Groarke D A, Wilson S, Krasel C, Milligan G
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Davidson Building, University of Glasgow, Glasgow G12 8QQ, United Kingdom.
J Biol Chem. 1999 Aug 13;274(33):23263-9. doi: 10.1074/jbc.274.33.23263.
A fusion protein (beta-arrestin-1-green fluorescent protein (GFP)) was constructed between beta-arrestin-1 and a modified form of the green fluorescent protein from Aequorea victoria. Expression in HEK293 cells allowed immunological detection of an 82-kDa cytosolic polypeptide with antisera to both beta-arrestin-1 and GFP. Transient expression of this construct in HEK293 cells stably transfected to express the rat thyrotropin-releasing hormone receptor-1 (TRHR-1) followed by confocal microscopy allowed its visualization evenly distributed throughout the cytoplasm. Addition of thyrotropin-releasing hormone (TRH) caused a profound and rapid redistribution of beta-arrestin-1-GFP to the plasma membrane followed by internalization of beta-arrestin-1-GFP into distinct, punctate, intracellular vesicles. TRH did not alter the cellular distribution of GFP transiently transfected into these cells nor the distribution of beta-arrestin-1-GFP following expression in HEK293 cells lacking the receptor. To detect potential co-localization of the receptor and beta-arrestin-1 in response to agonist treatment, beta-arrestin-1-GFP was expressed stably in HEK293 cells. A vesicular stomatitis virus (VSV)-tagged TRHR-1 was then introduced transiently. Initially, the two proteins were fully resolved. Short term exposure to TRH resulted in their plasma membrane co-localization, and sustained exposure to TRH resulted in their co-localization in punctate, intracellular vesicles. In contrast, beta-arrestin-1-GFP did not relocate or adopt a punctate appearance in cells that did not express VSV-TRHR-1. Reciprocal experiments were performed, with equivalent results, following transient expression of beta-arrestin-1 into cells stably expressing VSVTRHR-1-GFP. These results demonstrate the capacity of beta-arrestin-1-GFP to interact with the rat TRHR-1 and directly visualizes their recruitment from cytoplasm and plasma membrane respectively into overlapping, intracellular vesicles in an agonist-dependent manner.
在β-抑制蛋白1与一种来自维多利亚水母的绿色荧光蛋白的修饰形式之间构建了一种融合蛋白（β-抑制蛋白1-绿色荧光蛋白（GFP））。在HEK293细胞中表达后，可用针对β-抑制蛋白1和GFP的抗血清对一种82 kDa的胞质多肽进行免疫检测。将该构建体在稳定转染以表达大鼠促甲状腺激素释放激素受体-1（TRHR-1）的HEK293细胞中瞬时表达，随后通过共聚焦显微镜观察发现其均匀分布于整个细胞质中。添加促甲状腺激素释放激素（TRH）会导致β-抑制蛋白1-GFP迅速大量重新分布到质膜，随后β-抑制蛋白1-GFP内化到不同的、点状的细胞内小泡中。TRH不会改变瞬时转染到这些细胞中的GFP的细胞分布，也不会改变在缺乏该受体的HEK293细胞中表达后的β-抑制蛋白1-GFP的分布。为了检测激动剂处理后受体与β-抑制蛋白1的潜在共定位情况，在HEK293细胞中稳定表达β-抑制蛋白1-GFP。然后瞬时导入一种水疱性口炎病毒（VSV）标记的TRHR-1。最初，这两种蛋白完全分离。短期暴露于TRH会导致它们在质膜上共定位，而持续暴露于TRH会导致它们在点状的细胞内小泡中共定位。相反，在不表达VSV-TRHR-1的细胞中，β-抑制蛋白1-GFP不会重新定位或呈现点状外观。在稳定表达VSVTRHR-1-GFP的细胞中瞬时表达β-抑制蛋白1后进行了反向实验，结果相同。这些结果证明了β-抑制蛋白1-GFP与大鼠TRHR-1相互作用的能力，并直接观察到它们分别以激动剂依赖的方式从细胞质和质膜募集到重叠的细胞内小泡中。
