The overall goal of the present proposal is to delineate the structure/function regulation of PAF receptors. Stable transfectants expressing epitope-tagged PAF receptors in RBL-2H3 cells will be utilized throughout this study. First, the G proteins physically associated with PAF receptors will be identified and the functional consequence of this interaction will be characterized. The mechanisms regulating G protein switching will be elucidated, including the role of receptor phosphorylation. These studies will utilize both genetic and immunological techniques. Second, the structural determinants on the PAF receptor which interact with different G proteins will be identified. Third, whether receptor phosphorylation is a mechanism by which PAF receptors are desensitized will be tested. To do this, PAF receptor mutants laking phosphorylation sites for protein kinases will be constructed. Stable transfectants expressing mutated and wild-type epitope- tagged PAF receptors will be generated in RBL-2H3 cells. The relationship between receptor phosphorylation and desensitization will be evaluated. Fourth, the dynamics of PAF receptor movement following stimulation will be determined utilizing, fluorescent microscopy, flow cytometric analysis and binding of the PAF receptor antagonist [3H]WEB-2086 to intact cells membranes. Thus, the proposed studies will provide new information pertinent to structure/function regulation of G protein coupled receptors in general. In addition, elucidation of signal transduction pathways activated by PAF and understanding the mechanisms of its regulation at the molecular level will provide a better rationale for the development of therapy directed against PAF-mediated allergic, inflammatory and cardiovascular disorders.
