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FEBS Lett. 1997 Dec 8;419(1):141-6.

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

Author information

1
Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, UK.

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.

PMID:
9426237
DOI:
10.1016/s0014-5793(97)01431-2
[Indexed for MEDLINE]
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