act.iv
Platelet-
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
receptor
signal
transduc-
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
2296
CHARACTERIZATION LIGAND BINDING
Columbia,
Mmun
OF PAF STUDIES
6212, USA RECEPTOR
BY
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
TO
ISOLATE
THE
PAF
RECEPTOR
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.
0892-6638/92/0006-2296/$O1.50.
© FASEB
www.fasebj.org by Univ Louisiana Dupre Library/Serials Dept (130.70.8.131) on January 07, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber
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.
CLONING AND STRUCTURE PAF RECEPTOR
OF
PAF
RECEPTOR
SIGNALING
PATHWAYS
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
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
PAF RECEPTOR SIGNAL TRANSDUCTION
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.
I
Inside
I
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
2297
www.fasebj.org by Univ Louisiana Dupre Library/Serials Dept (130.70.8.131) on January 07, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber
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
A2-mediated
turnover
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.
2298
Vol. 6
March
1992
D-mediated
turnover
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.
POSSIBLE
ROLE
OF
G PROTEINS
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.
INVOLVEMENT OF TYROSINE PROTEIN KINASE C
KINASE
AND
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
The FASEB Journal
SH UKLA
www.fasebj.org by Univ Louisiana Dupre Library/Serials Dept (130.70.8.131) on January 07, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber
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.
REGULATION
OF
PAF
SIGNALING
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
SIGNALING
AND
GENE
FUTURE
PERSPECTIVES
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
Protein
t
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-
PAF RECEPTOR SIGNAL
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.
TRANSDUCTION
Translation
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.
2299
www.fasebj.org by Univ Louisiana Dupre Library/Serials Dept (130.70.8.131) on January 07, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber
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.
REFERENCES 1. Snyder, F. (1986) Chemical and biochemical aspects of platelet activating factor: a novel class of acylated ether-linked choline phospholipids. Med. Res. Rev. 5, 107-140 2. Hanahan, D. J. (1986) Platelet activating factor: a biologically active phosphoglyceride. Annu. Rev. Biochem. 55, 483-509 3. Braquet, P. L., Touqui, L., Shen, T. Y., and Vargaftig, B. B. (1987) Perspectives in platelet activating factor research. PharmacoL Rev. 39, 97-145 4. Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1990) Platelet-activating factor. J. BioL Chem. 265, 17381-17384 5. Hwang, S. (1990) Specific receptors of platelet-activating factor, receptor heterogeneity, and signal transduction mechanisms. j Lipid Mediators 2, 123-158 6. Honda, Z., Nakamura, M., Miki, I., Minami, M., Watanabe, T., Seyama, Y., Okado, H., Toh, H., Ito, K., Miyamoto, T., and Shimizu, T. (1991) Cloning functional expression of platelet activating factor receptor from guinea-pig lung. Nature (London) 349,
342-346
7. Hwang, S., Lam, M. H., and Pong, S. S. (1986) Ionic and GTP regulation of binding of platelet activating factor induced activation of GTPase in rabbit platelet membranes. J. Biol. Chem. 261, 532-537 8. Pinckard, R. N., Ludwig, J. C., and McManus, L. M. (1988) Platelet activating factor. In Inflammation: Basic Principles and Clinical Correlates (Gallins, J. I., Goldstein, I. M., and Snyderman, R., eds), pp. 139-167, Raven, New York 9. Smiley, P. L., Stremler, K. E., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1991) Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet activating factor. J. BioL Chem. 266, 11104-11110
10. Valone, F. H. (1984) Isolation of a platelet membrane protein which binds the platelet activating factor. Immunology 52, 169-174
11. Nishihara,J., Purification
2300
Vol. 6
Ishibashi, T., Imai, Y., and Muramatsu, T (1985) and characterization of the specific binding protein
March
1992
for PAF from human platelets. Toholcuj Exp. Med. 147, 145-152 12. Chau, L. Y., and Jii, Y. J. (1988) Characterization of [3H]labelled platelet-activating factor receptor complex solubilized for rabbit platelet membranes. Biochim. Biophys. Ada 970, 103-112
