Modulation of the affinity and selectivity of RGS protein interaction withG alpha subunits by a conserved asparagine serine residue

The crystal structure of the complex between a G protein alpha subunit (G(i alpha 1)) and its GTPase activating protein (RGS4) demonstrated that RGS4 acts predominantly by stabilization of the transition state for GTP hydroly sis [Tesmer, J. J., et al. (1997) Cell 89, 251]. However, attention was cal led to a conserved Asn residue (Asn(128)) 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 Asn(12 8) on the GAP activity of RGS4 toward four G(alpha) substrates (G(o), G(i), G(q), and G(z)) using two assay formats. The results substantiate the impo rtance of this residue but indicate that it is largely involved in substrat e binding and that its function may vary with different G(alpha) targets. V arious mutations decreased the apparent affinity of RGS4 for substrate G al pha proteins by several orders of magnitude, but had variable and modest ef fects on maximal rates of GTP hydrolysis when tested with different G(alpha ) subunits. One mutation, N128F, that differentially decreased the GAP acti vity toward G(alpha i) compared with that toward G(alpha q) could be partia lly suppressed by mutation of the nearby residue in G(alpha i) to that foun d in G(alpha q) (K180P). Detection of GAP activities of the mutants was enh anced in sensitivity up to 100-fold by assay at steady state in proteolipos omes that contain heterotrimeric G protein and receptor.