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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Some G protein heterotrimers physically dissociate in living cells
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Some G protein heterotrimers physically dissociate in living cells

机译：一些G蛋白异源三聚体在活细胞中物理解离

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摘要

Heterotrimeric G proteins mediate physiological processes ranging from phototransduction to cell migration. In the accepted model of G protein signaling, G alpha beta gamma heterotrimers physically dissociate after activation, liberating free G alpha subunits and G beta gamma dimers. This model is supported by evidence obtained in vitro with purified proteins, but its relevance in vivo has been questioned. Here, we show that at least some heterotrimeric G protein isoforms physically dissociate after activation in living cells. Ga subunits extended with a transmembrane (TM) domain and cyan fluorescent protein (CFP) were immobilized in the plasma membrane by biotinylation and cross-linking with avidin. Immobile CFP-TM-G alpha greatly decreased the lateral mobility of intracellular G beta(1 gamma 2)-YFP, indicating the formation of stable heterotrimers. A GTPase-deficient (constitutively active) mutant of CFP-TM-G alpha(oA) lost the ability to restrict G beta(1 gamma 2)-YFP mobility, whereas GTPase-deficient mutants of CFP-TM-G alpha(i3) and CFP-TM-G alpha(s), retained this ability. Activation of cognate G protein-coupled receptors partially relieved the constraint on G beta(1 gamma 2)-YFP mobility induced by immobile CFP-TM-GaoA and CFP-TM-Gai3 but had no effect on the constraint induced by CFP-TM-Gas. These results demonstrate the physical dissociation of heterotrimers containing G alpha(oA) and G alpha(i3) subunits in living cells, supporting the subunit dissociation model of G protein signaling for these subunits. However, these results are also consistent with the suggestion that G protein heterotrimers (e.g., G alpha(s)) may signal without physically dissociating.
机译：异三聚体G蛋白介导从光转导到细胞迁移的生理过程。在公认的G蛋白信号转导模型中，Gαβγ异源三聚体在激活后发生物理解离，释放出游离的Gα亚基和Gβγ二聚体。该模型得到体外纯化蛋白的证据的支持，但其在体内的相关性受到质疑。在这里，我们显示至少一些异三聚体G蛋白同工型在活细胞中激活后会物理解离。通过跨膜（TM）域和青色荧光蛋白（CFP）延伸的Ga亚基通过生物素化和与抗生物素蛋白的交联固定在质膜中。固定的CFP-TM-G alpha大大降低了细胞内G beta（1 gamma 2）-YFP的横向迁移性，表明形成了稳定的异三聚体。 CFP-TM-G alpha（oA）的GTPase缺陷型（组成型活性）突变体失去了限制G beta（1 gamma 2）-YFP迁移性的能力，而CFP-TM-G alpha（i3）的GTPase缺陷型突变型CFP-TM-G alpha保留了此功能。关联的G蛋白偶联受体的激活部分缓解了固定CFP-TM-GaoA和CFP-TM-Gai3诱导的对G beta（1 gamma 2）-YFP迁移的限制，但对CFP-TM-诱导的限制没有影响加油站。这些结果证明了活细胞中含有G alpha（oA）和G alpha（i3）亚基的异源三聚体的物理解离，支持了这些亚基的G蛋白信号转导亚基解离模型。但是，这些结果也与G蛋白异源三聚体（例如，Gα）可以发信号而不发生物理解离的暗示相一致。

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