Tiam1, a prototypical Rho-family GEF

Tiam1，一个典型的 Rho 家族 GEF

• 批准号: 6398501
• 负责人: JOHN E SONDEK
• 金额: $ 25.2万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6398501
• 关键词: X ray crystallography; biological signal transduction; cell growth regulation; cell line; cell transformation; chemical kinetics; conformation; crystallization; fluorescence spectrometry; fluorescent dye /probe; guanine nucleotide binding protein; guanine nucleotide exchange factors; guanine nucleotides; intermolecular interaction; model design /development; molecular site; phosphatidylinositols; physical model; protein purification; protein structure function; reporter genes; site directed mutagenesis; structural biology; thermodynamics; transfection

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.

G蛋白的RHO家族调节涉及细胞骨架重排和转录控制的坐标改变的各种细胞过程，最终对于像趋化性，吞噬作用以及细胞生长和分化的过程一样，最终是必需的。 相反，Rho-family 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DB1相关的鸟嘌呤核苷酸交换因子的大量多样化的类别通过促进与无活性G蛋白结合的GDP的释放，并催化GTP伴随GTP载荷以在下游信号中产生GTP的伴随载荷，从而促进GDP的释放，从而控制Rho-family G蛋白的调节激活。 因此，与DBL相关的GEF的功能失调的组成型激活异常提高了有助于肿瘤发生和发育障碍的活性，GTP结合的G蛋白的细胞内浓度。尽管针对Rho-family G蛋白的GEF在大小和域结构上差异很大，但都包含与并列的C端Pleckstrin同源域（ph）不变相关的DB1 - 同源域（DH）。 DH结构域催化鸟嘌呤核苷酸交换，而相关的pH结构域的功能较不明显且变化较大。 该提案描述了旨在通过原子分辨率阐明鸟嘌呤核苷酸交换的保守机制，该实验由DBL相关GEF催化。 最近，我们分别与它们的同源G蛋白Rac1和cdc42分别解决了TIAM1和DBS的DH/pH片段的晶体结构。 这些结构构成了理解与DBL相关GEF催化的鸟嘌呤核苷酸交换的基础，并指导许多生物物理，生化和体内实验。 该提案的互补方面旨在阐明由DBL相关的GEF催化的有效鸟嘌呤核苷酸交换所需的机械细节，包括将特定功能分配给与DH域不变相关的pH结构域。 可以预料，这些信息对于理解和控制与组成性活性G蛋白相关的病理学的改善时，将Rho-family G蛋白的激活最终激活将是无价的。