Abstract
A fusion protein was constructed between the porcine α 2A-adrenoceptor and a pertussis toxin-insensitive (Cys 351Gly) form of the α subunit of the G protein G i1. 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]GTPγS. By considering the fusion protein as an agonist-activated enzyme and measuring V max of GTP hydrolysis a range of agonist ligands displayed varying efficacy in their capacity to activate the receptor-associated G protein with adrenaline=noradrenaline=α-methylnoradrenaline>UK14304>BHT933≥xylazine=clonidine. A similar rank order was observed following independent co-expression of the α 2A-adrenoceptor and Cys 351Gly-G i1α. 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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