Some G protein heterotrimers physically dissociate in living cells
Some G protein heterotrimers physically dissociate in living cells
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DOI：
10.1073/pnas.0607116103
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发表时间：
2006-11-21
影响因子：
11.1
通讯作者：
Lambert, Nevin A.
中科院分区：
文献类型：
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作者：
Digby, Gregory J.;Lober, Robert M.;Lambert, Nevin A.
异源三聚体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，G α β γ异源三聚体在活化后物理解离，释放游离的G α亚基和G β γ二聚体。该模型得到了纯化蛋白体外实验证据的支持，但其在体内的相关性受到质疑。在这里，我们表明，至少有一些异源三聚体G蛋白异构体在活细胞中激活后物理解离。通过生物素化和与亲和素交联将具有跨膜（TM）结构域的Ga亚基和青色荧光蛋白（CFP）固定在质膜上。固定的CFP-TM-G α大大降低了细胞内G β（1 γ 2）-YFP的横向迁移率，表明形成了稳定的异源三聚体。CFP-TM-G α（oA）的GTP酶缺陷（组成型活性）突变体失去了限制G β（1 γ 2）-YFP迁移的能力，而CFP-TM-G α（i3）和CFP-TM-G α（s）的GTP酶缺陷突变体保留了这种能力。同源G蛋白偶联受体的激活部分地缓解了由固定的CFP-TM-GaoA和CFP-TM-Gai 3诱导的对G β（1 γ 2）-YFP移动性的限制，但对由CFP-TM-Gas诱导的限制没有影响。这些结果证明了活细胞中含有G α（α A）和G α（β 3）亚基的异源三聚体的物理解离，支持了这些亚基的G蛋白信号传导的亚基解离模型。然而，这些结果也与G蛋白异源三聚体（例如，G alpha（s））可以在没有物理解离的情况下发出信号。
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.