13. Chau, L., Tsai, Y., and Cheng, J. (1989) Photoaffinity labelling of PAF binding sites in rabbit platelet membranes. Biochem. Biophys. Res. Commun. 161, 1070-1076 14. Nakamura, M., Honda, Z., Izumi, T, Sakanaka, C., Mutoh, H., Minami, M., Bito, H., Seyama, Y., Matsumoto, T, Noma, M., and Shimizu, T. (1991) Molecular cloning and expression of platelet activating factor receptor from human leukocytes. j BioL Chem. 266, 20400-20405 15. Shukla, S. D. (1991) Inositol phospholipid turnover in PAF transmembrane signalling. Lipids 26, 1028-1033 16. Hallam, T. J., Sanchez, A., and Rink, T. J. (1984) Stimulus response coupling in platelets: changes evoked by PAF in cytoplasmic free calcium monitored with the fluorescent calcium indicator quin 2. Biochem. j 218, 819-827 17. Yue, T. L., Gleason, M. M., Hallenbeck, J., and Feurstein, G. (1991) Characterization of PAF induced elevation of cytosolic free-calcium level in neurohybrid NCB-20 cells. Neuroscience 41, 177-185 18. Avdonin, P. V., Cheglakov, I. B., and Tkachuk, V. A. (1991) Stimulation of non-selective cation channels providing Ca2 influx into platelets by platelet activating factor and other aggregation inducers. Eur. J. Biochein. 198, 267-273 19. Kester, M., Kumar, R., and Hanahan, D. J. (1986) Alkylacetylglycerophosphocholine stimulates Na-Ca2 exchange, protein phosphorylation and polyphosphoinositide turnover in rat ileal plasmalemmal vesicles. Biochim. Biophys. Ada 888, 306-315 20. Okayasu, T., Hasegawa, K., and Ishibashi, T. (1987) PAF stimulates metabolism of phosphoinositide via phospholipase A2 in primary cultured rat hepatocytes. j Lipid Res. 28, 760-767 21. Kawaguchi, H., and Yasuda, H. (1986) PAF stimulates prostaglandin synthesis in cultured cells. Hypertension 8, 192-197 22. Gandhi, C. R., Hanahan, D. J., and Olson, M. 5. (1990) Two distinct pathways of platelet activating factor induced hydrolysis of phosphoinositides in primary cultures of rat Kupfor cells. J. BioL Chem. 265, 18234-18241 23. Birkle, D. L., Kurian, P., Braquet, P., and Bazan, N. G. (1988) Platelet activating factor antagonist BN52021 decreases accumulation of free polyunsaturated fatty acid in mouse brain during ischemia and electroconvulsive shock. j Neurochem. 51, 1900-1905 24. Garcia-Sainz, J. A. (1989) Intracellular communication within the liver has clinical implications. Trends Pharmacol. Sci. 10, 10-11 25. Glaser, K. B., Asmis, R., and Dennis, E. A. (1990) Bacterial lipopolysaccharide priming of P338D, macrophage-like cells for enhanced arachidonic acid metabolism: PAF receptor activation and regulation of phospholipase A2. J. Biol. Chem. 265, 8658-8664 26. Shukla, S. D., and Halenda, S. P. (1991) Phospholipase D in cell signalling and its relationship to phospholipase C. Life Sci. 48, 851-866 27. Kanaho, Y., Kanoh, H., Saitoh, K., and Nozawa, Y. (1991) Phospholipase D activation by platelet-activating factor, leukotriene B4, and FMLP in rabbit neutrophils: phospholipase D activation is involved in enzyme release. j ImmunoL 146, 3536-3541 28. Uhing, R. J., Prpic, V., Hollenback, P. W., and Adams, D. 0. (1989) Involvement of protein kinase C in platelet activating factor stimulated diacylglycerol accumulation in murine peritoneal macrophages. J. BioL Chem. 264, 9224-9230 29. Houslay, M. D., Bojanic, D., and Wilson, A. (1986) Platelet activating factor and U44069 stimulate a GTPase activity in human platelets which is distinct from the guanine nucleotide regulatory proteins, N, and N1. Biochem. J. 234, 737-740 30. Dhar, A., Paul, A. K., and Shukla, S. D. (1990) Platelet activating factor stimulation of tyrosine kinase and its relationship to phospholipase C: studies with genistein and monoclonal antibody to phosphotyrosine. MoL Pharmacol. 37, 519-525 31. Dhar, A.. and Shukla, S. D. (1991) Involvement of pp6OcS in
The FASEB Journal
SHUKLA
www.fasebj.org by Univ Louisiana Dupre Library/Serials Dept (130.70.8.131) on January 07, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber
platelet activating factor stimulated platelets: evidence for translocation from cytosol to membrane.j Biol. Chem. 266, 18797-18801 32. Gomez-Cambronero, J., Wang, E., Johnson, G., Huang, C., and
Shaafi,
R. I. (1990) Platelet in human
sine phosphorylation 6240-6245
activating neutrophils.
