Vol. 180, No. 1, 1991 October 15, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages ]05-]]] CHARACTERIZATION OF A HUMAN eDNA THAT ENCODES A FUNCTIONAL RECEPTOR FOR PLATELET ACTIVATING FACTOR Richard D. Ye*, Eric R. Prossnitz, Aihua Zou, and Charles G. Cochrane Department of Immunology, The Scripps Research Institute, La/olla, CA 92037 Received August 28, 1991 SUMMARY: We have cloned a cDNA for the platelet activating factor (PAF) receptor by screening an HL-60 granulocyte eDNA library with degenerate oligonueleotide probes based on conserved sequences of rhodopsin-type receptors. The 342-amino acid receptor contains 7 putative transmembrane domains, but lacks sites for N-linked glycosylation at the N-terminus. Stably transfected fibroblasts expressing the cloned eDNA responded to sub-nanomolar PAF stimulation with calcium mobilization, which could be inhibited by the PAF antagonist L- 659,989. The PAF receptor message was detected as a single species of approximately 4 kb in human placenta, lung, and differentiated HL-60 granulocytes. Expression of the cloned eDNA in undifferentiated HL-60 cells devoid of endogenous PAF receptor resulted in specific and saturable binding of the PAF antagonist WEB 2086 with a dissociation constant of 30.7 nM. © 1991 Academic Press, Tnc. Platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a lipid mediator that possesses diverse and potent effects on a variety of cells and tissues. PAF was originally identified as a soluble substance released from IgE-stimulated basophils that could aggregate platelets (1-3). Its cellular effects have since been found to involve the activation of neutrophils, protein phosphorylation, glycogenolysis, induction of expression of regulatory genes, and activation of arachidonic acid and phosphoinositide metabolites (reviewed in Ref. 4,5). PAF actions are mediated by specific receptors on the plasma membrane of target cells. These receptors are high-affinity binding sites for PAF, with dissociation constants in the nanomolar or sub-nanomolar range (chapters 6 and 8 in Ref. 5). Binding of PAF to the receptor correlates with its action, as both can be inhibited by specific PAF receptor antagonists. PAF was found to stimulate GTPase activity in platelets and neutrophils (6,7), and GTP specifically inhibits PAF binding to platelet and neutrophil membranes (8). These findings suggest that the PAF receptor is coupled to guanine nueleotide regulatory proteins (G proteins). *To whom correspondence should be addressed. Abbreviations: PAF, platelet activating factor; G proteins, guanine nucleotide regulatory proteins. 105 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.