects on cells when added exogenously (2, 3). It has biological effects on, for example, platelets, neutrophils, endothelial cells, macrophges, monocytes, kidney, heart, lung, liver, brain, muscle, eye, etc. (3). The fact that lyso-PAF (the inactive metabolite) is without effect on cells indicates the structural specificity for the PAF molecule. Many structurally different PAF antagonists have also been developed (3, 5). Several studies have provided information on the nature of the interactions of PAF with membrane receptor and the signaling pathways involved in its cellular actions. With the recent cloning of the PAF receptor (6) the molecular architecture of the receptor protein is also emerging. This review highlights some of these new research developments in this area. PLATELET-ACTIVATING








Radioligand binding studies in many cells have provided the characteristics of specific PAF binding sites. This aspect has been covered in a recent article (5). It is abundantly clear that PAF receptors are present in cells responsive to PAF. In various studies the number of PAF receptors in a cell and the affinity of the ligand have been determined and indicate a range of values. Various ions, e.g., K, Rb, Mg2, Ca2, and Mn2 enhance [3H]PAF binding whereas Na and Li inhibit it. These ion effects are likely to be on interactions between PAF and its receptors. GTP specifically inhibited [3H]PAF binding to platelet membranes either at 37 or 0#{176}C and thus implicated G proteins (5, 7). One important outcome of ligand binding studies is the view that there may be heterogeneity in PAF receptors. With the availability of structurally different antagonists, several observations indicate that the K values are different among different cells isolated from the same species. High- and low-affinity PAF receptors have been identified in many cells and tissues (5). The fact that various ions can modulate these receptor binding properties and affinities has led to the proposal that the PAF receptor exists in different conformational states (5). The presence of heterogeneity in the PAF molecule itself (8) and of the recently noted oxidatively fragmented PAF molecule (9), all of which have PAF-like activity, are provocative observations in this regard. The question of transition from high to low affinity has also been raised (5). The mechanism by which these transitions can occur is unknown. It could be due to masking of the receptor or of its internalization or translocation into the inner membrane layer. Nevertheless, these studies have laid the groundwork for the existence of PAF receptors in a variety of biological systems. This necessitated the isolation of the PAF receptor. ATTEMPTS






Efforts to solubilize and isolate the PAF receptor have not been very rewarding. PAF is a phospholipid ligand, and

‘This article is based, in part, on an American Society for Pharmacology and Experimental Therapeutics (ASPET) symposium, “Platelet-Activating Factor Receptor Signal Mechanisms” held at the 75th Annual Meeting of FASEB in Atlanta, Georgia, April 22, 1991. 2Abbreviations: AA, arachidonic acid; DG, diglyceride; G protein, guanine nucleotide binding proteins: IP,, inositol trisphosphate; PAF, platelet-activating factor; PG,, prostaglandins; P1, phosphatidylinositol; PKC, protein kinase C; PLA,, phospholipase A2; PLC, phospholipase C; PLD, phospholipase D; PPI, phosphoinositides.


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hence the binding assays for the solubilized receptor are tedious. For example, in detergent-solubilized receptor PAF can interact with detergent micelles. In one study, human platelet plasma membranes were solubiized with 5% sodium dodecyl sulfate (SDS) and loaded on a column of PAF-human serum albumin Sepharose. The column was washed and eluted with fivefold molar excess of PAF. A protein with an apparent molecular weight of 180,000 was eluted from the column and was identified as the high-affinity platelet receptor for PAF (10). A protein with 160 kDa was also isolated from solubilized human platelet membranes by sequentially using DEAE cellulose, CM cellulose, and Sephadex G-200 chromatography (11). In another study, a PAF receptor complex of 220 kDa was isolated from rabbit platelet membranes. This was achieved by solubiization of rabbit platelet membranes with 2% digitonin followed by sucrose density gradient and gel filtration on Sephacryl S-300 (12). In this study, [3H]PAF bound to its receptor not only survived the detergent solubilization, but also remained in a high-affinity form. Unlabeled PAF was unable to replace the bound PAF in contrast to membrane receptor studies where such displacement can occur (12). GTP was observed to facilitate the dissociation of [3HJPAF from the PAF receptor complex and addition of Na2 enhanced this dissociation, suggesting a role for G protein (12). In their continuing investigation, these investigators used a photoreactive, radioiodinated derivative of PAF to label platelet membranes (13). SDS-PAGE analysis of the samples showed an iodinated protein of 52 kDa. Unlabeled PAF and the PAF antagonists SRI-63-675 and L-652-731 inhibited the labeling and this led to the suggestion that 52-kDa protein may represent a binding subunit of the PAF receptor complex. It is apparent from the preceding results that proteins of widely different molecular masses (e.g., 52, 160, 180, and 220 kDa) have been claimed to be a purified receptor. The molecular characteristics of these proteins remain to be established; progress has been slow. An important development toward this end is the recent cloning of the PAF receptor.







