Mutational analysis of the Asn residue essential for RGS protein binding to G-proteins.
Members of the RGS family serve as GTPase-activating proteins (GAPs) for heterotrimeric G-proteins and negatively regulate signaling via G-protein-coupled receptors. The recently resolved crystal structure of RGS4 bound to Gialpha1 suggests two potential mechanisms for the GAP activity of RGS proteins as follows: stabilization of the Gialpha1 switch regions by RGS4 and the catalytic action of RGS4 residue Asn128. To elucidate a role of the Asn residue for RGS GAP function, we have investigated effects of the synthetic peptide corresponding to the Galpha binding domain of human retinal RGS (hRGSr) containing the key Asn at position 131, and we have carried out mutational analysis of Asn131. Synthetic peptide hRGSr-(123-140) retained its ability to bind the AlF4--complexed transducin alpha-subunit, Gtalpha.AlF4-, but failed to elicit stimulation of Gtalpha GTPase activity. Wild-type hRGSr stimulated Gtalpha GTPase activity by approximately 10-fold with an EC50 value of 100 nM. Mutant hRGSr proteins with substitutions of Asn131 by Ser and Gln had a significantly reduced affinity for Gtalpha but were capable of substantial stimulation of Gtalpha GTPase activity, 80 and 60% of Vmax, respectively. Mutants hRGSr-Leu131, hRGSr-Ala131, and hRGSr-Asp131 were able to accelerate Gtalpha GTPase activity only at very high concentrations (>10 microM) which appears to correlate with a further decrease of their affinity for transducin. Two mutants, hRGSr-His131 and hRGSr-Delta131, had no detectable binding to transducin. Mutational analysis of Asn131 suggests that the stabilization of the G-protein switch regions rather than catalytic action of the Asn residue is a key component for the RGS GAP action.
Natochin
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Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
McEntaffer
R L
RL
Artemyev
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NO
D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism*; D001667:Binding, Competitive; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D015293:Transducin / Q000378:metabolism
D020690:GTPase-Activating Proteins; D011506:Proteins; D001216:Asparagine; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D015293:Transducin