Mechanism and specificity of G protein regulation by RGS proteins.

Abstract

Many extracellular stimuli convey information to cells through G protein-coupled receptors. Steady state activation of this pathway depends on GTPase activity of the Galpha subunit. Three advances in G protein function are addressed in this thesis: (1) The roles of tryptophans in fluorescent and functional properties of Galphao; (2) Rapid kinetics of RGS-mediated Galpha deactivation and (3) characterization of the RGS-insensitive Galpha mutants. Fluorescence of two mutant Galphao proteins (W132F and W212F) were examined to understand the determinants of the N-methyl-3<super>'</super>-<italic> O</italic>-anthraniloyl (Mant)-guanine nucleotide fluorescence signals. Trp212 in Galphao is essential for GTP-stimulated fluorescence, whereas resonance energy transfer from both Trp132 and Trp212 contributes to the enhanced Mant-guanine nucleotide fluorescence upon binding to Galphao. R&barbelow;egulator of G&barbelow; protein s&barbelow;ignaling (RGS) proteins function as GTPase activating proteins (GAPs) for Galpha and are thought to be responsible for rapid deactivation of G protein responses. Using stopped-flow spectrofluorimetry, I examined G protein deactivation on the millisecond time scale. RGS4 dramatically enhanced deactivation rates of Galphao and Galphai1. The rate increased linearly with RGS concentration to over 5 sec<super>-1</super>. These are the first <italic>in vitro</italic> measurements of G protein deactivation at rates comparable to <italic>in vivo </italic> turn-off of G-protein-mediated signals. In contrast to the rapid deactivation of Galpha when GTP was bound, Mant-GTP-bound Galphao was not deactivated rapidly by RGS4, suggesting that Mant-GTP prevents RGS regulation. Therefore, Mant-GTP may be a useful tool to generate active but RGS-insensitive Galpha to study the physiological role of RGS. G protein alpha subunit mutants insensitive to RGS proteins could be used to assess the combined role of all RGS proteins in the function of that subunit <italic>in vivo</italic>. Based on a yeast Galpha mutation, a single glycine residue was mutated to serine in Galphao or Galphai1. RGS4 and RGS7 were unable to stimulate GTP hydrolysis by the G > S mutant proteins. The ability of G183S Galphai1 to interact with receptor, Gbetagamma and adenylyl cyclase was similar to that wild type, however, the affinity for RGS4 was decreased >1000-fold. The G > S mutants will be useful in biochemical or expression studies to evaluate the role of endogenous RGS proteins in Galphai function.