Hynes Thomas R, Yost Evan A, Yost Stacy M, Berlot Catherine H
Weis Center for Research, Geisinger Clinic, Danville, PA, USA.
Methods Mol Biol. 2011;756:229-43. doi: 10.1007/978-1-61779-160-4_12.
Cells co-express multiple G protein β and γ subunit isoforms, but the extent to which individual subunits associate to form particular βγ complexes is not known. This issue is important because in vivo knockout experiments suggest that specific βγ complexes may have unique functions despite the fact that most complexes exhibit similar properties when assayed in reconstituted systems. This chapter describes how multicolor bimolecular fluorescence complementation (BiFC) can be used in living cells to study the association preferences of β and γ subunits. Multicolor BiFC determines the association preferences of these subunits by quantifying the two fluorescent complexes formed when β or γ subunits fused to amino terminal fragments of yellow fluorescent protein (YFP-N) and cyan fluorescent protein (CFP-N) compete for interaction with limiting amounts of a common γ or β subunit, respectively, fused to a carboxyl terminal fragment of CFP (CFP-C). One means by which βγ complexes may differ from each other and thereby mediate unique functions in vivo is in the kinetics and patterns of their internalization responses to stimulation of G protein-coupled receptors (GPCRs). Methods are described for imaging and quantifying the internalization of pairs of βγ complexes in response to GPCR stimulation in living cells.
细胞共表达多种G蛋白β和γ亚基异构体，但单个亚基形成特定βγ复合物的程度尚不清楚。这个问题很重要，因为体内敲除实验表明，尽管大多数复合物在重组系统中检测时表现出相似的特性，但特定的βγ复合物可能具有独特的功能。本章描述了如何利用多色双分子荧光互补（BiFC）在活细胞中研究β和γ亚基的结合偏好。多色BiFC通过量化当与黄色荧光蛋白（YFP-N）和青色荧光蛋白（CFP-N）的氨基末端片段融合的β或γ亚基分别与有限量的与CFP羧基末端片段（CFP-C）融合的共同γ或β亚基竞争相互作用时形成的两种荧光复合物，来确定这些亚基的结合偏好。βγ复合物彼此不同并因此在体内介导独特功能的一种方式是它们对G蛋白偶联受体（GPCR）刺激的内化反应的动力学和模式。本文描述了在活细胞中成像和量化βγ复合物对GPCR刺激的内化反应的方法。
