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. In addition, RGS16 was also shown to be palmitoylated on a novel cysteine residue in the RGS box, and mutation of this residue also abolished RGS16 catalytic activity in the same assay. RGS16 co-immunoprecipitated with and underwent tyrosine phosphorylation by the Epidermal Growth Factor (EGF) receptor kinase in vitro and in cultured cell lines. Mutation of RGS16 tyrosine residues blocked EGFR-induced RGS16 tyrosine phosphorylation and prevented RGS16 inhibition of Gi-mediated MAP kinase activation and adenylyl cyclase inhibition. RGS16 interacted with the G protein G-alpha 13 and inhibited G13-mediated stimulation of Rho-dependent serum response element (SRE)-induced transcription. In addition, RGS16 injection into astrocytoma cells inhibited G13-induced Rho-dependent cell rounding. The RGS16-G13 interaction and inhibition of SRE-dependent transcription required only the RGS16 N-terminus.
