SYNTHESIS IN ESCHERICHIA-COLI OF GTPASE-DEFICIENT MUTANTS OF GS-ALPHA

  • 15 September 1989
    • journal article
    • research article
    • Vol. 264  (26) , 15475-15482
Abstract
We have reduced the GTPase activity of the .alpha. subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase, by introduction of point mutations analogous to those described in p21ras. Mutants G49V and Q227L differ from the wild type protein in the substitution of Val for Gly49 and Leu for Gln 227, respectively (analogous to positions 12 and 61 in p21ras). Wild type and mutant proteins were synthesized in Escherichia coli, purified, and characterized. The rate constants for dissociation of GDP from G49V recombinant Gs.alpha. (rGs.alpha.) (0.47/min) and Q227L rGs.alpha. (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 rGs.alpha. (0.78/min) and Q227L rGs.alpha. (0.03-0.06/min) are 4-fold and roughly 100-fold slower than that for wild type rGs.alpha. (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 rGs.alpha. and 0.78 for Q227L rGs.alpha., compared to 0.05 for wild type rGs.alpha.. When reconstituted with cyc- (Gs.alpha.-deficient) S49 cell membranes or purified adenylyl cyclase, both mutant proteins stimulate adenylyl cyclase activity in the presence of GTP to much greater extent than does wild type Gs.alpha.; their maximal ability to activate the enzyme is largely unaltered. The fractional ability of a given Gs.alpha. 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 .beta..gamma. subunits, and their ability to activate adenylyl cyclase is enhanced by interaction with .beta.-adrenergic receptors. These results indicate that the structural analogy that has been inferred between the guanine nucleotide-binding domains of G proteins and the p21ras 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 .alpha. subunits.