Rahmatullah M, Robishaw J D
Geisinger Clinic, Weis Center for Research, Danville, Pennsylvania 17822.
J Biol Chem. 1994 Feb 4;269(5):3574-80.
The heterotrimeric G proteins are often regarded functionally as a heterodimer, consisting of a guanine nucleotide-binding alpha subunit and a beta gamma subunit complex. Since the tightly associated beta gamma subunit complex can be separated only under denaturing conditions, studies aimed at determining the individual contributions of the beta and gamma subunits in terms of binding to the various alpha subunits, interacting with receptors, and regulating effectors, have not been possible. To circumvent this problem, we have used baculovirus-infected cells to direct the individual expression of the beta 1 and gamma 2 subunits. Application of extracts from baculovirus-infected cells to an alpha subunit of G protein (G(o) alpha)-affinity matrix resulted in the selective retention and AMF-specific elution of the expressed gamma 2 subunit, but not the expressed beta 1 subunit. Overall, these and other data provide the first evidence of a direct association between the gamma and alpha subunits, which is dependent on prenylation of gamma. The apparent direct association between the gamma and alpha subunits was further probed by limited trypsin proteolysis. Upon addition of trypsin, the G(o) alpha subunit was rapidly cleaved to a 24-kDa fragment. However, in the presence of the purified gamma 2 subunit, trypsin cleavage of the G(o) alpha subunit was completely prevented. This demonstration of a direct association between the gamma and alpha subunits is particularly intriguing in light of the increasingly large number of known alpha, beta, and gamma subunits, which raises important questions regarding the assembly of these subunits into functionally distinct G proteins. Thus, a direct association between the gamma and alpha subunits, which exhibit the greatest structural diversity, may provide the basis for the selective assembly of these subunits into G proteins with functional diversity.
异源三聚体G蛋白在功能上常被视为由一个鸟嘌呤核苷酸结合α亚基和一个βγ亚基复合物组成的异二聚体。由于紧密结合的βγ亚基复合物只有在变性条件下才能分离，因此旨在确定β和γ亚基在与各种α亚基结合、与受体相互作用以及调节效应器方面各自贡献的研究一直无法进行。为了解决这个问题，我们利用杆状病毒感染的细胞来指导β1和γ2亚基的单独表达。将杆状病毒感染细胞的提取物应用于G蛋白α亚基（G(o)α）亲和基质，导致表达的γ2亚基被选择性保留并通过AMF特异性洗脱，但表达的β1亚基没有。总体而言，这些及其他数据首次证明了γ和α亚基之间的直接关联，这种关联依赖于γ的异戊二烯化。通过有限的胰蛋白酶蛋白水解进一步探究了γ和α亚基之间明显的直接关联。加入胰蛋白酶后，G(o)α亚基迅速被切割成一个24 kDa的片段。然而，在纯化的γ2亚基存在的情况下，胰蛋白酶对G(o)α亚基的切割被完全阻止。鉴于已知的α、β和γ亚基数量越来越多，γ和α亚基之间直接关联的这一证明尤其引人入胜，这就提出了关于这些亚基组装成功能不同的G蛋白的重要问题。因此，γ和α亚基之间的直接关联（它们表现出最大的结构多样性）可能为这些亚基选择性组装成具有功能多样性的G蛋白提供基础。
