Agonist occupation of an α2A-adrenoreceptor-Gi1α fusion protein results in activation of both receptor-linked and endogenous Gi proteins -: Comparisons of their contributions to GTPase activity and signal transduction and analysis of receptor-G protein activation stoichiometry

A fusion protein between a pertussis toxin-resistant (C351G) mutant of the alpha subunit of the G protein G(i1) and the porcine alpha(2A)-adrenoreceptor was stably expressed in Rat 1 fibroblasts, Agonists caused stimulation of high affinity GTPase activity, which was partially prevented by pertussis toxin treatment, demonstrating that the toxin-resistant component of the GTPase activity was derived from the receptor-fused G protein and the remainder hom endogenous G(i) alpha. Half-maximal stimulation of the GTPase activity of endogenous Gi was achieved with lower concentrations of agonist, Although the K-m for GTP of the fusion protein-linked G(i) was lower than for the endogenous G protein, V-max measurements demonstrated that adrenaline activated some 5 mol of endogenous G(i)/mol of fusion protein-linked G(i). The isolated alpha(2A)-adrenoreceptor could activate G(s); however, the fusion protein did not. Compared with adrenaline, the efficacy of a range of partial agonists to stimulate endogenous G(i) alpha was greater than for the fusion protein-constrained C351G G(i1)alpha. alpha(2A)-Adrenoreceptor agonists could stimulate both p44 mitogen-activated protein kinase and p70 S6 kinase and inhibit forskolin-amplified adenylyl cyclase activity in untreated alpha(2A)-adrenoreceptor-C351G G(i1)alpha fusion protein-expressing cells, but these signals were abolished following pertussis toxin treatment. These results demonstrate conclusively, and for the first time, that agonist occupancy of a receptor-G protein fusion protein can result in activation of G proteins other than that physically linked to the receptor. This was selective between G protein classes. Analysis of the contributions of fusion protein-linked and endogenous G proteins to agonist-stimulated GTPase activity provided a direct and original measure of receptor-G protein activation stoichiometry.