In the past few years it has become apparent that the interactions of PAF to its receptor activates several transmembrane signaling mechanisms. For example, PAF activates phospholipid turnover via phospholipase C, phospholipase A2, and phospholipase D. It also stimulates protein kinases, e.g., protein kinase C (PKC) and tyrosine kinase. G proteins have also been implicated in these responses. These developments are critically evaluated below.

I Outside I


The PAF receptor from guinea pig lung was recently cloned by functional expression (6). These investigators used a ligand-specific cloning strategy that involved gene expression in Xenopus laevis oocytes. A eDNA library was constructed from size-fractionated guinea pig lung poly(A)RNA that elicited an electrophysiological response to PAF when injected into the oocytes. A functional phage clone, Z-74, was converted to a plasmid Zp74. Transcript of Zp74 was shown to induce a PAF-dependent response in oocytes (6). The protein encoded by the insert of Zp74 (expressed in oocytes or cos-7) was identified as the PAF receptor on the basis of its pharmacological response. Lyso-PAF was ineffective, whereas PAF antagonists CV-6209 and Y-24180 inhibited the response. Membranes from the plasmid transfected cos-7 cells exhibited specific binding to [3H]WEB, a PAF receptor antagonist. These investigators identified the PAF-receptor mRNA and were able to observe its abundance in leukocytes and small amounts in spleen, lung, and kidney (6, 14) from various species including human (14). PAF receptor eDNA showed a 3020 nucleotide sequence. Based on the 342 amino acid sequence (deduced from the longest open reading frame of the cloned DNA), the molecular mass was calculated to be 38,982. Hydropathy analysis


showed seven hydrophobic putative transmembrane segments (Fig. 1) that have now become a characteristic feature of G protein-coupled receptors. When compared with other G protein receptors, several amino acids are highly conserved; for example: aspartic acid in the second transmembrane segment; one cysteine each in the second and third cxtracellular loops; and three proline in the sixth and seventh segment. The cytoplasmic tail of the PAF receptor contains four serines and five threonines. There are a total of 12 tyrosine residues including 2 in the cytoplasmic loops (Fig. 1). Several asparagine residues are present at the external surface of the receptor and could serve as sites for attachment of glycosylated residues. With this new development, the molecular interactions between PAF and the receptor, the mechanisms for desensitization, and the interactions between the receptor and transducers can be approached with ease in the future.




1. A diagrammatic representation of the PAF receptor. Seven transmembrane domains together with extracellular (outside) and intracellular (inside) segments of the receptor are schematically presented. The numbers indicate the location of the amino acids in the PAF receptor cloned from guinea pig lung (6). Figure

