Dias Sandra M G, Cerione Richard A
Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA.
Biochemistry. 2007 Jun 5;46(22):6547-58. doi: 10.1021/bi700035p. Epub 2007 May 12.
RhoC is a member of the Rho family of Ras-related (small) GTPases and shares significant sequence similarity with the founding member of the family, RhoA. However, despite their similarity, RhoA and RhoC exhibit different binding preferences for effector proteins and give rise to distinct cellular outcomes, with RhoC being directly implicated in the invasiveness of cancer cells and the development of metastasis. While the structural analyses of the signaling-active and -inactive states of RhoA have been performed, thus far, the work on RhoC has been limited to an X-ray structure for its complex with the effector protein, mDia1 (for mammalian Diaphanous 1). Therefore, in order to gain insights into the molecular basis for RhoC activation, as well as clues regarding how it mediates distinct cellular responses relative to those induced by RhoA, we have undertaken a structural comparison of RhoC in its GDP-bound (signaling-inactive) state versus its GTP-bound (signaling-active) state as induced by the nonhydrolyzable GTP analogues, guanosine 5'-(beta,gamma-iminotriphosphate) (GppNHp) and guanosine 5'-(3-O-thiotriphosphate) (GTPgammaS). Interestingly, we find that GppNHp-bound RhoC only shows differences in its switch II domain, relative to GDP-bound RhoC, whereas GTPgammaS-bound RhoC exhibits differences in both its switch I and switch II domains. Given that each of the nonhydrolyzable GTP analogues is able to promote the binding of RhoC to effector proteins, these results suggest that RhoC can undergo at least two conformational transitions during its conversion from a signaling-inactive to a signaling-active state, similar to what has recently been proposed for the H-Ras and M-Ras proteins. In contrast, the available X-ray structures for RhoA suggest that it undergoes only a single conformational transition to a signaling-active state. These and other differences regarding the changes in the switch domains accompanying the activation of RhoA and RhoC provide plausible explanations for the functional specificity exhibited by the two GTPases.
RhoC是Ras相关（小）GTP酶的Rho家族成员，与该家族的创始成员RhoA具有显著的序列相似性。然而，尽管它们相似，但RhoA和RhoC对效应蛋白表现出不同的结合偏好，并产生不同的细胞结果，RhoC直接参与癌细胞的侵袭和转移的发展。虽然已经对RhoA的信号激活和非激活状态进行了结构分析，但到目前为止，关于RhoC的研究仅限于其与效应蛋白mDia1（哺乳动物的Diaphanous 1）复合物的X射线结构。因此，为了深入了解RhoC激活的分子基础，以及关于它如何介导相对于RhoA诱导的不同细胞反应的线索，我们对RhoC在其GDP结合（信号非激活）状态与由不可水解的GTP类似物鸟苷5'-（β，γ-亚氨基三磷酸）（GppNHp）和鸟苷5'-（3-O-硫代三磷酸）（GTPγS）诱导的GTP结合（信号激活）状态进行了结构比较。有趣的是，我们发现与GDP结合的RhoC相比，GppNHp结合的RhoC仅在其开关II结构域显示差异，而GTPγS结合的RhoC在其开关I和开关II结构域均显示差异。鉴于每种不可水解的GTP类似物都能够促进RhoC与效应蛋白的结合，这些结果表明RhoC在从信号非激活状态转变为信号激活状态的过程中至少可以经历两个构象转变，这与最近对H-Ras和M-Ras蛋白的提议类似。相比之下，RhoA的现有X射线结构表明它仅经历一次构象转变至信号激活状态。这些以及关于RhoA和RhoC激活时开关结构域变化的其他差异为这两种GTP酶表现出的功能特异性提供了合理的解释。
