Measurement of agonist efficacy using an alpha2A-adrenoceptor-Gi1alpha fusion protein

FEBS Letters
A WiseG Milligan

Abstract

A fusion protein was constructed between the porcine alpha2A-adrenoceptor and a pertussis toxin-insensitive (Cys351Gly) form of the alpha subunit of the G protein Gi1. Addition of agonist ligands to membranes of COS-7 cells transiently transfected to express this construct, and treated with pertussis toxin prior to cell harvest, resulted in stimulation of both high affinity GTPase activity and enhanced binding of [35S]GTPgammaS. By considering the fusion protein as an agonist-activated enzyme and measuring Vmax of GTP hydrolysis a range of agonist ligands displayed varying efficacy in their capacity to activate the receptor-associated G protein with adrenaline = noradrenaline = alpha-methylnoradrenaline > UK14304 > BHT933 > or = xylazine = clonidine. A similar rank order was observed following independent co-expression of the alpha2A-adrenoceptor and Cys351Gly-Gi1alpha. These data demonstrate the utility and applicability of using a receptor-G protein fusion protein approach, in which the stoichiometry of receptor and G protein is fixed at 1:1, to measure and further understand the nature of agonist efficacy.

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Citations

Jan 19, 2000·Trends in Pharmacological Sciences·G Milligan
Nov 5, 1999·Biochimica Et Biophysica Acta·D R Flower
Apr 15, 2008·Trends in Endocrinology and Metabolism : TEM·G Milligan
Aug 8, 2001·Journal of Pharmacological and Toxicological Methods·T Wurch, P J Pauwels
Sep 25, 2008·FEMS Microbiology Reviews·Chaoyang XueJoseph Heitman
Dec 25, 2003·The Journal of Physiology·D Walter WrayMikael Sander
Oct 20, 2000·Molecular Pharmacology·K Wenzel-Seifert, R Seifert
Aug 27, 1999·Trends in Pharmacological Sciences·R SeifertB K Kobilka

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