Grygorczyk R, Abramovitz M, Boie Y, Bastien L, Adam M
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada.
Anal Biochem. 1995 May 1;227(1):27-31. doi: 10.1006/abio.1995.1248.
To detect heterologous expression of receptors coupled via G proteins to the stimulation of adenylate cyclase in Xenopus laevis oocytes, the receptor of interest is coexpressed with the cystic fibrosis transmembrane conductance regulator (CFTR)--a cAMP-dependent Cl- channel. The binding of an agonist to the expressed receptor stimulates adenylate cyclase resulting in intracellular cAMP elevation, which in turn activates the CFTR. The CFTR-mediated Cl- current response is then measured using the standard two-electrode voltage-clamp technique. This method has allowed us to detect functional expression in oocytes of the human EP2 and IP prostanoid receptors. This method should prove valuable for expression and identification of putative G protein-coupled receptors signaling through stimulation of adenylate cyclase, for structure/function studies, and for analysis of receptor antagonists and agonists.
为了检测在非洲爪蟾卵母细胞中通过G蛋白偶联刺激腺苷酸环化酶的受体的异源表达，将感兴趣的受体与囊性纤维化跨膜电导调节因子（CFTR）——一种cAMP依赖性氯离子通道共表达。激动剂与表达的受体结合会刺激腺苷酸环化酶，导致细胞内cAMP升高，进而激活CFTR。然后使用标准的双电极电压钳技术测量CFTR介导的氯离子电流反应。该方法使我们能够检测人EP2和IP前列腺素受体在卵母细胞中的功能性表达。该方法对于通过刺激腺苷酸环化酶进行信号传导的假定G蛋白偶联受体的表达和鉴定、结构/功能研究以及受体拮抗剂和激动剂的分析应该是有价值的。
