Burford N T, Wang D, Sadée W
Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, School of Pharmacy, Box 0446, University of California, San Francisco, CA 94143-0446, USA.
Biochem J. 2000 Jun 15;348 Pt 3(Pt 3):531-7.
To determine mu-opioid receptor (OP(3)) signalling activity, guanosine 5'-[gamma-[(35)S]thio]triphosphate (GTP[(35)S]) binding to G-proteins was measured in the membranes of human embryonic kidney cells (HEK-293) transfected with mu-opioid receptor (HEK-mu). GTP[(35)S] binding to HEK-mu membranes was significantly elevated compared with HEK-293 control membranes (without OP(3)), and this was abolished by pertussis-toxin pretreatment. The irreversible antagonist beta-chlornaltrexamine (beta-CNA) dose-dependently decreased elevated basal G-protein coupling of HEK-mu to control levels in cells devoid of OP(3). This characterizes beta-CNA as an inverse OP(3) agonist. Immunoprecipitation of solubilized G-proteins with G(i3)alpha antisera demonstrated that basal GTP[(35)S] binding to G(i3)alpha was also substantially elevated in HEK-mu membranes over the control, whereas G(i3)alpha protein levels were unchanged. Basal GTP[(35)S] binding to G(i1)alpha/G(i2)alpha and G(o)alpha was also increased twofold in HEK-mu membranes over the control. Morphine further increased coupling to each of these Galpha proteins with similar potency, but not to G(q)/(11)alpha or G(s)alpha. These results indicate that the wild-type OP(3) can couple constitutively to endogenously expressed G(i3)alpha, G(i1)alpha/G(i2)alpha and G(o)alpha subunits of G-proteins in HEK-293 cells.
为了确定μ-阿片受体（OP(3)）的信号传导活性，我们在转染了μ-阿片受体（HEK-mu）的人胚肾细胞（HEK-293）膜中测量了鸟苷5'-[γ-[(35)S]硫代]三磷酸（GTP[(35)S]）与G蛋白的结合。与HEK-293对照膜（无OP(3)）相比，GTP[(35)S]与HEK-mu膜的结合显著升高，且这一现象被百日咳毒素预处理所消除。不可逆拮抗剂β-氯诺美沙明（β-CNA）在缺乏OP(3)的细胞中，剂量依赖性地将HEK-mu升高的基础G蛋白偶联降低至对照水平。这表明β-CNA是一种反向OP(3)激动剂。用G(i3)α抗血清对溶解的G蛋白进行免疫沉淀表明，与对照相比，HEK-mu膜中基础GTP[(35)S]与G(i3)α的结合也显著升高，而G(i3)α蛋白水平未变。HEK-mu膜中基础GTP[(35)S]与G(i1)α/G(i2)α和G(o)α的结合也比对照增加了两倍。吗啡以相似的效力进一步增强了与这些Gα蛋白中每一种的偶联，但对G(q)/(11)α或G(s)α则无此作用。这些结果表明，野生型OP(3)可在HEK-293细胞中组成性地与内源性表达的G蛋白的G(i3)α、G(i1)α/G(i2)α和G(o)α亚基偶联。
