ALPHA-RECPTOR PURIFICATION & ROLE IN NEUROTRANSMISSION

α-受体纯化

• 批准号: 3399686
• 负责人: ROBERT M GRAHAM
• 金额: $ 16.57万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3399686
• 关键词: G protein; affinity chromatography; alpha adrenergic receptor; antireceptor antibody; biological signal transduction; calcium metabolism; complementary DNA; computer simulation; gene expression; guanosine monophosphate; immunochemistry; laboratory mouse; laboratory rabbit; laboratory rat; molecular cloning; neural transmission; phosphatidylinositols; protein metabolism; protein sequence; protein structure; proteins; site directed mutagenesis

Alpha1-adrenergic receptors play a critical role in sympathetic neurotransmission. They mediate a variety of responses including those involved in central nervous system functions, circulatory homeostasis and metabolism, such as alterations in locomotor activity, vascular smooth muscle contraction and glycogenolysis, respectively. Recent evidence suggest that alpha1-receptors are a heterogenous group of distinct but related membrane glycoproteins and are members of a superfamily of receptors that have a common structural motif. They are all composed of single polypeptide chains containing 466 to 560 amino acids with seven hydrophobic domains that most likely represent alpha-helical membrane spanning regions. Alpha1-Adrenergic receptors also interact with a heterogenous group of effectors and are coupled to these effectors via guanine nucleotide-binding regulatory proteins (G-proteins). In most instances receptor activation results in membrane polyphosphoinositide breakdown, although the G-protein involved in this signalling pathway has not been defined. During the present funding period a cDNA clone encoding the alpha1b-adrenergic receptor has been expressed and the ligand-binding properties of the receptor characterized; a cDNA clone encoding a previously undescribed putative subtype of the alpha1-adrenergic receptor has been isolated; a computer model of the alpha1b-receptor has been developed, and thermodynamic studies have been undertaken to evaluate receptor conformation. Additionally, a unique 74 kDa G-protein (Gh) that functionally couples to the alpha1-adrenergic receptor has been identified, characterized, and purified, and considerable progress has been made in isolating a cDNA clone encoding this protein. To gain further insights into the molecular mechanisms involved in signal transduction by alpha1- adrenergic receptors, we propose now to continue efforts to clone and sequence the genes and cDNAs for these proteins. We also propose to undertake a multifaceted approach to understanding receptor structure and function. This involves gene synthesis, site-directed mutagenesis, combinatorial cassette mutagenesis, thermodynamic analyses, macromolecular modeling, and Fourier-transform infrared difference spectroscopy. Each of these approaches should provide unique but complementary information aimed at addressing the following issues: i) which residues are critical for the formation of the ligand binding pocket, and for receptor interactions with agonists and antagonists; ii) which residues are responsible for subtype selectivity; iii) what is the degeneracy of the message encoded by the amino acid sequence that underlies receptor shape and function; iv) what are the major domains and amino acid determinants of receptor G-protein interactions; and v) what is the molecular basis for the actions of agonists and antagonists. Finally, we propose to further test the hypothesis that Gh mediates alpha1-adrenergic receptor signalling. These studies will be aimed at understanding, in more detail, the kinetics and stoichiometry of receptor-G protein interactions and at isolating a cDNA clone for Gh. With the availability of a cDNA for Gh, efforts will be directed at developing a null phenotype for Gh, and at over-expressing this protein to obtain larger quantities for the more detailed evaluation of the functional properties of receptor mutants.

α1-肾上腺素能受体在交感神经中起关键作用 神经传递。它们调节各种反应，包括 参与中枢神经系统功能、循环内稳态和 新陈代谢，如运动活动的改变，血管通畅 肌肉收缩和糖原分解。最近的证据 提示α1受体是一组不同的不同的 相关的膜糖蛋白，是一个超家族的成员 具有共同结构基序的受体。它们都是由 含466-560个氨基酸的单链多肽链 最有可能代表α-螺旋膜的疏水结构域 跨越多个区域。α1-肾上腺素能受体也与 异质效应器组，并通过以下方式耦合到这些效应器 鸟嘌呤核苷酸结合调节蛋白(G蛋白)。在大多数 受体激活导致膜多磷肌醇 崩溃，尽管参与这一信号通路的G蛋白已经 未定义。在目前的资助期内，编码 已经表达了α1b-肾上腺素能受体，并与配体结合 确定了受体的性质；编码一个 先前未描述的α1-肾上腺素能受体亚型 已经被分离出来；α1b受体的计算机模型已经被分离出来 已经开发，并进行了热力学研究，以评估 受体构象。此外，一种独特的74 kDa G蛋白(Gh) 已经确定了与α1肾上腺素能受体的功能偶联， 特色化和提纯，并已在 分离编码该蛋白的cDNA克隆。为了获得更深入的见解 与α1信号转导有关的分子机制- 肾上腺素能受体，我们现在建议继续努力克隆和 对这些蛋白质的基因和cDNA进行测序。我们还建议 采取多方面的方法来了解受体结构和 功能。这涉及到基因合成，定点突变， 组合盒诱变、热力学分析、大分子 建模和傅里叶变换红外差分光谱分析。每一个 这些方法应提供独特但具有互补性的信息 解决以下问题：i)哪些残留物对 形成配基结合袋，并与受体相互作用 激动剂和拮抗剂；ii)哪些残基是亚型 选择性；iii)编码的消息的简并度是多少 构成受体形状和功能的氨基酸序列；iv)什么 是受体G蛋白的主要结构域和氨基酸决定因素 相互作用；以及v)作用的分子基础是什么 激动剂和拮抗剂。最后，我们建议进一步测试 假设GH介导α1-肾上腺素能受体信号传导。这些 研究的目的将是更详细地了解动力学和 受体-G蛋白相互作用的化学计量学及其在分离cDNA时的作用 为GH克隆。随着Gh的cDNAs的问世，将努力 旨在开发Gh的无效表型，并过度表达这一点 以获得更大数量的蛋白质，以便更详细地评估 受体突变体的功能特性。