Abstract
A fusion protein between a pertussis toxin-resistant (C351G) mutant of the alpha subunit of the G protein Gi1 and the porcine alpha2A-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 from endogenous Gialpha. Half-maximal stimulation of the GTPase activity of endogenous Gi was achieved with lower concentrations of agonist. Although the Km for GTP of the fusion protein-linked Gi was lower than for the endogenous G protein, Vmax measurements demonstrated that adrenaline activated some 5 mol of endogenous Gi/mol of fusion protein-linked Gi. The isolated alpha2A-adrenoreceptor could activate Gs; however, the fusion protein did not. Compared with adrenaline, the efficacy of a range of partial agonists to stimulate endogenous Gialpha was greater than for the fusion protein-constrained C351G Gi1alpha. alpha2A-Adrenoreceptor agonists could stimulate both p44 mitogen-activated protein kinase and p70 S6 kinase and inhibit forskolin-amplified adenylyl cyclase activity in untreated alpha2A-adrenoreceptor-C351G Gi1alpha 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.
Highlights
A fusion protein between a pertussis toxin-resistant (C351G) mutant of the ␣ subunit of the G protein Gi1 and the porcine ␣2A-adrenoreceptor was stably expressed in Rat 1 fibroblasts
A fusion protein (␣2AR-C351G Gi1␣) in which the N terminus of a pertussis toxin-resistant (C351G) mutant of the ␣-subunit of the G protein Gi1␣ was fused to the C-terminal tail of the porcine ␣2A-adrenoreceptor was stably expressed in Rat 1 fibroblasts. 3H-Labeled ligand binding studies using the high affinity and highly selective ␣2-adrenoreceptor antagonist [3H]RS-79948-197 were performed in membranes derived from a number of individual clones and demonstrated the presence of an apparently uniform population of sites
Membranes from a clonal cell line (TAG WT17) derived from Rat 1 cells that express some 4 pmol/mg protein of the isolated porcine ␣2A-adrenoreceptor produced similar levels of stimulation of high affinity GTPase upon the addition of adrenaline or UK14304. These results demonstrated both that adrenaline and UK14304 were capable of causing guanine nucleotide exchange and subsequent hydrolysis of GTP by the G protein-coupled receptors (GPCRs)-linked G protein and that the GPCR within the fusion protein had the capacity to activate endogenous Gi-like G proteins as well as the linked G protein
Summary
Adrenaline was able to activate some 6 mol of G protein/mol of receptor. studies that have assumed that agonistdependent prevention of proliferation of cancer cell lines following expression of a 2-adrenoreceptor-Gs␣ fusion protein reflects activation of the receptor-attached G protein [9] may require careful reanalysis.
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