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 cell-free systems, yet they may discriminate between G-protein-coupled receptors (GPCRs) linked to the same G-alpha. To address the issue of receptor specificity directly, fusion proteins consisting of different GPCRs fused to a single G-alpha subunit were constructed and expressed in cell lines, and receptor-stimulated GTPase activity in the presence of RGS proteins is studied. Although no GTPase accelerating (GAP) activity of any RGS protein for Gs-alpha has been detected, RGS16 interacted unexpectedly with Gs-alpha and inhibited Gs-induced cyclic AMP (cAMP) generation in mammalian cells. Regulation of RGS16 activity was studied by examining whether the protein underwent post-translational modification. RGS16 shares amino terminal cysteine residues with RGS4 and RGS5 that were hypothesized to be sites of palmitoylation. RGS16 underwent palmitoylation, and palmitoylation-defective RGS16 mutants demonstrated impaired ability to inhibit both Gi- and Gq-coupled signaling in mammalian cells. RGS16 also underwent tyrosine phosphorylation in response to both GPCR or receptor tyrosine kinase (Epidermal Growth Factor, EGF) stimulation. Mutation of a conserved tyrosine residue blocked tyrosine phosphorylation of RGS16, and the tyrosine mutant was quantitatively less able to inhibit Gi and Gq- induced mitogen activated protein (MAP) kinase activation. RGS16 co- immunoprecipitated with the EGF receptor complex and inhibited EGF- induced MAP kinase activation. A novel RGS protein with a divergent RGS box was cloned, D-AKAP2, which also contains a protein kinase A anchoring (AKAP) domain. Expression of D-AKAP2 in human embryonic kidney (HEK)293 cells resulted in inhibition of signaling induced by Gs-coupled GPCRs but not those coupled to Gi or Gq. - GTP, G proteins, RGS proteins, signal transduction