factor induces tyroj Biol. Chem. 266,
33. O’Flaherty, J. T., and Nishihara, J. (1987) Arachidonate metabolites, platelet activating factor, and the mobilization of protein kinase C in human polymorphonuclear neutrophils. j ImmunoL 138, 1889-1895 34. O’Flaherty, J. T., Redman, J. F., Jacobson, D. P., and Rossi, A. G. (1990) Stimulation and priming of protein kinase C translocation by a Ca2 transient-independent mechanism: studies in human neutrophils challenged with platelet-activating factor and other receptor agonists. j Biol. Chem. 265, 21619-21623 35. Homma, H., Tokumura, A., and Hanahan, D. J. (1987) Binding and internalization of platelet activating factor 1-O-alkyl2-acetyl-sn-glycero-3-phosphocholine in washed rabbit platelets. j Biol. C/tern. 262, 10582-10587 36. Marcheselli, V. L., Rossowska, M. J., Domingo, M., Braquet, P., and Bazan, N. G. (1990) Distinct platelet activating factor binding sites in synaptic endings and in intracellular membranes of rat cerebral cortex. j Biol. Chem. 265, 9140-9145
PAF RECEPTOR SIGNAL
TRANSDUCTION
37. Hausdorif, W. P., Caron, M. G., and Lefkowitz, R. J. (1990) Turning off the signal: desensitization of 13-adrenergic receptor function. FASEB J. 4, 2881-2889 38. Haslam, R. J., and Vanderwel, M. (1982) Inhibition of platelet adenylate cyclase by 1-O-alkyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (platelet activating factor). j Biol. C/tern. 257, 6879-6885 39. Ho, Y., Lee, W. M. F., and Snyderman, R. (1987) Chemoattractant-induced activation of c-fos gene expression in human monocytes. j Exp. Med. 165, 1524-1538 40. Squinto, S. P., Block, A. L., Braquet, P., and Bazan, N. G. (1989) Platelet-activating factor stimulates a FOS/JUN/AP-l transcriptional
signaling
system
in human
neuroblastoma
cells.
Neumsci. Res. 24, 558-566 41. Tripathi, Y. B., Kandala, J., Guntaka, j
R. V., Lim, R. W., and Shukla, S. D. (1991) Platelet-activating factor induces expression of early response genes c-fos and T15-1 in human epidermoid carcinoma A-431 cells. Lfe Sci. 49, 1761-1767
42. Mazer, B., Doinenico, J., Sawami, H., and Gelfand, E. W. (1991) Platelet-activating factor induces an increase in intracellular calcium and expression of regulatory genes in human B lymphoblastoid cells. j ImmunoL 146, 1914-1920
2301
www.fasebj.org by Univ Louisiana Dupre Library/Serials Dept (130.70.8.131) on January 07, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber
Platelet-
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
receptor
signal
transduc-
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
2296
CHARACTERIZATION LIGAND BINDING
Columbia,
Mmun
OF PAF STUDIES
6212, USA RECEPTOR
BY
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
TO
ISOLATE
THE
PAF
RECEPTOR
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.
