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Activation and Regulation of Platelet-Activating Factor Receptor: Role of G_i and G_q in Receptor-Mediated Chemotactic, Cytotoxic, and Cross-Regulatory Signals¹

Stephan L. Brown, Venkatakrishna R. Jala, Sandeep K. Raghuwanshi^*, Mohd W. Nasser^*, Bodduluri Haribabu and Ricardo M. Richardson^2,*

^* Julius L. Chambers Biomedical/Biotechnology Research Institute and Department of Biology, North Carolina Central University, Durham, NC 27707; Department of Biochemistry, Meharry Medical College, Nashville, TN 37208; and James Graham Brown Cancer Center and Department of Microbiology and Immunology, University of Louisville Health Sciences Center, Louisville, KY 40202

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycerolphosphocholine; PAF) induces leukocyte accumulation and activation at sites of inflammation via the activation of a specific cell surface receptor (PAFR). PAFR couples to both pertussis toxin-sensitive and pertussis toxin-insensitive G proteins to activate leukocytes. To define the role(s) of G_i and G_q in PAF-induced leukocyte responses, two G-protein-linked receptors were generated by fusing G_i3 (PAFR-G_i3) or G_q (PAFR-G_q) at the C terminus of PAFR. Rat basophilic leukemia cell line (RBL-2H3) stably expressing wild-type PAFR, PAFR-G_i3, or PAFR-G_q was generated and characterized. All receptor variants bound PAF with similar affinities to mediate G-protein activation, intracellular Ca²⁺ mobilization, phosphoinositide (PI) hydrolysis, and secretion of -hexosaminidase. PAFR-G_i3 and PAFR-G_q mediated greater GTPase activity in isolated membranes than PAFR but lower PI hydrolysis and secretion in whole cells. PAFR and PAFR-G_i3, but not PAFR-G_q, mediated chemotaxis to PAF. All three receptors underwent phosphorylation and desensitization upon exposure to PAF but only PAFR translocated arrestin to the cell membrane and internalized. In RBL-2H3 cells coexpressing the PAFRs along with CXCR1, IL-8 (CXCL8) cross-desensitized Ca²⁺ mobilization to PAF by all the receptors but only PAFR-G_i3 activation cross-inhibited the response of CXCR1 to CXCL8. Altogether, the data indicate that G_i exclusively mediates chemotactic and cross-regulatory signals of the PAFR, but both G_i and G_q activate PI hydrolysis and exocytosis by this receptor. Because chemotaxis and cross-desensitization are exclusively mediated by G_i, the data suggest that differential activation of both G_i and G_q by PAFR likely mediate specific as well as redundant signaling pathways.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants AI-38910 (to R.M.R.) and AI-52381 (to B.H.).

² Address correspondence and reprint requests to Dr. Ricardo M. Richardson, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707. E-mail address: mrrichardson{at}nccu.edu

³ Abbreviations used in this paper: PAF, platelet-activating factor; GPCR, G protein-coupled receptor; PLC, phospholipase C; Ptx, pertussis toxin; PI, phosphoinositide; FR, formylpeptide receptor; HA, hemagglutinin; arr, arrestin; IP, inositol phosphate.

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