A point mutation in Gα(o) and Gα(i1) blocks interaction with regulator of G protein signaling proteins

Keng Li Lan, Noune A. Sarvazyan, Ronald Taussig, Robert G. Mackenzie, Paul R. DiBello, Henrik G. Dohlman, Richard R. Neubig

Research output: Contribution to journalArticle

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Abstract

Regulator of G protein-signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits and are thought to be responsible for rapid deactivation of enzymes and ion channels controlled by G proteins. We wanted to identify and characterize G(i)-family a subunits that were insensitive to RGS action. Based on a glycine to serine mutation in the yeast Gα subunit Gpa1(sst) that prevents deactivation by Sst2 (DiBello, P. R., Garrison, T. R., Apanovitch, D. M., Hoffman, G., Shuey, D. J., Mason, K., Cockett, M. I., and Dohlman, H. G. (1998) J. Biol. Chem. 273, 5780-5784), site-directed mutagenesis of α(o) and α(i1) was done. G184S α(o) and G183S α(i1) show kinetics of GDP release and GTP hydrolysis similar to wild type. In contrast, GTP hydrolysis by the G → S mutant proteins is not stimulated by RGS4 or by a truncated RGS7. Quantitative flow cytometry binding studies show IC50 values of 30 and 96 nM, respectively, for aluminum fluoride-activated wild type α(o) and all to compete with fluorescein isothiocyanate-α(o) binding to glutathione S-transferase-RGS4. The G → S mutant proteins showed a greater than 30-100-fold lower affinity for RGS4. Thus, we have defined the mechanism of a point mutation in α(o) and all that prevents RGS binding and GTPase activating activity. These mutant subunits should be useful in biochemical or expression studies to evaluate the role of endogenous RGS proteins in G(i) function.

Original languageEnglish (US)
Pages (from-to)12794-12797
Number of pages4
JournalJournal of Biological Chemistry
Volume273
Issue number21
DOIs
StatePublished - May 22 1998

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RGS Proteins
Guanosine Triphosphate
GTP-Binding Protein Regulators
Point Mutation
Hydrolysis
Mutant Proteins
GTP-Binding Proteins
Mutagenesis
Flow cytometry
GTP Phosphohydrolases
Site-Directed Mutagenesis
Glutathione Transferase
Fluorescein
Ion Channels
Protein Binding
Yeast
Glycine
Serine
Inhibitory Concentration 50
Flow Cytometry

ASJC Scopus subject areas

  • Biochemistry

Cite this

Lan, K. L., Sarvazyan, N. A., Taussig, R., Mackenzie, R. G., DiBello, P. R., Dohlman, H. G., & Neubig, R. R. (1998). A point mutation in Gα(o) and Gα(i1) blocks interaction with regulator of G protein signaling proteins. Journal of Biological Chemistry, 273(21), 12794-12797. https://doi.org/10.1074/jbc.273.21.12794

A point mutation in Gα(o) and Gα(i1) blocks interaction with regulator of G protein signaling proteins. / Lan, Keng Li; Sarvazyan, Noune A.; Taussig, Ronald; Mackenzie, Robert G.; DiBello, Paul R.; Dohlman, Henrik G.; Neubig, Richard R.

In: Journal of Biological Chemistry, Vol. 273, No. 21, 22.05.1998, p. 12794-12797.

Research output: Contribution to journalArticle

Lan, Keng Li ; Sarvazyan, Noune A. ; Taussig, Ronald ; Mackenzie, Robert G. ; DiBello, Paul R. ; Dohlman, Henrik G. ; Neubig, Richard R. / A point mutation in Gα(o) and Gα(i1) blocks interaction with regulator of G protein signaling proteins. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 21. pp. 12794-12797.
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