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.

The Journal of Biological Chemistry
A R BurtGraeme Milligan

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 untrea...Continue Reading

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Citations

Sep 7, 2001·Biochemical Pharmacology·J NasmanK E Akerman
Dec 22, 1999·Trends in Pharmacological Sciences·I Waelbroeck
Jan 19, 2000·Trends in Pharmacological Sciences·G Milligan
Nov 5, 1999·Biochimica Et Biophysica Acta·D R Flower
Aug 8, 2001·Journal of Pharmacological and Toxicological Methods·T Wurch, P J Pauwels
Jun 18, 2002·Journal of Neurochemistry·Dominique MassotteGraeme Milligan
Oct 29, 2008·Neuroreport·Billy C ChiengDominique Massotte
Jan 16, 2009·The Journal of Pharmacology and Experimental Therapeutics·A I KayaH O Onaran
Jan 8, 2013·Molecular Pharmacology·Feng ShenDavid R Manning
Jan 25, 2002·Annual Review of Pharmacology and Toxicology·Terry Kenakin
Dec 8, 2000·Biochemical Pharmacology·P J Pauwels
Aug 16, 2003·The Journal of Biological Chemistry·Juan J CarrilloGraeme Milligan
Jul 18, 2002·Nature Reviews. Drug Discovery·Terry Kenakin
Jan 27, 2006·Molecular Pharmacology·John D Hildebrandt
Nov 24, 1999·The Journal of Biological Chemistry·K Wenzel-SeifertR Seifert
May 15, 2015·Molecular Pharmacology·William G RobichauxJoe B Blumer
Jul 5, 2002·Journal of Biomolecular Screening·Renée S MartinStephen J MacLennan
Aug 27, 1999·Trends in Pharmacological Sciences·R SeifertB K Kobilka

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