Quantitative analysis of a cysteine 351glycine mutation in the G protein Gi1α:: effect on α2A-adrenoceptor-Gi1α fusion protein activation

Fusion proteins mere constructed between the porcine alpha(2A)-adrenoceptor and either wild-type (Cys(351)) Or a pertussis toxin-resistant (Gly(351)) form of the G protein G(i1)alpha. Addition of adrenaline to membranes expressing the fusion proteins resulted in concentration-dependent stimulation of their high affinity GTPase activity. The alpha(2A)-adrenoceptor-wild type G(i1)alpha fusion protein produced substantially higher maximal stimulation of GTPase activity in response to adrenaline than that containing Gly(351) G(i1)alpha. Treatment of the fusion proteins as agonist-regulated enzymes allowed measurement of V-max and turnover number for adrenaline-stimulation of the GTPase activity of each fusion construct. The turnover number of the alpha(2A)-adrenoceptor-Cys(351) Gly G(i1)alpha fusion protein was only 44% of that for the alpha(2A)-adrenoceptor-wild type G(i1)alpha fusion protein. These data provide the first direct quantitative evaluation of the effects of a mutation of a G protein on the capacity of an agonist-occupied receptor to activate the mutant. (C) 1998 Federation of European Biochemical Societies.