Synthesis in Escherichia coli of GTPase-deficient mutants of G(sα)

M. P. Graziano, A. G. Gilman

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Abstract

We have reduced the GTPase activity of the α subunit of G(s), the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase, by introduction of point mutations analogous to those described in p21(ras). Mutants G49V and Q227L differ from the wild type protein in the substitution of Val for Gly49 and Leu for Gln227, respectively (analogous to positions 12 and 61 in p21(ras)). Wild type and mutant proteins were synthesized in Escherichia coli, purified, and characterized. The rate constants for dissociation of GDP from G49V recombinant G(sα) (rG(sα)) (0.47/min) and Q227L rG(sα) (0.23/min) differ by no more than 2-fold from that observed for the wild type protein (0.5/min). In marked contrast, the rate constants for hydrolysis of GTP by G49V rG(sα) (0.78/min) and Q227L rG(sα) (0.03-0.06/min) are 4-fold and roughly 100-fold slower than that for wild type rG(sα) (3.5/min). These reductions in the rate of hydrolysis of GTP result in significant fractional occupancy of these proteins by GTP in the presence of the nucleotide, 0.37 for G49V rG(sα) and 0.78 for Q227L rG(sα), compared to 0.05 for wild type rG(sα). When reconstituted with cyc- (G(sα)-deficient) S49 cell membranes or purified adenylyl cyclase, both mutant proteins stimulate adenylyl cyclase activity in the presence of GTP to a much greater extent than does wild type G(sα); their maximal ability to activate the enzyme is largely unaltered. The fractional ability of a given G(sα) polypeptide to active adenylyl cyclase in the presence of GTP correlates well with the fractional occupancy of the protein by the nucleotide. The mutant subunits appear to interact normally with G protein βγ subunits, and their ability to activate adenylyl cyclase is enhanced by interaction with β-adrenergic receptors. These results indicate that the structural analogy that has been inferred between the guanine nucleotide-binding domains of G proteins and the p21(ras) family is at least generally correct. They also provide confirmation of the kinetic model of G protein function and document mutations that permit the expression in vivo of constitutively activated G protein α subunits.

Original languageEnglish (US)
Pages (from-to)15475-15482
Number of pages8
JournalJournal of Biological Chemistry
Volume264
Issue number26
StatePublished - 1989

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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