Measurement of agonist efficacy using an α2A‐adrenoceptor‐Gi1α fusion protein

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Abstract
A fusion protein was constructed between the porcine α2A‐adrenoceptor and a pertussis toxin‐insensitive (Cys351Gly) form of the α 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]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 α2A‐adrenoceptor and Cys351Gly‐Gi1α. 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.