Heterotrimeric G proteins form stable complexes with adenylyl cyclase and Kir3.1 channels in living cells
Heterotrimeric G proteins form stable complexes with adenylyl cyclase and Kir3.1 channels in living cells
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DOI：
10.1242/jcs.03021
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发表时间：
2006-07-01
影响因子：
4
通讯作者：
Hebert, Terence E.
中科院分区：
文献类型：
--
作者：
Rebois, R. Victor;Robitaille, Melanie;Hebert, Terence E.
生物发光共振能量转移（BRET）和免疫共沉淀实验表明，异源三聚体G蛋白和它们的效应器被发现在稳定的复合物，持续在信号转导过程中。腺苷酸环化酶、Kir3.1通道亚基和几种G蛋白亚基（G α（s）、G α（i）、G β（1）和G γ（2））用荧光素酶（RLuc）或GFP或YFP的互补片段（特别是G β（1）-YFP 1 -158和G γ（2）-YFP 159 -238，其异源二聚化产生荧光YFP-G β（1）γ（2））标记。在腺苷酸环化酶-RLuc或Kir3.1-RLuc与GFP-G γ（2）、GFP-G β（1）或YFP-G β（1 γ 2）之间观察到BRET。G α亚基也与两种效应子稳定相关，无论信号转导是否由受体激动剂启动。虽然BRET效应和G β γ受体刺激增加，我们的数据表明，这些变化很可能是构象的性质。此外，受体敏感的G蛋白效应复合物可以被检测到之前被运送到质膜，提供了第一个直接证据的细胞内网站的组装。
Bioluminescence resonance energy transfer (BRET) and co-immunoprecipitation experiments revealed that heterotrimeric G proteins and their effectors were found in stable complexes that persisted during signal transduction. Adenylyl cyclase, Kir3.1 channel subunits and several G-protein subunits (G alpha(s), G alpha(i), G beta(1) and G gamma(2)) were tagged with luciferase (RLuc) or GFP, or the complementary fragments of YFP (specifically G beta(1)-YFP1-158 and G gamma(2)-YFP159-238, which heterodimerize to produce fluorescent YFP-G beta(1)gamma(2)). BRET was observed between adenylyl-cyclase-RLuc or Kir3.1-RLuc and GFP-G gamma(2), GFP-G beta(1) or YFP-G beta(1 gamma 2). G alpha subunits were also stably associated with both effectors regardless of whether or not signal transduction was initiated by a receptor agonist. Although BRET between effectors and G beta gamma was increased by receptor stimulation, our data indicate that these changes are likely to be conformational in nature. Furthermore, receptor-sensitive G-protein-effector complexes could be detected before being transported to the plasma membrane, providing the first direct evidence for an intracellular site of assembly.