MOLECULAR ANALYSIS OF G PROTEIN ALPHA SUBUNIT FUNCTIONS

异源三聚体（α，β，γ）G蛋白，一个GTP酶家族， 传递细胞表面受体接收的激素和感觉信号 产生细胞反应的效应蛋白。G蛋白成为 当受体催化GDP的替代时， α亚基与GTP结合。α亚基具有高度的相似性 在它们的氨基酸序列中。然而，阿尔法之间的差异 亚基在决定蛋白质的特异性和性质方面起着关键作用， 受体、G蛋白和效应子之间的相互作用。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 将产生α亚基并进行生物化学表征， 鉴定指定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： 最近解决的α亚基的X射线晶体结构 transducin是一种调节视觉的G蛋白。结合结构和 功能数据将使详细的分子模型的发展 G蛋白的信号转导机制。 确定 受体之间有效相互作用的要求，α 亚基和效应子可能导致合理的治疗设计， 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)以确定α-s如何与β-肾上腺素能受体相互作用。 突变体α-s构建体将在cyc S49中表达和表征 淋巴瘤细胞，这是遗传缺陷的α-s，以确定 所述α-S残基指定受体相互作用。为了阐明 α亚单位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，α亚单位区域， 响应GTP结合的构象，以及先前鉴定的 腺苷酸环化酶激活的α-S表面。 (II)鉴定与磷脂酶相互作用的α-q的残基 C（PLC）。突变体α-q构建体将瞬时表达， 特征在于人胚肾成纤维细胞系（HEK-293）， 以确定哪些α-q残基指定PLC相互作用。PLC不同 与腺苷酸环化酶不同，因为它不是一种完整的膜蛋白。 此外，与腺苷酸环化酶不同，PLC可以激活GT3活性 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的表面将揭示结构上保守的 α亚单位使用共同的机制与不同的效应物相互作用。

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.