ALPHA-RECPTOR PURIFICATION & ROLE IN NEUROTRANSMISSION

α 1-肾上腺素能受体在交感神经系统中起关键作用。 神经传递 它们介导各种反应，包括 参与中枢神经系统功能、循环稳态和 代谢，如自发活动的改变，血管平滑 肌肉收缩和糖原分解。 最近的证据 表明α 1受体是一组不同但 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 含有466至560个氨基酸的单链多肽， 最可能代表α-螺旋膜疏水结构域 跨越区域。 α 1-肾上腺素能受体也与 效应物的异质性基团，并通过 鸟嘌呤核苷酸结合调节蛋白（G蛋白）。 在大多数 受体激活导致膜聚磷酸肌醇 尽管参与这一信号通路的G蛋白 没有被定义。 在目前的资助期间， 已经表达了α 1b-肾上腺素能受体， 表征的受体的性质;编码 先前未描述的α 1-肾上腺素能受体的假定亚型 已经被分离出来; alpha 1b受体的计算机模型已经被分离出来。 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）， 已经鉴定出与α 1-肾上腺素能受体功能性偶联， 特点，并纯化，并取得了相当大的进展， 分离编码该蛋白质的cDNA克隆。 为进一步厘清 参与信号转导的分子机制， 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。 我们亦建议 采取多方面的方法来了解受体结构， 功能 这包括基因合成，定点突变， 组合盒式诱变，热力学分析，大分子 建模和傅里叶变换红外差光谱。 中的每 这些方法应提供独特但互补的信息， （i）哪些残留物对生物体的生长至关重要， 配体结合口袋的形成，以及受体与 激动剂和拮抗剂; ii）哪些残基负责亚型 选择性; iii）由编码的消息的简并性是什么？ 构成受体形状和功能基础的氨基酸序列; iv）什么 是受体G蛋白的主要结构域和氨基酸决定簇 （5）什么是分子基础的行动， 激动剂和拮抗剂。 最后，我们建议进一步测试 Gh介导α 1-肾上腺素能受体信号传导的假说。 这些 研究将旨在更详细地了解动力学， 受体-G蛋白相互作用的化学计量和分离cDNA 克隆Gh. 随着Gh cDNA的可用性，将努力 目的是产生Gh的无效表型，并过度表达这种表型。 蛋白质，以获得更大的数量，用于更详细的评估， 受体突变体的功能特性。

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.