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Comparing the Affinity of GTPase-binding Proteins using Competition Assays
Published on: October 8, 2015
G protein selectivity is a determinant of RGS2 function
S P Heximer1, S P Srinivasa, L S Bernstein
1Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
The Journal of Biological Chemistry
|November 24, 1999
Summary
Regulator of G protein signaling (RGS) proteins like RGS2 and RGS4 exhibit distinct potencies against different G protein families. Structural differences explain RGS2
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Signaling
Background:
- Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins, modulating heterotrimeric G protein signaling.
- The precise mechanisms governing RGS protein biological functions, including G protein selectivity and structure-activity relationships, remain largely uncharacterized.
Purpose of the Study:
- To investigate the functional differences between RGS2 and RGS4 regarding their GTPase-activating protein (GAP) activity toward G(q) and G(i) protein families.
- To elucidate the structural basis for the observed selectivity of RGS proteins toward specific Galpha subunits.
Main Methods:
- In vivo assays measuring G(q)-stimulated phosphoinositide hydrolysis and G(i)-mediated signaling.
- Site-directed mutagenesis of RGS2 to identify key residues influencing G protein interaction.
- Structural analysis of RGS-G protein complexes.
Main Results:
- RGS2 demonstrated 5-fold greater potency than RGS4 in inhibiting G(q)-mediated signaling.
- RGS4 was 8-fold more potent than RGS2 in inhibiting G(i)-mediated signaling.
- RGS2 mutants were generated with enhanced potency against G(i) proteins, without altering G(q) activity, suggesting specific structural determinants for selectivity.
Conclusions:
- The functional divergence of RGS proteins, exemplified by RGS2, is significantly influenced by quantitative differences in their GTPase-activating protein activity across distinct Galpha subunit classes.
- Structural features, including the switch I binding pocket geometry and loop interactions, dictate the selectivity of RGS proteins for specific G protein families.
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