Awaji T, Hirasawa A, Kataoka M, Shinoura H, Nakayama Y, Sugawara T, Izumi S, Tsujimoto G
Department of Molecular and Cell Pharmacology, National Children's Medical Research Center, Tokyo, Japan.
Mol Endocrinol. 1998 Aug;12(8):1099-111. doi: 10.1210/mend.12.8.0149.
The study of G protein-coupled receptor signal transduction and behavior in living cells is technically difficult because of a lack of useful biological reagents. We show here that a fully functional alphalb-adrenoceptor tagged with the green fluorescent protein (alphalbAR/GFP) can be used to determine the molecular mechanism of intemalization of alphalbAR/ GFP in living cells. In mouse alphaT3 cells, alpha1bAR/GFP demonstrates strong, diffuse fluorescence along the plasma membrane when observed by confocal laser scanning microscope. The fluorescent receptor binds agonist and antagonist and stimulates phosphatidylinositol/Ca2+ signaling in a similar fashion to the wild receptor. In addition, alpha1bAR/ GFP can be internalized within minutes when exposed to agonist, and the subcellular redistribution of this receptor can be determined by measurement of endogenous fluorescence. The phospholipase C inhibitor U73,122, the protein kinase C activator PMA, and inhibitor staurosporine, and the Ca2+-ATPase inhibitor thapsigargin were used to examine the mechanism of agonist-promoted alphalbAR/GFP redistribution. Agonist-promoted internalization of alphalbAR/GFP was closely linked to phospholipase C activation and was dependent on protein kinase C activation, but was independent of the increase in intracellular free Ca2+ concentration. This study demonstrated that real-time optical monitoring of the subcellular localization of alphalbAR (as well as other G protein-coupled receptors) in living cells is feasible, and that this may provide a valuable system for further study of the biochemical mechanism(s) of agonist-induced receptor endocytosis.
由于缺乏有用的生物试剂，对活细胞中G蛋白偶联受体信号转导和行为的研究在技术上存在困难。我们在此表明，标记有绿色荧光蛋白的完全功能性α1b -肾上腺素能受体（α1bAR/GFP）可用于确定活细胞中α1bAR/GFP内化的分子机制。在小鼠αT3细胞中，当通过共聚焦激光扫描显微镜观察时，α1bAR/GFP在质膜上呈现强烈的、弥漫性荧光。荧光受体结合激动剂和拮抗剂，并以与野生型受体相似的方式刺激磷脂酰肌醇/Ca2+信号传导。此外，α1bAR/GFP在暴露于激动剂后几分钟内即可内化，并且该受体的亚细胞重新分布可通过测量内源性荧光来确定。使用磷脂酶C抑制剂U73122、蛋白激酶C激活剂PMA、抑制剂星形孢菌素以及Ca2+ -ATP酶抑制剂毒胡萝卜素，来研究激动剂促进的α1bAR/GFP重新分布的机制。激动剂促进的α1bAR/GFP内化与磷脂酶C激活密切相关，并依赖于蛋白激酶C激活，但与细胞内游离Ca2+浓度的增加无关。这项研究表明，对活细胞中α1bAR（以及其他G蛋白偶联受体）的亚细胞定位进行实时光学监测是可行的，并且这可能为进一步研究激动剂诱导的受体内吞作用的生化机制提供一个有价值的系统。