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PHOSPHOLIPID Phospholipase

TURNOVER C-mediated

Phospholipase turnover

PAF stimulates turnover of polyphosphoinositide (PPI) in a number of cells (15). Detailed investigations have indicated that, in general, within 5-10 s of PAF challenge the level of IP3 increases several fold with a concomitant increase in the level of diglyceride. This increase in IP3 appears to be independent of extracellular Ca2. Further, increases in 1P4 and 1P5 have also been observed. PAF stimulation of phospholipase C-mediated PPI turnover is a receptor-dependent process as several antagonists (e.g., CV-6209, SRI-63441, SRI 63-675, BN-52021) were shown to inhibit this process. A variety of agents/drugs have been used to examine their effect on PAF stimulation of PLC (15). Jne of the important functions of PPI turnover is that it causes generation of the second messenger, 1P3, which elicits an increase in intracellular Ca2. Several studies have documented that in the absence of extracellular Ca2, PAF caused an increase in intracellular Ca2, presumably via an IP3-mediated mechanism (16, 17; M. James-Kracke, R. Sexe, and S. D. Shukla, unpublished results). Of the total increase in cellular Ca2 caused by PAF, about one-fourth is due to the 1P3-induced mobilization of Ca2. Thus, the PAFinduced increase in cell Ca2 is predominantly by influx of Ca2 (15, 16). The mechanism for this influx is presently unknown. In this context, it is noteworthy that PAF has been proposed to stimulate Ca2 influx through receptor-operated channel in platelets that are not strictly selective to cation because Na and Li are also permeable (18). A Na-Ca2 exchange pathway is also activated by PAF (19). Obviously, the effect of PAF on membrane ion transport systems in relation to the PAF receptor awaits investigation. Phospholipase



In many cells and tissues, PAF causes production of arachidonic acid and its prostaglandin metabolites (20-23). This occurs via activation of PLA2, which deacylates fatty acid at the carbon-2 position of phospholipids. PAF receptor antagonists inhibit the responses and thus suggest a close relationship between the PAF receptor and the PLA2. The production of thromboxane A2 by PAF in liver has been considered to play a key role in the effects of PAF on liver hemodynamics (24). In another study, PAF activated phospholipase A2 in primed P388D1 macrophage-like cells. Priming was achieved by treatment of P388D1 cells with lipopolysaccharide. PAF caused a two- to threefold increase in PGE2 production in unprimed vs. 9- to 12-fold in primed cells. In primed cells, PAF stimulation of PGE2 production was inhibited by the PLA2 inhibitor manoalogue and the tyrosine kinase inhibitor, genistein, but not by the PKC inhibitor, H7 (25). These investigators observed that in primed cells the enzymatic activity of cyclooxygenase or PLA2 was not changed, and therefore the increases in PGE2 are not a result of change in these enzyme activities. Actinomycin D (a transcriptional inhibitor) and cycloheximide (a protein synthesis inhibitor) both substantially inhibited the PAF-stimulated increase in PGE2, indicating a role for expression of certain genes in this process. It was further established that this enhanced production of PGE2 is a PAF receptor-dependent event because the PAF antagonist L-695-989 blocked it. It was concluded that the activation of PLA2 by PAF in primed P388D1 cells is dependent on the PAF receptor transduction pathways and that protein tyrosine kinase may play a role.


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In addition to the above two pathways, recent observations have offered a role for PLD in the PAF signaling (26-28). One of the biochemical markers for this activity is the production of phosphatidylethanol in the presence of ethanol, a result of transphosphatidylation reaction of PLD. Phosphatidic acid (PA), a PLD-catalyzed product, may have a role in the cell and can also be metabolized by phosphatidic acid phosphohydrolase to generate diacylglycerol. This can activate PKC or be metabolized to free fatty acid (e.g., arachidonic acid). The relative contribution by PLC and PLD in the generation of diacylglycerol by PAF remains to be established. The preceding section provides support for PAF actions via PLC, PLA2, and PLD pathways and that one or more of these enzymes may be activated in a given cell. As a result of the activation of these pathways, several lipid mediators are generated and are involved in PAF responses.





Cloning studies with the PAF receptor indicated that the receptor shows homology to G protein-coupled receptors. Several other lines of evidence also support this view, as follows: 1) PAF stimulates GTPase activity (5, 29); 2) GTP causes a shift in the binding of PAF (5, 7); and 3) in many systems (but not all) pertussis toxin inhibits PAF-stimulated responses, e.g., phospholipid turnover. In this latter case, both toxin-sensitive and -insensitive responses have been documented (see citations in ref 15). Nevertheless, current trends in the field clearly favor the involvement of G proteins in PAF receptor function. A G protein that is distinct from G, or G has been suggested to be involved in PAF-mediated phosphoinositide metabolism in platelets (29). Recent advances indicate the presence of several high and low molecular weight GTP binding proteins in biological systems. With this information, the search for the G protein involved in PAF receptor function could be feasible in the future.




