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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Intramural Research (Z01)
Project #
1Z01AI000840-05
Application #
6809055
Study Section
(LAD)
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2003
Total Cost
Indirect Cost
Name
Niaid Extramural Activities
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Bahia, Daljit S; Sartania, Nana; Ward, Richard J et al. (2003) Concerted stimulation and deactivation of pertussis toxin-sensitive G proteins by chimeric G protein-coupled receptor-regulator of G protein signaling 4 fusion proteins: analysis of the contribution of palmitoylated cysteine residues to the GAP activity o J Neurochem 85:1289-98
Osterhout, James L; Waheed, Abdul A; Hiol, Abel et al. (2003) Palmitoylation regulates regulator of G-protein signaling (RGS) 16 function. II. Palmitoylation of a cysteine residue in the RGS box is critical for RGS16 GTPase accelerating activity and regulation of Gi-coupled signalling. J Biol Chem 278:19309-16
Hiol, Abel; Davey, Penelope C; Osterhout, James L et al. (2003) Palmitoylation regulates regulators of G-protein signaling (RGS) 16 function. I. Mutation of amino-terminal cysteine residues on RGS16 prevents its targeting to lipid rafts and palmitoylation of an internal cysteine residue. J Biol Chem 278:19301-8
Derrien, Alexandrine; Zheng, Bin; Osterhout, James L et al. (2003) Src-mediated RGS16 tyrosine phosphorylation promotes RGS16 stability. J Biol Chem 278:16107-16
Moratz, C; Kang, V H; Druey, K M et al. (2000) Regulator of G protein signaling 1 (RGS1) markedly impairs Gi alpha signaling responses of B lymphocytes. J Immunol 164:1829-38
Cavalli, A; Druey, K M; Milligan, G (2000) The regulator of G protein signaling RGS4 selectively enhances alpha 2A-adreoreceptor stimulation of the GTPase activity of Go1alpha and Gi2alpha. J Biol Chem 275:23693-9
Sullivan, B M; Harrison-Lavoie, K J; Marshansky, V et al. (2000) RGS4 and RGS2 bind coatomer and inhibit COPI association with Golgi membranes and intracellular transport. Mol Biol Cell 11:3155-68