AGONIST OCCUPATION OF AN ALPHA(2A)-ADRENORECEPTOR-G(I1)ALPHA FUSION PROTEIN RESULTS IN ACTIVATION OF BOTH RECEPTOR-LINKED AND ENDOGENOUS G(I) 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)-ad renoreceptor was stably expressed in Rat 1 fibroblasts, Agonists cause d stimulation of high affinity GTPase activity, which was partially pr evented by pertussis toxin treatment, demonstrating that the toxin-res istant component of the GTPase activity was derived from the receptor- fused G protein and the remainder hom endogenous G(i) alpha. Half-maxi mal stimulation of the GTPase activity of endogenous Gi was achieved w ith lower concentrations of agonist, Although the K-m for GTP of the f usion 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 alp ha(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 activi ty in untreated alpha(2A)-adrenoreceptor-C351G G(i1)alpha fusion prote in-expressing cells, but these signals were abolished following pertus sis toxin treatment. These results demonstrate conclusively, and for t he first time, that agonist occupancy of a receptor-G protein fusion p rotein can result in activation of G proteins other than that physical ly linked to the receptor. This was selective between G protein classe s. Analysis of the contributions of fusion protein-linked and endogeno us G proteins to agonist-stimulated GTPase activity provided a direct and original measure of receptor-G protein activation stoichiometry.