Functional analysis of a human A1 adenosine receptor/green fluorescent protein/Gi1α fusion protein following stable expression in CHO cells

FEBS Letters

Palmer, Tim¹  Drmota, Tomas¹  Coote, Jim³  Rees, Stephen⁴  Wise, Alan²  Milligan, Graeme¹  Bevan, Nicola⁴ 

[1] Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK;Molecular Pharmacology, Glaxo Wellcome Research and Development, Gunnels Wood Road, Stevenage, Herts. SG1 2NY, UK;Biomolecular Structure, Glaxo Wellcome Research and Development, Gunnels Wood Road, Stevenage, Herts. SG1 2NY, UK;Biological Chemistry, Glaxo Wellcome Research and Development, Gunnels Wood Road, Stevenage, Herts. SG1 2NY, UK

关键词: G-protein coupled receptor;    A1 adenosine receptor;    Green fluorescent protein;    Fusion protein;    A1/Gi;    fusion protein between the human A1 adenosine receptor and rat Gi1α;    A1/GFP/Gi;    fusion protein between the human A1 adenosine receptor;    GFP and rat Gi1α;    CHO;    Chinese hamster ovary;    GFP;    green fluorescent protein;    G-protein;    guanine nucleotide binding protein;    GPCR;    G-protein coupled receptor;    [35S]GTPγS;    guanosine 5′-[γ35S]triphosphate;    HEK;    human embryonic kidney;    MAP kinase;    mitogen activated protein kinase;    NECA;    5′-N-ethylcarboxamidoadenosine;   

DOI  :  10.1016/S0014-5793(99)01467-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Fusion proteins between the human A₁ adenosine receptor and the pertussis toxin resistant (Cys351Gly) mutant of the G-protein α subunit G_i1α (A1/Gi), and between the human A₁ adenosine receptor, the Aequorea victoria green fluorescent protein (GFP) and Cys351Gly G_i1α (A1/GFP/Gi), were expressed in CHO cells. The agonist NECA caused a stimulation of [³⁵S]GTPγS binding at both fusion proteins with similar concentration dependence as at the native receptor. However in the presence of pertussis toxin NECA stimulation of [³⁵S]GTPγS binding was only seen at the A1/GFP/Gi fusion protein. The regulation of the adenylyl cyclase and MAP kinase effector systems by both fusion proteins was attenuated following pertussis toxin treatment. These studies demonstrate for the first time the characterisation of a fusion protein between a G-protein coupled receptor, GFP and a G-protein α subunit.

【 授权许可】

Unknown