Mutational analysis of the Asn residue essential for RGS protein binding to G-proteins

Michael Natochin, Randall L. McEntaffer, Nikolai O. Artemyev

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Abstract

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 G1α1 suggests two potential mechanisms for the GAP activity of RGS proteins as follows: stabilization of the G1α1 switch regions by RGS4 and the catalytic action of RGS4 residue Asn128. To elucidate a role of the Ash residue for RGS GAP function, we have investigated effects of the synthetic peptide corresponding to the Gα binding domain of human retinal RGS (hRGSr) containing the key Ash 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 α-subunit, G(t)α- AlF4-, but failed to elicit stimulation of Gtα GTPase activity. Wild-type hRGSr stimulated G(t)α GTPase activity by ~ 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 G(t)α but were capable of substantial stimulation of G(t)α GTPase activity, 80 and 60% of V(max) respectively. Mutants hRGSr-Leu131 hRGSr-Ala191 and hRGSr-Asp131 were able to accelerate G(t)α GTPase activity only at very high concentrations (>10 μM) which appears to correlate with a further decrease of their affinity for transducin. Two mutants, hRGSr-His131 and hRGSr- Δ131 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 Ash residue is a key component for the RGS GAP action.

Original languageEnglish (US)
Pages (from-to)6731-6735
Number of pages5
JournalJournal of Biological Chemistry
Volume273
Issue number12
DOIs
StatePublished - Mar 20 1998

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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