Quantitative analysis of a cysteine351glycine mutation in the G protein Gi1α: effect on α2A‐adrenoceptor‐Gi1α fusion protein activation

FEBS Letters

Wright, Jason¹  Carr, I.Craig¹  Burt, Andrew R¹  Rees, Stephen²  Wise, Alan²  Jackson, Vicky N¹  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, Scotland, UK;Receptor Systems Unit, Glaxo-Wellcome Research and Development, Stevenage, Hertfordshire SG1 2NY, UK

关键词: Receptor;    G protein;    Agonist;    Adrenaline;    α2AR-Gi1α;    fusion protein containing the porcine α2A-adrenoceptor linked to the α subunit of Gi1;    α2AR-Cys351GlyGi1α;    fusion protein containing the porcine α2A-adrenoceptor linked to the α subunit of Gi1 in which cysteine351 was converted to glycine;   

DOI  :  10.1016/S0014-5793(98)00476-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Fusion proteins were constructed between the porcine α_2A-adrenoceptor and either wild-type (Cys³⁵¹) or a pertussis toxin-resistant (Gly³⁵¹) form of the G protein G_i1α. Addition of adrenaline to membranes expressing the fusion proteins resulted in concentration-dependent stimulation of their high affinity GTPase activity. The α_2A-adrenoceptor-wild type G_i1α fusion protein produced substantially higher maximal stimulation of GTPase activity in response to adrenaline than that containing Gly³⁵¹ G_i1α. Treatment of the fusion proteins as agonist-regulated enzymes allowed measurement of V _max and turnover number for adrenaline-stimulation of the GTPase activity of each fusion construct. The turnover number of the α_2A-adrenoceptor-Cys³⁵¹Gly G_i1α fusion protein was only 44% of that for the α_2A-adrenoceptor-wild type G_i1α fusion protein. These data provide the first direct quantitative evaluation of the effects of a mutation of a G protein on the capacity of an agonist-occupied receptor to activate the mutant.

【 授权许可】

Unknown