FEBS Letters | |
Measurement of agonist efficacy using an α2A‐adrenoceptor‐Gi1α fusion protein | |
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 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.
【 授权许可】
Unknown
【 预 览 】
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RO201912020305326ZK.pdf | 979KB | download |