A COLORIMETRIC ASSAY FOR MEASURING ACTIVATION OF G(S)-COUPLED AND G(Q)-COUPLED SIGNALING PATHWAYS
A COLORIMETRIC ASSAY FOR MEASURING ACTIVATION OF G(S)-COUPLED AND G(Q)-COUPLED SIGNALING PATHWAYS
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DOI：
10.1006/abio.1995.1235
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发表时间：
1995-04-10
影响因子：
2.9
通讯作者：
CONE, RD
中科院分区：
文献类型：
--
作者：
CHEN, WB;SHIELDS, TS;CONE, RD
目前 G(s) 偶联受体功能激活的测定通常涉及腺苷酸环化酶的定量或通过放射免疫测定测量 cAMP 浓度。 G(q) 偶联受体的激活通常通过测量由磷脂酰肌醇 4,5-二磷酸产生的肌醇三磷酸或二酰甘油或细胞内钙的变化来测定。这些测定通常需要大量细胞 (10(5)-10(6)) 和/或使用放射性材料。我们开发了一种快速非放射性比色测定法，利用与五个拷贝的环 AMP 反应元件 (CRE) 融合的 β-半乳糖苷酶 (lacZ) 基因来检测由于细胞内 cAMP 或钙的增加而导致的 CRE 结合蛋白的激活。该测定可以使用 96 孔格式的少至 30,000 个细胞进行，并在酶标仪中同时测量最终产物。因此，一个人每天可以轻松检测 1000 个样本。使用该测定，β-半乳糖苷酶活性的倍数增加在幅度上与cAMP或腺苷酸环化酶活性的增加相似，并且在细胞内cAMP的0.01至0.27fmol/细胞之间近似线性。此外，使用该测定法对黑皮质素受体之一 mMC5-R 进行药理学表征，结果发现几种黑皮质素肽的效力顺序与通过常用的腺苷酸环化酶测定法获得的效力顺序相似。该测定对于 G(q) 偶联受体的表征也很有用，正如此处使用小鼠铃蟾肽受体转染的细胞所证明的那样。该测定的大规模能力使其成为筛选作用于 G(s) 和 G(q) 偶联受体的感兴趣分子的绝佳方法。 (C) 1995 学术出版社
Current assays for functional activation of G(s)-coupled receptors usually involve quantitation of adenylyl cyclase or measurement of cAMP concentration by radioimmunoassay. The activation of G(q)-coupled receptors is commonly assayed by measurement of the production of inositol triphosphate or diacylglycerol from phosphatidylinositol 4,5-bisphosphate or of changes in intracellular calcium. These assays generally require large numbers of cells (10(5)-10(6)) and/or the use of radioactive materials. We have developed a rapid nonradioactive colorimetric assay that utilizes a beta-galactosidase (lacZ) gene fused to five copies of the cyclic AMP response element (CRE) to detect the activation of CRE-binding protein that results from an increase in intracellular cAMP or calcium. This assay can be performed using as few as 30,000 cells in a 96-well format with the end products measured simultaneously in a microplate reader. Consequently, a single individual can readily assay 1000 samples a day. Using this assay, the fold increase in beta-galactosidase activity was similar in magnitude to increases in cAMP or adenylyl cyclase activity and was approximately linear from 0.01 to 0.27 fmol/cell of intracellular cAMP. Furthermore, pharmacological characterization of one of the melanocortin receptors, mMC5-R, using this assay resulted in a similar order of potency for several melanocortin peptides to that obtained with a commonly used adenylyl cyclase enzyme assay. This assay is also useful for the characterization of G(q)-coupled receptors as is demonstrated here using cells transfected with the mouse bombesin receptor. The large-scale capacity of this assay makes it an excellent method for screening molecules of interest acting on G(s)- and G(q)-coupled receptors. (C) 1995 Academic Press, Inc.