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

FEBS Letters

Carr, I.Craig¹  Groarke, D.Alex¹  Wise, Alan¹  Milligan, Graeme¹ 

[1] Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute Of Biomedical and Life Sciences, Davidson Building, University of Glasgow, Glasgow G12 8QQ, UK

关键词: G protein;    Receptor;    Efficacy;    Adrenaline;    GPCR;    G protein-coupled receptor;    G protein;    guanine nucleotide binding protein;    ORF;    open reading frame;    DMEM;    Dulbecco's modification of Eagle's medium;    Cys351Gly;    codon 351 converted from cysteine to glycine;   

DOI  :  10.1016/S0014-5793(97)01431-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A fusion protein was constructed between the porcine α_2A-adrenoceptor and a pertussis toxin-insensitive (Cys³⁵¹Gly) 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 Cys³⁵¹Gly-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.

【 授权许可】

Unknown