Stables J, Green A, Marshall F, Fraser N, Knight E, Sautel M, Milligan G, Lee M, Rees S
Receptor Systems Unit, Glaxo Wellcome Research and Development, Stevenage, Herts, United Kingdom.
Anal Biochem. 1997 Oct 1;252(1):115-26. doi: 10.1006/abio.1997.2308.
Transient expression of apoaequorin in Chinese hamster ovary (CHO) cells and reconstitution with the co-factor coelenterazine resulted in a large, concentration-dependent agonist-mediated luminescent response following cotransfection with the endothelin ETA, angiotensin ATII, thyrotropin-releasing hormone (TRH), and neurokinin NK1 receptors, all of which interact pre-dominantly with the G alpha q-like phosphoinositidase-linked G-proteins. A substantially greater luminescence was obtained with mitochondrially targeted apoaequorin compared to cytoplasmically expressed apoaequorin. To generate a system amenable for the study of agonist activity at virtually any G-protein-coupled receptor the alpha subunit of the receptor promiscuous G-protein G alpha 16 was either transiently or stably expressed in CHO cells together with apoaequorin. In cells expressing G alpha 16, but not in its absence, agonists at a series of receptors which normally interact with either G alpha s or G alpha i were now able to cause a luminescent response from mitochondrially targeted apoaequorin. In the case of the A1 adenosine receptor, this response was clearly a result of activation of G alpha 16 and not a consequence of the release of the G alpha i-associated beta/gamma complex, as the luminescent response was unaffected by pertussis toxin treatment of the cells, whereas agonist-mediated inhibition of adenylyl cyclase activity was attenuated. These studies describe the use of coexpressed apoaequorin as a reporter for G-protein-coupled receptor-mediated calcium signaling. Furthermore, coexpression of G alpha 16 and apoaequorin provides a basis for a generic mammalian cell microplate assay for the assessment of agonist action at virtually any G-protein-coupled receptor, including orphan receptors for which the physiological signal transduction mechanism may be unknown.
在中国仓鼠卵巢（CHO）细胞中瞬时表达脱辅基水母发光蛋白，并与辅因子腔肠素重组，在与内皮素ETA、血管紧张素ATII、促甲状腺激素释放激素（TRH）和神经激肽NK1受体共转染后，会产生一种强烈的、浓度依赖性的激动剂介导的发光反应，所有这些受体主要与Gαq样磷酸肌醇酶偶联的G蛋白相互作用。与细胞质中表达的脱辅基水母发光蛋白相比，线粒体靶向的脱辅基水母发光蛋白产生的发光要大得多。为了构建一个几乎适用于研究任何G蛋白偶联受体激动剂活性的系统，受体通用G蛋白Gα16的α亚基与脱辅基水母发光蛋白一起在CHO细胞中瞬时或稳定表达。在表达Gα16的细胞中，而不是在缺乏Gα16的细胞中，一系列通常与Gαs或Gαi相互作用的受体的激动剂现在能够引起线粒体靶向的脱辅基水母发光蛋白产生发光反应。就A1腺苷受体而言，这种反应显然是Gα16激活的结果，而不是与Gαi相关的β/γ复合物释放的结果，因为发光反应不受百日咳毒素处理细胞的影响，而激动剂介导的腺苷酸环化酶活性抑制作用减弱。这些研究描述了共表达的脱辅基水母发光蛋白作为G蛋白偶联受体介导的钙信号报告物的用途。此外，Gα16和脱辅基水母发光蛋白的共表达为一种通用的哺乳动物细胞微孔板测定法提供了基础，用于评估几乎任何G蛋白偶联受体的激动剂作用，包括其生理信号转导机制可能未知的孤儿受体。
