MOLECULAR ANALYSIS OF G PROTEIN ALPHA SUBUNIT FUNCTIONS

G 蛋白 α 亚基功能的分子分析

• 批准号: 2188161
• 负责人: CATHERINE H BERLOT
• 金额: $ 11.85万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2188161
• 关键词: G protein; X ray crystallography; beta adrenergic receptor; biological signal transduction; chimeric proteins; conformation; density gradient ultracentrifugation; guanine nucleotide binding protein; membrane proteins; mutant; neoplastic cell; phospholipase C; polymerase chain reaction; protein isoforms; protein structure; receptor coupling

The heterotrimeric (alpha, beta, gamma) G proteins, a family of GTPases, transmit hormonal and sensory signals received by cell surface receptors to effector proteins that generate cellular responses. G proteins become activated when receptors catalyze the replacement of GDP bound to the alpha subunit with GTP. Alpha subunits share a high degree of similarity in their amino acid sequences. However, the differences among alpha subunits play a critical role in determining the specificity and nature of the interactions between receptors, G proteins, and effectors. Aberrant or subunits can cause pituitary, adrenal cortical, thyroid, and ovarian tumors, as well as McCune-Albright syndrome, type-1 pseudohypoparathyroidism, whooping cough, and cholera. The overall goal of the proposed studies is to understand the molecular mechanisms by which G protein alpha subunits transmit signals. Mutant alpha subunits will be generated and characterized biochemically to identify residues that specify interactions with particular receptors and effectors. These studies will be interpreted in the context of the recently solved X-ray crystal structure of the alpha subunit of transducin, the G protein that mediates vision. Combining structural and functional data will enable the development of a detailed molecular model of the mechanism of signal transduction by G proteins. Determining the requirements for productive interactions between receptors, alpha subunits, and effectors may lead to the rational design of therapeutic agents for use in treating diseases caused by aberrant G protein signaling pathways. The Specific Aims of this project are: (I) To establish how alpha-s interacts with the beta-adrenergic receptor. Mutant alpha-s constructs will be expressed and characterized in cyc S49 lymphoma cells, which are genetically deficient in alpha-s, to determine which alpha-s residues specify receptor interactions. To elucidate how alpha subunits transmit signals between receptors and effectors, the receptor-interacting surface defined by these residues will be related to the guanine nucleotide binding site, the alpha subunit regions that change conformation in response to GTP binding, and the previously identified adenylyl cyclase-activating surface of alpha-s. (II) To identify the residues of alpha-q that interact with phospholipase C (PLC). Mutant alpha-q constructs will be transiently expressed and characterized in a human embryonic kidney fibroblast cell line (HEK-293) to determine which alpha-q residues specify PLC interaction. PLC differs from adenylyl cyclase in that it is not an integral membrane protein. Furthermore, unlike adenylyl cyclase, PLC can activate the GTPase activity of the alpha subunit that stimulates it. Therefore, comparing the PLC- interacting surface of alpha-q with the adenylyl cyclase-activating surface of alpha-s will reveal the extent to which structurally conserved alpha subunits use common mechanisms to interact with diverse effectors.

异三聚体（α、β、γ）G 蛋白，GTP 酶家族， 传输细胞表面受体接收到的激素和感觉信号 产生细胞反应的效应蛋白。 G蛋白变成 当受体催化取代与结合的 GDP 时被激活 带有 GTP 的 α 亚基。 Alpha 亚基具有高度相似性 在它们的氨基酸序列中。然而，阿尔法之间的差异 亚基在决定特异性和性质方面发挥着关键作用 受体、G 蛋白和效应器之间的相互作用。异常或 亚单位可引起垂体、肾上腺皮质、甲状腺和卵巢 肿瘤，以及 McCune-Albright 综合征，1 型 假性甲状旁腺功能减退症、百日咳和霍乱。 拟议研究的总体目标是了解分子 G蛋白α亚基传递信号的机制。突变体 α亚基将被生成并进行生化表征 识别与特定受体相互作用的残基 效应器。这些研究将在以下背景下进行解释： 最近解决了α亚基的X射线晶体结构 转导蛋白，介导视觉的 G 蛋白。结合结构和 功能数据将有助于开发详细的分子模型 G蛋白的信号转导机制。 确定 受体、α 之间有效相互作用的要求 亚基和效应子可能导致治疗方案的合理设计 用于治疗由异常G蛋白信号传导引起的疾病的药物 途径。 该项目的具体目标是： (I) 确定α-s如何与β-肾上腺素能受体相互作用。 突变的 alpha-s 构建体将在 cyc S49 中表达和表征 淋巴瘤细胞在遗传上缺乏α-S，以确定 哪些α-S残基指定受体相互作用。阐明如何 α亚基在受体和效应器之间传递信号， 由这些残基定义的受体相互作用表面将与 鸟嘌呤核苷酸结合位点，改变的α亚基区域 响应 GTP 结合的构象，以及先前确定的 α-S 的腺苷酸环化酶激活表面。 (II) 鉴定与磷脂酶相互作用的α-q残基 C（PLC）。突变的 alpha-q 构建体将被瞬时表达并 人胚胎肾成纤维细胞系 (HEK-293) 的特征 确定哪些 alpha-q 残基指定 PLC 相互作用。 PLC不同 与腺苷酸环化酶不同，因为它不是完整的膜蛋白。 此外，与腺苷酸环化酶不同，PLC 可以激活 GTPase 活性 刺激它的α亚基。因此，比较PLC- α-q 与腺苷酸环化酶激活的相互作用表面 α-S 的表面将揭示结构保守的程度 α 亚基使用共同的机制与不同的效应器相互作用。