Hollins Bettye, Kuravi Sudhakiranmayi, Digby Gregory J, Lambert Nevin A
Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912, USA.
Cell Signal. 2009 Jun;21(6):1015-21. doi: 10.1016/j.cellsig.2009.02.017. Epub 2009 Mar 1.
Signals mediated by heterotrimeric G proteins often develop over the course of tens of milliseconds, and could require either conformational rearrangement or complete physical dissociation of Galphabetagamma heterotrimers. Although it is known that some active heterotrimers are dissociated (into Galpha and Gbetagamma) at steady-state, it is not clear that dissociation occurs quickly enough to participate in rapid signaling. Here we show that fusion proteins containing the c-terminus of GPCR kinase 3 (GRK3ct) and either the fluorescent protein cerulean or Renilla luciferase bind to venus-labeled Gbetagamma dimers (Gbetagamma-V), resulting in Förster or bioluminescence resonance energy transfer (FRET or BRET). GRK3ct fusion proteins are freely-diffusible, and do not form preassembled complexes with G proteins. GRK3ct fusion proteins bind to free Gbetagamma-V dimers but not to rearranged heterotrimers, and thus can report G protein dissociation with high temporal resolution. We find that heterotrimer dissociation can occur in living cells in less than 100 ms. Under the conditions of these experiments diffusion and collision of masGRK3ct fusion proteins and Gbetagamma-V were not rate-limiting. These results indicate that G protein heterotrimers can dissociate quickly enough to participate in rapid signaling.
由异源三聚体G蛋白介导的信号通常在几十毫秒内产生，可能需要Gαβγ异源三聚体进行构象重排或完全物理解离。虽然已知一些活性异源三聚体在稳态下会解离（成Gα和Gβγ），但尚不清楚解离是否足够快以参与快速信号传导。在这里，我们表明，含有GPCR激酶3（GRK3ct）的c末端以及荧光蛋白天蓝蛋白或海肾荧光素酶的融合蛋白与金星标记的Gβγ二聚体（Gβγ-V）结合，导致Förster或生物发光共振能量转移（FRET或BRET）。GRK3ct融合蛋白可自由扩散，不会与G蛋白形成预组装复合物。GRK3ct融合蛋白与游离的Gβγ-V二聚体结合，但不与重排的异源三聚体结合，因此可以高时间分辨率报告G蛋白解离。我们发现异源三聚体解离可在不到100毫秒内在活细胞中发生。在这些实验条件下，masGRK3ct融合蛋白与Gβγ-V的扩散和碰撞不是限速因素。这些结果表明，G蛋白异源三聚体可以足够快地解离以参与快速信号传导。
