Activation of p115-RhoGEF Requires Direct Association of Gα13 and the Dbl Homology Domain
Activation of p115-RhoGEF Requires Direct Association of Gα13 and the Dbl Homology Domain
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DOI：
10.1074/jbc.m111.333716
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发表时间：
2012-07-20
影响因子：
4.8
通讯作者：
Sternweis, Paul C.
中科院分区：
文献类型：
--
作者：
Chen, Zhe;Guo, Liang;Sternweis, Paul C.
含rgs的RhoGEFs （RGS-RhoGEFs）代表了G（12）类异三聚体G蛋白与单体gtp酶之间的直接联系。除了对RhoA具有鸟嘌呤核苷酸交换因子（GEF）活性的典型Dbl同源结构域（DH）和pleckstrin同源结构域外，这些rhogef还具有与G（12）和G（13）的活化α亚基相互作用的RGS同源结构域（RH）。虽然p115- rhogef (p115)，一种RGS-RhoGEF的GEF活性可以被G α刺激（13），但刺激的确切机制尚不清楚。通过小角度x射线散射、生物化学和诱变的综合研究，我们在p115的DH区域发现了活化的G α(13)的另一个结合位点。小角度x射线散射显示，G α(13)的螺旋结构域停靠在DH结构域上，与结合RhoA的DH表面相反。在DH的α 3b螺旋中单个色氨酸残基的突变减少了与活化的G α的结合（13），并减弱了G α对p115的刺激（13）。G α螺旋结构域α b - α C环中预测的dh结合位点的互补突变（13）也会影响G α对p115的刺激（13）。虽然p115的GAP活性不是G α刺激所必需的（13），但RH中两个在共识RGS框之外的疏水基序对这一过程至关重要。因此，G α(13)与RH结构域的结合促进了G α(13)与DH结构域的直接结合，从而调节其交换活性。本研究对RGS-RhoGEF的调控机制有了新的认识，拓宽了我们对G蛋白信号传导的认识。
RGS-containing RhoGEFs (RGS-RhoGEFs) represent a direct link between the G(12) class of heterotrimeric G proteins and the monomeric GTPases. In addition to the canonical Dbl homology (DH) and pleckstrin homology domains that carry out the guanine nucleotide exchange factor (GEF) activity toward RhoA, these RhoGEFs also possess RGS homology (RH) domains that interact with activated alpha subunits of G(12) and G(13). Although the GEF activity of p115-RhoGEF (p115), an RGS-RhoGEF, can be stimulated by G alpha(13), the exact mechanism of the stimulation has remained unclear. Using combined studies with small angle x-ray scattering, biochemistry, and mutagenesis, we identify an additional binding site for activated G alpha(13) in the DH domain of p115. Small angle x-ray scattering reveals that the helical domain of G alpha(13) docks onto the DH domain, opposite to the surface of DH that binds RhoA. Mutation of a single tryptophan residue in the alpha 3b helix of DH reduces binding to activated G alpha(13) and ablates the stimulation of p115 by G alpha(13). Complementary mutations at the predicted DH-binding site in the alpha B-alpha C loop of the helical domain of G alpha(13) also affect stimulation of p115 by G alpha(13). Although the GAP activity of p115 is not required for stimulation by G alpha(13), two hydrophobic motifs in RH outside of the consensus RGS box are critical for this process. Therefore, the binding of G alpha(13) to the RH domain facilitates direct association of G alpha(13) to the DH domain to regulate its exchange activity. This study provides new insight into the mechanism of regulation of the RGS-RhoGEF and broadens our understanding of G protein signaling.