In PAF-responsive cells where phosphoinositide turnover occurs, the activation of PKC by DG is also observed. In a recent development it was observed that PAF also stimulated the tyrosine kinase as monitored by tyrosinephosphorylated proteins. In platelets, PAF caused tyrosine phosphorylation of several proteins including 50-, 60-, 71-, 82-, and 300-kDa proteins. This response was rapid and could be blocked by PAF receptor antagonists (30, 31). Further, when control and PAF-treated rabbit platelet samples (lysates) were fractionated using a phosphotyrosine monoclonal antibody agarose column, it was observed that PAF caused an increase in the immunoreactivity of 50- and 60-kDa proteins to the pp6Osrc antibody. Immunoprecipitation with pp6Orc confirmed that PAF caused an increase in phosphorylation of the 60- and 50-kDa protein (31). PAF also caused translocation of the pp6oc-src from cytosol to membrane fraction (31). These studies point to a role for pp6ocsc (a tyrosine kinase) in PAF signal transduction. In human neutrophils PAF stimulated tyrosine phosphorylation of several proteins (41, 54, 66, 104, and 116 kDa). Phosphorylation of 66-, 116-, and 104-kDa proteins was inhibited by pertussis toxin treatment whereas that of 41 kDa remained

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unchanged. This may indicate a role of toxin-sensitive G protein in the phosphorylation of some of these proteins. These studies have led to the conclusion that PAF directly or indirectly affects tyrosine kinase and/or phosphoryl tyrosine phosphatase and that two different sets of kinases and/or phosphatases may be involved (32). Platelet-activating factor activates and primes polymorphonuclear neutrophils and has been shown to cause translocation of PKC in neutrophils pretreated with cytochalasin B (33, 34). PAF caused a loss in the PMA binding sites and PKC activity in the soluble fraction and an increase in the particulate fraction, although the loss from soluble fraction was only partially recovered in membrane fraction (34). It was suggested that PKC may be down-regulated in the membranes after translocation. PAF can bypass intracellular [Ca2), to translocate protein kinase C. However, some Ca2 pool other than [Ca2] per se may regulate partitioning of the enzyme between the cytosol and membranes.





In many systems, responses to PAF are desensitized. This has also led to studies of the regulation of PAF receptor and its responses. PAF receptors are down-regulated as monitored by ligand binding. This can occur via internalization of the receptor (35). The presence of intracellular PAF receptor has been reported in rat cerebral cortex (36) and is therefore worthy of consideration in other systems too. In PAFdesensitized platelets, the binding of [3HIPAF is effected. It is also documented that, for example in PAF-treated platelets, several responses are desensitized, e.g., aggregation, PLC activation (PPI turnover), protein phosphorylation, and Ca2 mobilization (see ref 15). In neutrophils desensitization of granular secretion and Ca2 mobilization occur also (33, 34). In liver, PAF-stimulated glycogenolysis also exhibits this phenomenon (2). Thus, a homologous desensitization to PAF is common in a variety of cells and tissues. The mechanism for this remains to be established. With the recent cloning of the PAF receptor and our understanding of its structure, it will now be possible to dissect out the role of phosphorylation of the receptor in desensitization, as has been extensively studied with adrenergic receptors (37). As discussed previously, the importance of G proteins, PKC, or tyrosine kinase in the regulation of PAF receptor signal transduction mechanisms can also be specifically addressed. An interrelationship (or interrelationships) among these pathways is poorly understood. In this regard, a role for cyclic AMP also cannot be overlooked as PAF has been demonstrated to inhibit adenylate cyclase activity (38). Elevation of cellular cAMP inhibits PAF signaling responses, e.g., phosphoinositide turnover (15). PAF RECEPTOR EXPRESSION






