MOLECULAR ANALYSIS OF G PROTEIN ALPHA SUBUNIT FUNCTIONS

异三聚体(α、β、γ)G蛋白，一个GTP酶家族， 传递细胞表面受体接收的荷尔蒙和感觉信号 到产生细胞反应的效应器蛋白。G蛋白变成 当受体催化结合的GDP替换时被激活 含GTP的Alpha亚基。阿尔法亚单位有很高的相似性 在它们的氨基酸序列中。然而，阿尔法和阿尔法之间的区别 亚基在决定白血病的特异性和性质方面起着至关重要的作用。 受体、G蛋白和效应器之间的相互作用。反常的或 亚基可引起脑下垂体、肾上腺皮质、甲状腺和卵巢 肿瘤以及1型麦克库恩-奥尔布赖特综合征 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。突变型 将产生α亚基，并对其进行生化表征，以 确定与特定受体相互作用的残基和 效应器。这些研究将在以下背景下解释 最近解决了α亚基的X射线晶体结构 转导蛋白，调节视觉的G蛋白。将结构和 功能数据将使开发详细的分子模型成为可能 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 亚单位和效应器可能导致治疗的合理设计 用于治疗G蛋白信号异常引起的疾病的药物 小路。 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α-S结构将在Cyc S49中表达和鉴定 缺乏α-S的淋巴瘤细胞，以确定 哪些α-S残基规定了受体的相互作用。为了阐明如何 阿尔法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，即改变的阿尔法亚单位区域 响应GTP结合的构象，以及先前识别的 α-S的腺酰环化酶激活表面。 (Ii)鉴定与磷脂酶相互作用的α-Q残基 C(PLC)。突变的α-Q结构将被瞬时表达并 人胚胎肾成纤维细胞系(HEK-293)的特性 以确定哪些α-Q残基指定PLC相互作用。PLC不同 从腺酰环化酶中分离出来，因为它不是完整的膜蛋白。 此外，与腺酰环化酶不同，PLC可以激活GTP酶的活性 刺激它的阿尔法亚单位。因此，比较PLC- α-Q与腺苷环化酶激活表面的相互作用 α-S的表面将揭示结构保守的程度 Alpha子单元使用公共机制来与不同的效应器交互。

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.