0892-6638/92/0006-2296/$O1.50.
© FASEB
www.fasebj.org by Univ Louisiana Dupre Library/Serials Dept (130.70.8.131) on January 07, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber
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.
CLONING AND STRUCTURE PAF RECEPTOR
OF
PAF
RECEPTOR
SIGNALING
PATHWAYS
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
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
PAF RECEPTOR SIGNAL TRANSDUCTION
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.
I
Inside
I
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
2297
www.fasebj.org by Univ Louisiana Dupre Library/Serials Dept (130.70.8.131) on January 07, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber
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
A2-mediated
turnover
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.
2298
Vol. 6
March
1992
D-mediated
turnover
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.
POSSIBLE
ROLE
OF
G PROTEINS
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.
INVOLVEMENT OF TYROSINE PROTEIN KINASE C
KINASE
AND
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
The FASEB Journal
SH UKLA
www.fasebj.org by Univ Louisiana Dupre Library/Serials Dept (130.70.8.131) on January 07, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber
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.
REGULATION
OF
PAF
SIGNALING
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
SIGNALING
AND
GENE
FUTURE
PERSPECTIVES
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
Protein
t
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-
PAF RECEPTOR SIGNAL
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.
TRANSDUCTION
Translation
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.
2299
www.fasebj.org by Univ Louisiana Dupre Library/Serials Dept (130.70.8.131) on January 07, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber
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.
REFERENCES 1. Snyder, F. (1986) Chemical and biochemical aspects of platelet activating factor: a novel class of acylated ether-linked choline phospholipids. Med. Res. Rev. 5, 107-140 2. Hanahan, D. J. (1986) Platelet activating factor: a biologically active phosphoglyceride. Annu. Rev. Biochem. 55, 483-509 3. Braquet, P. L., Touqui, L., Shen, T. Y., and Vargaftig, B. B. (1987) Perspectives in platelet activating factor research. PharmacoL Rev. 39, 97-145 4. Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1990) Platelet-activating factor. J. BioL Chem. 265, 17381-17384 5. Hwang, S. (1990) Specific receptors of platelet-activating factor, receptor heterogeneity, and signal transduction mechanisms. j Lipid Mediators 2, 123-158 6. Honda, Z., Nakamura, M., Miki, I., Minami, M., Watanabe, T., Seyama, Y., Okado, H., Toh, H., Ito, K., Miyamoto, T., and Shimizu, T. (1991) Cloning functional expression of platelet activating factor receptor from guinea-pig lung. Nature (London) 349,
342-346
7. Hwang, S., Lam, M. H., and Pong, S. S. (1986) Ionic and GTP regulation of binding of platelet activating factor induced activation of GTPase in rabbit platelet membranes. J. Biol. Chem. 261, 532-537 8. Pinckard, R. N., Ludwig, J. C., and McManus, L. M. (1988) Platelet activating factor. In Inflammation: Basic Principles and Clinical Correlates (Gallins, J. I., Goldstein, I. M., and Snyderman, R., eds), pp. 139-167, Raven, New York 9. Smiley, P. L., Stremler, K. E., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1991) Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet activating factor. J. BioL Chem. 266, 11104-11110
10. Valone, F. H. (1984) Isolation of a platelet membrane protein which binds the platelet activating factor. Immunology 52, 169-174
11. Nishihara,J., Purification
2300
Vol. 6
Ishibashi, T., Imai, Y., and Muramatsu, T (1985) and characterization of the specific binding protein
March
1992
for PAF from human platelets. Toholcuj Exp. Med. 147, 145-152 12. Chau, L. Y., and Jii, Y. J. (1988) Characterization of [3H]labelled platelet-activating factor receptor complex solubilized for rabbit platelet membranes. Biochim. Biophys. Ada 970, 103-112
