Tiam1, a prototypical Rho-family GEF

G蛋白的Rho家族调节涉及细胞骨架重排和转录控制中的协调改变的多种细胞过程，并且最终对于如趋化性、吞噬作用以及细胞生长和分化等多种过程是必需的。 相反，Rho家族G蛋白的异常功能促进了从不规则发育到细胞转化和肿瘤发生的各种恶性肿瘤。 与Db 1相关的大量不同种类的鸟嘌呤核苷酸交换因子通过促进与失活G蛋白结合的GDP的释放并催化GTP的伴随加载以产生在下游信号传导中有活性的G蛋白来控制Rho家族G蛋白的调节活化。 因此，Dbl相关GEF的功能失调的组成性激活异常地升高了活性的、GTP结合的G蛋白的细胞内浓度，从而导致肿瘤发生和发育障碍。虽然对Rho家族G蛋白特异性的GEF在大小和结构域结构上变化很大，但所有GEF都含有与并列的C-末端普列克底物蛋白同源结构域（PH）恒定相关的Db 1-同源结构域（DH）。 DH结构域催化鸟嘌呤核苷酸交换，而相关的PH结构域的功能定义不太明确，而且变化更大。 该提案描述了旨在照亮，在原子分辨率，鸟嘌呤核苷酸交换DBL相关的GEF催化的保守机制的实验。 我们最近已经解决了的DH/PH片段的Tiam 1和DBS的复合物与它们的同源G蛋白，Rac 1和Cdc 42，分别的晶体结构。 这些结构形成了理解DBL相关GEF催化的鸟嘌呤核苷酸交换的基础，并指导了许多所述的生物物理、生物化学和体内实验。 该建议的补充方面的目的是阐明所需的机制的细节，有效的鸟嘌呤核苷酸交换DBL相关的GEF催化，包括分配特定的功能，PH域不变地与DH域。 预计这些信息对于理解和控制Rho家族G蛋白的活化将是非常宝贵的，最终改善与组成性活性G蛋白相关的病理。

The Rho family of G proteins regulate diverse cellular processes involving coordinate alterations in cytoskeletal rearrangements and transcriptional control, and ultimately are necessary for processes as varied as chemotaxis, phagocytosis, and cellular growth and differentiation. Conversely the aberrant functioning of Rho-family G proteins promotes various malignancies from irregular development to cellular transformation and oncogenesis. A large and diverse class of guanine nucleotide exchange factors related to Db1 control the regulated activation of Rho-family G proteins by facilitating the release of GDP bound to inactive G proteins and catalyzing the concomitant loading of GTP to produce G proteins active in downstream signaling. Consequently, dysfunctional constitutive activation of Dbl-related GEFs abnormally elevates intracellular concentrations of active, GTP-bound G proteins contributing to tumorigenesis and developmental disorders. Although GEFs specific for Rho-family G proteins vary greatly in size and domain architecture, all contain a Db1-homology domain (DH) invariantly associated with a juxtaposed, C-terminal pleckstrin homology domain (PH). DH domains catalyze guanine nucleotide exchange, while functions for the associated PH domains are less well defined and more variable. This proposal describes experiments designed to illuminate, at atomic resolution, the conserved mechanism of guanine nucleotide exchange catalyzed by Dbl-related GEFs. We have recently solved the crystal structures of the DH/PH fragments of Tiam1 and Dbs in complex with their cognate G proteins, Rac1 and Cdc42, respectively. These structures form the basis for understanding guanine nucleotide exchange catalyzed by Dbl-related GEFs and guide many of the biophysical, biochemical, and in vivo experiments described. Complementary aspects of this proposal are designed to elucidate mechanistic details required for efficient guanine nucleotide exchange catalyzed by Dbl-related GEFs, including assigning specific functions to PH domains invariantly associated with DH domains. It is anticipated that this information will be invaluable for understanding and controlling the activation of Rho-family G proteins culminating in the amelioration of pathologies associated with constitutively active G proteins.