Document Type
Article
Publication Title
Journal of Biological Chemistry
Publication Date
8-21-1998
Volume
273
Issue
34
Disciplines
Biology | Life Sciences
Abstract
Agonist-bound heptahelical receptors activate heterotrimeric G proteins by catalyzing exchange of GDP for GTP on their α subunits. In search of an approximation of the receptor-α subunit complex, we have considered the properties of A326S G(iα1), a mutation discovered originally in G(sα) (Iiri, T., Herzmark, P., Nakamoto, J. M., Van Dop, C., and Bourne, H. R. (1994) Nature 371, 164-168) that mimics the effect of receptor on nucleotide exchange. The mutation accelerates dissociation of GDP from the α(i1)β1γ2 heterotrimer by 250-fold. Nevertheless, affinity of mutant G(iα1) for GTPγS is high in the presence of Mg2+, and the mutation has no effect on the intrinsic GTPase activity of the α subunit. The mutation also uncouples two activities of βγ: stabilization of the GDP-bound α subunit (which is retained) and retardation of GDP dissociation heterotrimer (which is lost). For wild-type and mutant G(iαl), βγ prevents irreversible inactivation of the α subunit at 30 °C. However, the mutation accelerates irreversible inactivation of α at 37 °C despite the presence of βγ. Structurally, the mutation weakens affinity for GTPγS by steric crowding: a 2-fold increase in the number of close contacts between the protein and the purine ring of the nucleotide. By contrast, we observe no differences in structure at the GDP binding site between wild-type heterotrimers and those containing A326S G(i 1/2 ). However, the GDP binding site is only partially occupied in crystals of G protein heterotrimers containing A326S G(iα1). In contrast to original speculations about the structural correlates of receptor-catalyzed nucleotide exchange, rapid dissociation of GDP can be observed in the absence of substantial structural alteration of a G(α) subunit in the GDP-bound state.
DOI
https://doi.org/10.1074/jbc.273.34.21752
Rights
© 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.
Recommended Citation
Posner, Bruce A.; Mixon, Mark B.; Wall, Mark A.; Sprang, Stephen R.; and Gilman, Alfred G., "The A326S mutant of G(iα1) as an approximation of the receptor-bound state" (1998). Biological Sciences Faculty Publications. 533.
https://scholarworks.umt.edu/biosci_pubs/533