Modulation of the affinity and selectivity of RGS protein interaction with Gα subunits by a conserved asparagine/serine residue

The crystal structure of the complex between a G protein α subunit (G(iα1)) and its GTPase-activating protein (RGS4) demonstrated that RGS4 acts predominantly by stabilization of the transition state for GTP hydrolysis [Tesmer, J. J., et al. (1997) Cell 89, 251]. However, attention was called to a conserved Asn residue (Asn¹²⁸) that could play a catalytic role by interacting, directly or indirectly, with the hydrolytic water molecule. We have analyzed the effects of several disparate substitutions for Asn¹²⁸ on the GAP activity of RGS4 toward four G(α) substrates (G(o), G(i), G(q), and G(z)) using two assay formats. The results substantiate the importance of this residue but indicate that it is largely involved in substrate binding and that its function may vary with different G(α) targets. Various mutations decreased the apparent affinity of RGS4 for substrate G(α) proteins by several orders of magnitude, but had variable and modest effects on maximal rates of GTP hydrolysis when tested with different G(α) subunits. One mutation, N128F, that differentially decreased the GAP activity toward G(αi) compared with thai toward G(αq) could be partially suppressed by mutation of the nearby residue in G(αi) to that found in G(αq) (K180P). Detection of GAP activities of the mutants was enhanced in sensitivity up to 100-fold by assay at steady state in proteoliposomes that contain heterotrimeric G protein and receptor.