title: Agonist occupation of an alpha2A-adrenoreceptor-Gi1alpha 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.
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.
authors.affiliation: Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: A R
authors.initials: AR
authors.lastname: Burt
authors.forename: M
authors.initials: M
authors.lastname: Sautel
authors.forename: M A
authors.initials: MA
authors.lastname: Wilson
authors.forename: S
authors.initials: S
authors.lastname: Rees
authors.forename: A
authors.initials: A
authors.lastname: Wise
authors.forename: G
authors.initials: G
authors.lastname: Milligan
mesh_terms: D058647:Adrenergic alpha-2 Receptor Agonists; D000818:Animals; D002460:Cell Line; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000819:agonists*; D015398:Signal Transduction; D013379:Substrate Specificity
chemical_list: C547770:Adra2a protein, rat; D058647:Adrenergic alpha-2 Receptor Agonists; D018341:Receptors, Adrenergic, alpha-2; D011993:Recombinant Fusion Proteins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins