The c-terminus of GRK3 indicates rapid dissociation of G protein heterotrimers.
The c-terminus of GRK3 indicates rapid dissociation of G protein heterotrimers.
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DOI：
10.1016/j.cellsig.2009.02.017
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发表时间：
2009-06
影响因子：
4.8
通讯作者：
Lambert NA
中科院分区：
文献类型：
--
作者：
Hollins B;Kuravi S;Digby GJ;Lambert NA
由异源三聚体G蛋白介导的信号通常在几十毫秒的过程中发展，可能需要G-αβγ异源三聚体的构象重排或完全物理解离。虽然已知一些活性的杂三聚体在稳定状态下解离(分为Gα和Gβγ)，但尚不清楚解离发生的速度是否足够快，以参与快速信号传递。在这里，我们发现包含GPCRkinase3(GRK3ct)c-末端和荧光蛋白天青素或Renilla荧光素酶的融合蛋白与Venus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融合蛋白是可自由扩散的，不与G蛋白形成预组装的复合体。GRK3ct融合蛋白与游离的Gβγ-V二聚体结合，但不与重排的异三聚体结合，因此可以高时间分辨率地报道G蛋白的解离。我们发现，在活细胞中，异三聚体可以在不到100毫秒内发生解离。在本实验条件下，MasGRK3ct融合蛋白与Gβγ-V的扩散和碰撞不受速率限制。这些结果表明，G蛋白异源三聚体可以足够快地解离，从而参与快速信号转导。
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 Gα βγ heterotrimers. Although it is known that some active heterotrimers are dissociated (into Gα and Gβγ) 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 Gβγ dimers (Gβγ-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 Gβγ-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 milliseconds. Under the conditions of these experiments diffusion and collision of masGRK3ct fusion proteins and Gβγ-V were not rate-limiting. These results indicate that G protein heterotrimers can dissociate quickly enough to participate in rapid signaling.
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通讯作者：
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