THE 3RD INTRACELLULAR DOMAIN OF THE PLATELET-ACTIVATING-FACTOR RECEPTOR IS A CRITICAL DETERMINANT IN RECEPTOR COUPLING TO PHOSPHOINOSITIDE PHOSPHOLIPASE-C ACTIVATING G-PROTEINS - STUDIES USING INTRACELLULAR DOMAIN MINIGENES AND RECEPTOR CHIMERAS

Platelet activating factor (PAF) is a potent phospholipid mediator whi ch elicits a diverse array of biological actions by interacting with G protein-coupled PAF receptors (PAFR). Binding of PAF to PAFRs leads t o activation of G protein(s) that stimulate phosphoinositide phospholi pase C and subsequent intracellular signaling responses. To identify t he potential role of intracellular domains of the rat PAFR (rPAFR) in signaling, we examined effects of transfecting minigenes encompassing rPAFR intracellular domains 1 (1i), 2 (2i), and 3 (3i) on inositol pho sphate (IP) production mediated by the cotransfected rPAFR cDNA. Altho ugh transfection of the rPAFR1i and rPAFR2i minigenes had no effects o n PAF-stimulated signaling, transfection of the rPAFR3i minigene inhib ited PAF-stimulated IP production by approximately 50% compared to con trols. The rPAFR3i domain did not inhibit IP production mediated by th e multifunctional rat pituitary adenylate cyclase-activating polypepti de receptor (rPACAPR), demonstrating the specificity of the competitio n by the rPAFR3i domain, In further experiments, the rPAFR3i domain wa s engineered onto the homologous domain of a monofunctional transmembr ane variant of the rPACAPR (rPACAPR(2)) that activates only adenylyl c yclase. The rPACAPR(2)/rPAFR3i chimera responded to PACAP with increas es in IP production which were attenuated nearly completely in cells c otransfected with the rPAFR3i domain. In contrast, PACAP had no effect s on IP production in a receptor chimera expressing a mutated form of the rPAFR3i domain (rPACAPR(2)/rPAFR3i(mut)). These results demonstrat e the ability of the rPAFR3i domain to confer a phospholipase C-signal ing phenotype to a receptor deficient in this activity and show that t his activity is specific for the engineered rPAFR3i domain. These resu lts suggest that the third intracellular loop of the rPAFR is a primar y determinant in its coupling to phosphoinositide phospholipase C-acti vating G proteins, providing the first insight into the molecular basi s of interaction of PAFRs with signal transducing G proteins.