Pharmacological analysis of a dopamine D2Short:Gαo fusion protein expressed in Sf9 cells

FEBS Letters

Strange, Philip G.¹  Lopéz-Giménez, Juan F.¹  Wurch, Thierry²  Gazi, Lucien¹  Pauwels, Petrus J.² 

[1] School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 6AJ, UK;Department of Cellular and Molecular Biology, Centre de Recherche Pierre Fabre, Castres, France

关键词: D2 dopamine receptor;    G protein-coupled receptor;    Fusion protein;    Baculovirus;    [3H]Spiperone binding;    [35S]Guanosine-5′-O-(3-thio)triphosphate binding;    D2;    dopamine D2 receptor;    Gi/o;    heterotrimeric GTP binding protein type i or o;    GPCR;    G protein-coupled receptor;    GTPγS;    guanosine-5′-O-(3-thio)triphosphate;    NPA;    10;    11-dihydroxy-N-n-propylnorapomorphine;    PCR;    polymerase chain reaction;    PTX;    Bordetella pertussis toxin;    3-PPP;    3-(3-hydroxyphenyl)-N-propylpiperidine;   

DOI  :  10.1016/S0014-5793(03)00520-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A dopamine D_2Short receptor:G_αo fusion protein was expressed in Sf9 cells using the baculovirus expression system. [³H]Spiperone bound to D_2Short:G_αo with a pK _d≈10. Dopamine stimulated the binding of [³⁵S]guanosine-5′-O-(3-thio)triphosphate (GTPγS) to D_2Short:G_αo expressed with Gβ₁γ₂ (E _max>460%; pEC₅₀ 5.43±0.06). Most of the putative D₂ antagonists behaved as inverse agonists (suppressing basal [³⁵S]GTPγS binding) at D_2Short:G_αo/Gβ₁γ₂ although (−)-sulpiride and ziprasidone were neutral antagonists. Competition of [³H]spiperone binding by dopamine and 10,11-dihydroxy-N-n-propylnorapomorphine revealed two binding sites of different affinities, even in the presence of GTP (100 μM). The D_2Short:G_αo fusion protein is therefore a good model for characterising D₂ receptors.

【 授权许可】

Unknown