13. Chau, L., Tsai, Y., and Cheng, J. (1989) Photoaffinity labelling of PAF binding sites in rabbit platelet membranes. Biochem. Biophys. Res. Commun. 161, 1070-1076 14. Nakamura, M., Honda, Z., Izumi, T, Sakanaka, C., Mutoh, H., Minami, M., Bito, H., Seyama, Y., Matsumoto, T, Noma, M., and Shimizu, T. (1991) Molecular cloning and expression of platelet activating factor receptor from human leukocytes. j BioL Chem. 266, 20400-20405 15. Shukla, S. D. (1991) Inositol phospholipid turnover in PAF transmembrane signalling. Lipids 26, 1028-1033 16. Hallam, T. J., Sanchez, A., and Rink, T. J. (1984) Stimulus response coupling in platelets: changes evoked by PAF in cytoplasmic free calcium monitored with the fluorescent calcium indicator quin 2. Biochem. j 218, 819-827 17. Yue, T. L., Gleason, M. M., Hallenbeck, J., and Feurstein, G. (1991) Characterization of PAF induced elevation of cytosolic free-calcium level in neurohybrid NCB-20 cells. Neuroscience 41, 177-185 18. Avdonin, P. V., Cheglakov, I. B., and Tkachuk, V. A. (1991) Stimulation of non-selective cation channels providing Ca2 influx into platelets by platelet activating factor and other aggregation inducers. Eur. J. Biochein. 198, 267-273 19. Kester, M., Kumar, R., and Hanahan, D. J. (1986) Alkylacetylglycerophosphocholine stimulates Na-Ca2 exchange, protein phosphorylation and polyphosphoinositide turnover in rat ileal plasmalemmal vesicles. Biochim. Biophys. Ada 888, 306-315 20. Okayasu, T., Hasegawa, K., and Ishibashi, T. (1987) PAF stimulates metabolism of phosphoinositide via phospholipase A2 in primary cultured rat hepatocytes. j Lipid Res. 28, 760-767 21. Kawaguchi, H., and Yasuda, H. (1986) PAF stimulates prostaglandin synthesis in cultured cells. Hypertension 8, 192-197 22. Gandhi, C. R., Hanahan, D. J., and Olson, M. 5. (1990) Two distinct pathways of platelet activating factor induced hydrolysis of phosphoinositides in primary cultures of rat Kupfor cells. J. BioL Chem. 265, 18234-18241 23. Birkle, D. L., Kurian, P., Braquet, P., and Bazan, N. G. (1988) Platelet activating factor antagonist BN52021 decreases accumulation of free polyunsaturated fatty acid in mouse brain during ischemia and electroconvulsive shock. j Neurochem. 51, 1900-1905 24. Garcia-Sainz, J. A. (1989) Intracellular communication within the liver has clinical implications. Trends Pharmacol. Sci. 10, 10-11 25. Glaser, K. B., Asmis, R., and Dennis, E. A. (1990) Bacterial lipopolysaccharide priming of P338D, macrophage-like cells for enhanced arachidonic acid metabolism: PAF receptor activation and regulation of phospholipase A2. J. Biol. Chem. 265, 8658-8664 26. Shukla, S. D., and Halenda, S. P. (1991) Phospholipase D in cell signalling and its relationship to phospholipase C. Life Sci. 48, 851-866 27. Kanaho, Y., Kanoh, H., Saitoh, K., and Nozawa, Y. (1991) Phospholipase D activation by platelet-activating factor, leukotriene B4, and FMLP in rabbit neutrophils: phospholipase D activation is involved in enzyme release. j ImmunoL 146, 3536-3541 28. Uhing, R. J., Prpic, V., Hollenback, P. W., and Adams, D. 0. (1989) Involvement of protein kinase C in platelet activating factor stimulated diacylglycerol accumulation in murine peritoneal macrophages. J. BioL Chem. 264, 9224-9230 29. Houslay, M. D., Bojanic, D., and Wilson, A. (1986) Platelet activating factor and U44069 stimulate a GTPase activity in human platelets which is distinct from the guanine nucleotide regulatory proteins, N, and N1. Biochem. J. 234, 737-740 30. Dhar, A., Paul, A. K., and Shukla, S. D. (1990) Platelet activating factor stimulation of tyrosine kinase and its relationship to phospholipase C: studies with genistein and monoclonal antibody to phosphotyrosine. MoL Pharmacol. 37, 519-525 31. Dhar, A.. and Shukla, S. D. (1991) Involvement of pp6OcS in
The FASEB Journal
SHUKLA
www.fasebj.org by Univ Louisiana Dupre Library/Serials Dept (130.70.8.131) on January 07, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber
platelet activating factor stimulated platelets: evidence for translocation from cytosol to membrane.j Biol. Chem. 266, 18797-18801 32. Gomez-Cambronero, J., Wang, E., Johnson, G., Huang, C., and
Shaafi,
R. I. (1990) Platelet in human
sine phosphorylation 6240-6245
activating neutrophils.
