Mechanism of RGS4, a GTPase-activating Protein for G Protein α Subunits

Abstract

GTP hydrolysis by guanine nucleotide-binding proteins, an essential step in many biological processes, is stimulated by GTPase-activating proteins (GAPs). The mechanisms whereby GAPs stimulate GTP hydrolysis are unknown. We have used mutational, biochemical, and structural data to investigate how RGS4, a GAP for heterotrimeric G protein alpha subunits, stimulates GTP hydrolysis. Many of the residues of RGS4 that interact with Gi alpha 1 are important for GAP activity. Furthermore, optimal GAP activity appears to require the additive effects of interactions along the RGS4-G alpha interface. GAP-defective RGS4 mutants invariably were defective in binding G alpha subunits in their transition state; furthermore, the apparent strengths of GAP and binding defects were correlated. Thus, none of these residues of RGS4, including asparagine 128, the only residue positioned at the active site of Gi alpha 1, is required exclusively for catalyzing GTP hydrolysis. These results and structural data (Tesmer, J. G. G., Berman, D. M., Gilman, A. G., and Sprang, S. R. (1997) Cell 89, 251-261) indicate that RGS4 stimulates GTP hydrolysis primarily by stabilizing the transition state conformation of the switch regions of the G protein, favoring the transition state of the reactants. Therefore, although monomeric and heterotrimeric G proteins are related, their GAPs have evolved distinct mechanisms of action.

PIP 3 inhibition of RGS protein and its reversal by Ca 2+ /calmodulin mediate voltage-dependent control of the G protein cycle in a cardiac K + channel

“…The GST construct alone had no effect ( Fig. 1 Aa); neither did boiled GST-RGS4 (n ϭ 4, not shown) nor did the mutant GST-RGS4 (N128H) (n ϭ 4, not shown), which lacks GTPaseactivating protein activity (13,21). Inhibition of K G channel activity by 1 M GST-RGS4 was essentially irreversible upon washout of the protein (Fig.…”

Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae

“…Structure-function analysis of RGS proteins has revealed that the RGS homology domain or core domain is sufficient for GAP activity in vitro (12)(13)(14). In this study, we define a second functional domain of RGS4.…”

“…1A), is sufficient for the GAP activity of RGS4 in vitro (12)(13)(14). To define the minimal sequences required for RGS4 function in vivo, we used the ability of RGS4 protein to inhibit pheromone signaling in the budding yeast S. cerevisiae as an assay (11).…”

“…This conserved RGS domain is sufficient to stimulate GTPase activity of G protein ␣ subunits in vitro (12)(13)(14). Expression of the RGS homology domain of RET-RGS1 or RGS4 yields a recombinant protein that is a functional GAP (GTPase-activating protein).…”

“…In the crystal structure of RGS4 bound to AlF 4 Ϫ -activated G i␣1 , only the core domain is visible (15). The RGS homology domain binds to the switch regions of G i␣1 and appears to catalyze GTP hydrolysis by stabilizing the switch regions of the G protein in a conformation that favors the transition state of the reactants (14,15). Sequences outside the RGS homology domain exhibit considerable diversity among family members.…”