Analysis of G protein βγ dimer formation in live cells using multicolor bimolecular fluorescence complementation demonstrates preferences of β1 for particular γ subunits
Analysis of G protein βγ dimer formation in live cells using multicolor bimolecular fluorescence complementation demonstrates preferences of β1 for particular γ subunits
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DOI：
10.1124/mol.106.022616
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发表时间：
2006-07-01
影响因子：
3.6
通讯作者：
Berlot, Catherine H.
中科院分区：
文献类型：
--
作者：
Mervine, Stacy M.;Yost, Evan A.;Berlot, Catherine H.
体内敲除证明的G蛋白β - γ信号的特异性大于基于β - γ功能的体外测定的预期。在这项研究中，我们通过比较七种含有γ(1)、γ(2)、γ(5)、γ(7)、γ(10)、γ(11)或γ(12)的β (1) γ复合物与α(5)相互作用的能力，以及这些γ亚基在活的人胚胎肾（HEK） 293细胞中与β(1)相互作用的竞争能力，研究了这种差异的基础。利用双分子荧光互补对β - γ复合物进行成像，其中荧光是由青色荧光蛋白（CFP）或黄色荧光蛋白（YFP）的两个非荧光片段（N和C）通过融合到每个片段的蛋白质聚集在一起时产生的。α (s)-CFP的质膜靶向性与其表达水平成反比，而yfp - n - β (1) yfp - c - γ复合物增加这种靶向性的能力变化幅度为2倍或更少。然而，在cfp - n - γ亚基与cfp - c - β竞争关联的能力方面存在较大差异(1)。当在cfp - c - β(1)有限的条件下比较cfp - c - β(1)、cfp - n - γ（青色）和cfp - c - β (1) yfp - n - γ(2)（黄色）复合物的共表达强度时，cfp - n - γ(12)亚基在与cfp - c - β(1)的关联方面表现出与yfp - n - γ(2)竞争能力的4.5倍范围。CFP-N-gamma（12）和CFP-N-gamma(1)分别是最强和最弱的竞争者。结合先前对β - γ在受体信号传导特异性中的作用的证明，这些结果表明，共同表达的β和γ亚基相互关联偏好的差异可以决定哪些复合物在完整细胞中占主导地位并参与信号传导途径。
The specificity of G protein beta gamma signaling demonstrated by in vivo knockouts is greater than expected based on in vitro assays of beta gamma function. In this study, we investigated the basis for this discrepancy by comparing the abilities of seven beta(1)gamma complexes containing gamma(1), gamma(2), gamma(5), gamma(7), gamma(10), gamma(11), or gamma(12) to interact with alpha(s) and of these gamma subunits to compete for interaction with beta(1) in live human embryonic kidney (HEK) 293 cells. beta gamma complexes were imaged using bimolecular fluorescence complementation, in which fluorescence is produced by two nonfluorescent fragments (N and C) of cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) when brought together by proteins fused to each fragment. Plasma membrane targeting of alpha(s)-CFP varied inversely with its expression level, and the abilities of YFP-N-beta(1) YFP-C-gamma complexes to increase this targeting varied by 2-fold or less. However, there were larger differences in the abilities of the CFP-N-gamma subunits to compete for association with CFP-C-beta(1). When the intensities of coexpressed CFP-C-beta(1) CFP-N-gamma (cyan) and CFP-C-beta(1) YFP-N-gamma(2) ( yellow) complexes were compared under conditions in which CFP-C-beta(1) was limiting, the CFP-N-gamma(12) subunits exhibited a 4.5-fold range in their abilities to compete with YFP-N-gamma(2) for association with CFP-C-beta(1). CFP-N-gamma(12) and CFP-N-gamma(1) were the strongest and weakest competitors, respectively. Taken together with previous demonstrations of a role for beta gamma in the specificity of receptor signaling, these results suggest that differences in the association preferences of coexpressed beta and gamma subunits for each other can determine which complexes predominate and participate in signaling pathways in intact cells.