Digby Gregory J, Lober Robert M, Sethi Pooja R, Lambert Nevin A
Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA, 30912, USA.
Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17789-94. doi: 10.1073/pnas.0607116103. Epub 2006 Nov 9.
Heterotrimeric G proteins mediate physiological processes ranging from phototransduction to cell migration. In the accepted model of G protein signaling, Galphabetagamma heterotrimers physically dissociate after activation, liberating free Galpha subunits and Gbetagamma 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. Galpha 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-Galpha greatly decreased the lateral mobility of intracellular Gbeta1gamma2-YFP, indicating the formation of stable heterotrimers. A GTPase-deficient (constitutively active) mutant of CFP-TM-GalphaoA lost the ability to restrict Gbeta1gamma2-YFP mobility, whereas GTPase-deficient mutants of CFP-TM-Galphai3 and CFP-TM-Galphas retained this ability. Activation of cognate G protein-coupled receptors partially relieved the constraint on Gbeta1gamma2-YFP mobility induced by immobile CFP-TM-GalphaoA and CFP-TM-Galphai3 but had no effect on the constraint induced by CFP-TM-Galphas. These results demonstrate the physical dissociation of heterotrimers containing GalphaoA and Galphai3 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., Galphas) may signal without physically dissociating.
异源三聚体G蛋白介导从光转导到细胞迁移等多种生理过程。在公认的G蛋白信号传导模型中，Gαβγ异源三聚体在激活后会发生物理解离，释放出游离的Gα亚基和Gβγ二聚体。该模型得到了体外纯化蛋白实验证据的支持，但其在体内的相关性一直受到质疑。在这里，我们表明至少一些异源三聚体G蛋白亚型在活细胞激活后会发生物理解离。通过生物素化和与抗生物素蛋白交联，将带有跨膜（TM）结构域和青色荧光蛋白（CFP）的Gα亚基固定在质膜中。固定不动的CFP-TM-Gα极大地降低了细胞内Gβ1γ2-YFP的侧向流动性，表明形成了稳定的异源三聚体。CFP-TM-GαoA的GTP酶缺陷型（组成型激活）突变体失去了限制Gβ1γ2-YFP流动性的能力，而CFP-TM-Gαi3和CFP-TM-Gαs的GTP酶缺陷型突变体保留了这种能力。同源G蛋白偶联受体的激活部分缓解了由固定不动的CFP-TM-GαoA和CFP-TM-Gαi3对Gβ1γ2-YFP流动性的限制，但对CFP-TM-Gαs诱导的限制没有影响。这些结果证明了在活细胞中含有GαoA和Gαi3亚基的异源三聚体发生了物理解离，支持了这些亚基的G蛋白信号传导亚基解离模型。然而，这些结果也与G蛋白异源三聚体（如Gαs）可能在不发生物理解离的情况下进行信号传导的观点一致。
