Tiam1, a prototypical Rho-family GEF

Rho家族G蛋白调节多种细胞过程，包括细胞骨架重排和转录控制的协调变化，最终是趋化、吞噬、细胞生长和分化等各种过程所必需的。相反，Rho家族G蛋白的异常功能促进了从不规则发展到细胞转化和肿瘤发生的各种恶性肿瘤。一大类与DB1相关的鸟嘌呤核苷酸交换因子通过促进与非活性G蛋白结合的GDP的释放和催化GTP的伴随负载来控制Rho家族G蛋白的调节激活，从而产生在下游信号转导中活跃的G蛋白。因此，与DBL相关的GEF的功能失调的结构性激活异常地增加了细胞内活性的、GTP结合的G蛋白的浓度，导致肿瘤发生和发育障碍。尽管针对Rho家族G蛋白的GEF在大小和结构上有很大的差异，但它们都包含一个DB1-同源结构域(DH)，总是与一个并列的C-末端Pleckstrin同源结构域(PH)相关联。NH结构域催化鸟嘌呤核苷酸交换，而相关PH结构域的功能定义较少，且更具变异性。这项建议描述了旨在阐明在原子分辨率下，由DBL相关的GEF催化的鸟嘌呤核苷酸交换的保守机制的实验。我们最近解决了Tiam1和DBS的DH/PH片段分别与其同源G蛋白rac1和Cdc42形成的复合体的晶体结构。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，并指导了所描述的许多生物物理、生化和体内实验。这一建议的补充方面旨在阐明DBL相关GEF催化有效的鸟嘌呤核苷酸交换所需的机制细节，包括为与DH域不变的PH结构域分配特定的功能。预计这些信息对于了解和控制Rho家族G蛋白的激活将是非常有价值的，最终达到改善与结构性活性G蛋白相关的病理学的目的。

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.