FACTORS DETERMINING SPECIFICITY OF SIGNAL-TRANSDUCTION BY G-PROTEIN-COUPLED RECEPTORS - REGULATION OF SIGNAL TRANSFER FROM RECEPTOR TO G-PROTEIN

Among subfamilies of G-protein-coupled receptors, agonists initiate se veral cell signaling events depending on the receptor subtype (R) and the type of G-protein (G) or effector molecule (E) expressed in a part icular cell, Determinants of signaling specificity/efficiency may oper ate at the R-G interface, where events are influenced by cell architec ture or accessory proteins found in the receptor's microenvironment, T his issue was addressed by characterizing signal transfer from R to G following stable expression of the alpha(2A/D) adrenergic receptor in two different membrane environments (NIH-3T3 fibroblasts and the pheoc hromocytoma cell line, PC-12), Receptor coupling to endogenous Gr-prot eins in both cell types was eliminated by pertussis toxin pretreatment and R-G signal transfer restored by reconstitution of cell membranes with purified brain G-protein, Thus, the receptor has access to the sa me population of G-proteins in the two different environments, In this signal restoration assay, agonist-induced activation of G was 3-9-fol d greater in PC-12 as compared with NIH-3T3 alpha(2)-adrenergic recept or transfectants. The cell-specific differences in signal transfer wer e observed over a range of receptor densities or G-protein concentrati on, The augmented signal transfer in PC-12 versus NIH-3T3 transfectant s occurred despite a BS-fold lower level of receptors existing in the R-G-coupled state (high affinity, guanyl-5'-yl imidodiphosphate-sensit ive agonist binding), suggesting the existence of other membrane facto rs that influence the nucleotide binding behavior of G-protein in the two cell types, Detergent extraction of PC-12 but not NIH-3T3 membrane s yielded a heat-sensitive, macromolecular entity that increased S-35- labeled guanosine 5'-O-(thiotriphosphate) binding to brain G-protein i n a concentration-dependent manner. These data indicate that the trans fer of signal from R to G is regulated by a cell type-specific, membra ne-associated protein that enhances the agonist-induced activation of G.