Platelet-activating factor is the first phospholipid agonist for which the receptor has been cloned, and some information on the transmembrane signaling pathways is also available. There is ample evidence to raise the issue of heterogeneity or subtypes of the PAF receptor. PAF may also serve as a messenger inside the cells and could elicit its actions through interaction with intracellular receptors. Are PAF receptors on the plasma membrane different from intracellular receptors? It is increasingly being recognized that the PAF receptor activates multiple signaling pathways, and in a given cell several of these mechanisms may exist. Activation of phospholipid turnover (via PLC, or PLD or PLA2), protein kinase (e.g., PKC, tyrosine kinase), and Ca2 mobilization are important components of the PAF receptor system. Recent pharmacological evidence and the homology of the cloned PAF receptor to G protein-coupled receptors suggest that the G protein (or proteins) play a key role as transducers. The identity of the G protein remains unknown. A



One of the exciting facets in PAF research has emerged from the finding that this mediator induces expression of early response genes, e.g., c-fos, in human monocytes (39), neuroblastoma cells (40), A-431 cells (41), and lymphoblastoid cells (42). The inactive metabolite lyso-PAF does not induce this expression whereas PAF receptor antagonists blocked it. Thus, it is evident that PAF-induced expression of these genes is a PAF receptor-dependent phenomenon (see Fig. 2). In studies with neuroblastoma cells, the PKC activator, PMA, and PAF showed a synergistic effect on c-fos expres-


sion, suggesting the involvement of PKC (40). In A-431 cells, the PKC inhibitor, staurosporine, and a tyrosine kinase inhibitor, genistein, both inhibited PAF-stimulated c-fos gene expression (Y. B. Tripathi, and S. D. Shukia, unpublished results). This indicates a role for both PKC and tyrosine kinase. As pointed out previously in this article, PAF has been shown to stimulate tyrosine kinase. It therefore appears that PAF receptor-coupled signal transduction pathways may provide the signal for the gene expression. On the other hand, any role for the intracellular PAF receptor (or receptors) in the gene expression has yet to be ascertained. Its mechanism, therefore, remains undetermined (Fig. 2). Further, the identity of the gene product and how it may influence the PAF responses also remains to be studied.



Figure 2. An overview of the PAF receptor-coupled signal transduction pathways. The figure represents a simplified scheme of signaling pathways stimulated by PAE The dotted box indicates that the details of the interactions among various components are not known. AA, arachidonic acid; PG,, prostaglandins; PLA2, phospholipase A2; PLC, phospholipase C; PLD, phospholipase D.

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further interesting dimension is added by recent developments that suggest a role for tyrosine kinase in PAF signaling. Obviously, the relationship between tyrosine kinase and the G protein, both of which may mediate PAF actions, would be a topic of considerable interest. The PAF receptor has tyrosine residues at the cytoplasmic side (Fig. 1). Whether these residues are phosphorylated or not and whether the PAF receptor has tyrosine kinase activity is not known. If one assumes that PAF has no intrinsic tyrosine kinase activity, then it may indirectly stimulate tyrosine phosphorylation of proteins through an as yet unknown mechanism. Members of the src gene family (which are tyrosine kinases) could also have a role in these mechanisms. Regulation of the PAF receptor activity via protein phosphorylation (e.g., by PKC) is also worthy of consideration. In cells, PAF levels could be influenced by the presence of intracellular inhibitors which may also modulate PAF responses. With our recent knowledge of the structure of the PAF receptor and with the availability of novel molecular biological approaches, we expect to witness exciting developments in the near future. The regulation of the PAF receptor and of its signal transduction mechanism can now be addressed with precision at the molecular level. The author acknowledges the contribution of several investigators in this field whose work could not be cited because of the length and scope of this article. I am thankful to Professor Takao Shimizu for reviewing Fig. 1. Work from the author’s laboratory was supported by the National Institutes of Health grants #DK35170 and RCDA DK01782. Skillful typing by Judy Richey is gratefully acknowledged.

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Platelet-activating factor receptor and signal transduction mechanisms.

Platelet-activating factor (PAF) is the most potent phospholipid agonist known to date. Radioligand binding studies using [3H]PAF and structurally dif...
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