factor induces tyroj Biol. Chem. 266,
33. O’Flaherty, J. T., and Nishihara, J. (1987) Arachidonate metabolites, platelet activating factor, and the mobilization of protein kinase C in human polymorphonuclear neutrophils. j ImmunoL 138, 1889-1895 34. O’Flaherty, J. T., Redman, J. F., Jacobson, D. P., and Rossi, A. G. (1990) Stimulation and priming of protein kinase C translocation by a Ca2 transient-independent mechanism: studies in human neutrophils challenged with platelet-activating factor and other receptor agonists. j Biol. Chem. 265, 21619-21623 35. Homma, H., Tokumura, A., and Hanahan, D. J. (1987) Binding and internalization of platelet activating factor 1-O-alkyl2-acetyl-sn-glycero-3-phosphocholine in washed rabbit platelets. j Biol. C/tern. 262, 10582-10587 36. Marcheselli, V. L., Rossowska, M. J., Domingo, M., Braquet, P., and Bazan, N. G. (1990) Distinct platelet activating factor binding sites in synaptic endings and in intracellular membranes of rat cerebral cortex. j Biol. Chem. 265, 9140-9145
PAF RECEPTOR SIGNAL
TRANSDUCTION
37. Hausdorif, W. P., Caron, M. G., and Lefkowitz, R. J. (1990) Turning off the signal: desensitization of 13-adrenergic receptor function. FASEB J. 4, 2881-2889 38. Haslam, R. J., and Vanderwel, M. (1982) Inhibition of platelet adenylate cyclase by 1-O-alkyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (platelet activating factor). j Biol. C/tern. 257, 6879-6885 39. Ho, Y., Lee, W. M. F., and Snyderman, R. (1987) Chemoattractant-induced activation of c-fos gene expression in human monocytes. j Exp. Med. 165, 1524-1538 40. Squinto, S. P., Block, A. L., Braquet, P., and Bazan, N. G. (1989) Platelet-activating factor stimulates a FOS/JUN/AP-l transcriptional
signaling
system
in human
neuroblastoma
cells.
Neumsci. Res. 24, 558-566 41. Tripathi, Y. B., Kandala, J., Guntaka, j
R. V., Lim, R. W., and Shukla, S. D. (1991) Platelet-activating factor induces expression of early response genes c-fos and T15-1 in human epidermoid carcinoma A-431 cells. Lfe Sci. 49, 1761-1767
42. Mazer, B., Doinenico, J., Sawami, H., and Gelfand, E. W. (1991) Platelet-activating factor induces an increase in intracellular calcium and expression of regulatory genes in human B lymphoblastoid cells. j ImmunoL 146, 1914-1920
2301
www.fasebj.org by Univ Louisiana Dupre Library/Serials Dept (130.70.8.131) on January 07, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber
