Journal article
Mutational Analysis of the Asn Residue Essential for RGS Protein Binding to G-proteins
The Journal of biological chemistry, Vol.273(12), pp.6731-6735
03/1998
DOI: 10.1074/jbc.273.12.6731
PMID: 9506972
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 Giα1 suggests two potential mechanisms for the GAP activity of RGS proteins as follows: stabilization of the Giα1 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 Gα 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 α-subunit, Gtα·AlF4−, but failed to elicit stimulation of Gtα GTPase activity. Wild-type hRGSr stimulated Gtα 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 Gtα but were capable of substantial stimulation of Gtα GTPase activity, 80 and 60% of Vmax, respectively. Mutants hRGSr-Leu131, hRGSr-Ala131, and hRGSr-Asp131 were able to accelerate Gtα 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-His131and 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 Asn residue is a key component for the RGS GAP action.
Details
- Title: Subtitle
- Mutational Analysis of the Asn Residue Essential for RGS Protein Binding to G-proteins
- Creators
- Michael NatochinRandall L McEntafferNikolai O Artemyev
- Resource Type
- Journal article
- Publication Details
- The Journal of biological chemistry, Vol.273(12), pp.6731-6735
- DOI
- 10.1074/jbc.273.12.6731
- PMID
- 9506972
- NLM abbreviation
- J Biol Chem
- ISSN
- 0021-9258
- eISSN
- 1083-351X
- Language
- English
- Date published
- 03/1998
- Academic Unit
- Molecular Physiology and Biophysics; Iowa Neuroscience Institute; Physics and Astronomy; Ophthalmology and Visual Sciences
- Record Identifier
- 9984070788702771
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