Numerous cell surface receptors transduce signals through heterotrimeric GTP binding proteins (G proteins). The alpha subunit of these proteins is a molecular switch, cycling between GDP-bound (inactive) and GTP-bound (active) forms. The purpose of this study is to characterize the intracellular regulation of G-protein-mediated signal transduction. GTPase activity of the alpha subunit is enhanced by a novel family of regulators of G protein signaling (RGS proteins), resulting in inhibition of Gi and Gq-coupled signaling. This project studies specifically the interaction between RGS proteins and G proteins and the resultant control of G protein function. RGS proteins demonstrate little specificity for Gi and Gq subunits in vitro, yet they aparently discriminate between G-protein- coupled receptors (GPCRs) linked to the same G-alpha in some cells. Fusion proteins consisting of different GPCRs fused to various G-alpha subunits were constructed and expressed in mammalian cell lines. Receptor-stimulated GTPase activity of membrane preparations was determined in the presence or absence of RGS proteins. RGS proteins were previously shown to augment agonist-stimulated GTPase activity of the receptor-G-alpha fusion proteins. This system is utilized to study the regulation of RGS activity by covalent modification. RGS16 was previously shown to undergo palmitoylation on conserved N-terminal cysteine residues (C2, C12). Mutation of these residues to alanine and expression in cellular membranes prevented RGS16 enhancement of serotonin-induced GTPase activity of a serotonin-G-alpha o fusion protein. Furthermore, these residues were shown to be dispensible for plasma membrane localization but required for targeting of RGS16 to lipid rafts. N-terminal RGS16 palmitoylation was shown to permit palmitoylation of a conserved cysteine residue in the RGS box. Mutation of this residue (C98)abolished RGS16 catalytic activity in the same assay and its ability to regulate Gi-mediated adenylyl cyclase inhibition. RGS16 was shown to be phosphorylated on a conserved tyrosine residue by src family kinases. Such phosphorylation could be induced by stimulation of G-protein-coupled receptors or by engagement of the B cell antigen receptor in B lymphocytes transfected with RGS16. Src-induced RGS16 tyrosine phosphorylation prolonged the half life of RGS16. RGS13 expression was shown to inhibit beta-adrenergic receptor-induced cAMP generation through an unknown mechanism.
