REGULATION OF STABLY TRANSFECTED PLATELET-ACTIVATING-FACTOR RECEPTOR IN RBL-2H3 CELLS - ROLE OF MULTIPLE G-PROTEINS AND RECEPTOR PHOSPHORYLATION

Platelet activating factor (PAF) interacts with cell surface receptors to mediate inflammatory responses. To determine the mechanisms of PAF receptor regulation, we constructed epitope-tagged human PAF receptor cDNA (ET-PAFR) and generated stable transfectants in a rat basophilic cell line (RBL-2H3 cells). The expressed receptors displayed ligand b inding and functional properties similar to the native receptors in ne utrophils. PAF-stimulated intracellular Ca2+ mobilization was not inhi bited by pertussis toxin (PTx), whereas phosphoinositide hydrolysis an d secretion were blocked by similar to 40%. The PTx-resistant secretio n mediated by PAF was, however, inhibited by guanosine 5'-O-(2-thiodip hosphate) in permeabilized RBL-2H3 cells, indicating a role for PTx-in sensitive G protein. In contrast to the PAF receptor, responses mediat ed by formylpeptide and C5a chemoattractants were inhibited by PTx. PA F stimulated a dose- and time dependent phosphorylation of its recepto r. ET-PAFR was also phosphorylated by phorbol la-myristate 13-acetate (PMA) and dibutyryl cyclic AMP. Staurosporine caused complete inhibiti on of ET-PAFR phosphorylation by PMA but only partial inhibition by PA F Receptor phosphorylation by PAF and PMA correlated with desensitizat ion as measured by a decrease in both PAF-stimulated GTPase activity i n membranes and Ca2+ mobilization in intact cells. Phosphorylation of ET-PAFR by dibutyryl cyclic AMP was not, however, associated with dese nsitization. These data demonstrate that a single PAF receptor populat ion interacts with multiple G proteins to mediate its biological respo nses. Moreover, ET-PAFR, unlike the formylpeptide or C5a receptors, is phosphorylated by at least three kinases (most likely protein kinases A and C and a receptor kinase). The functional consequences of cellul ar activation by various chemoattractants may depend upon the G protei n to which their receptor is coupled.