Quantitative analysis of a cysteine351glycine mutation in the G protein Gi1α: effect on α2A‐adrenoceptor‐Gi1α fusion protein activation

摘要

>Fusion proteins were constructed between the porcine α_2A-adrenoceptor and either wild-type (Cys351) or a pertussis toxin-resistant (Gly351) form of the G protein G_i1α. Addition of adrenaline to membranes expressing the fusion proteins resulted in concentration-dependent stimulation of their high affinity GTPase activity. The α_2A-adrenoceptor-wild type G_i1α fusion protein produced substantially higher maximal stimulation of GTPase activity in response to adrenaline than that containing Gly351 G_i1α. 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 α_2A-adrenoceptor-Cys351Gly G_i1α fusion protein was only 44% of that for the α_2A-adrenoceptor-wild type G_i1α 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.