EXPRESSION AND MUTAGENESIS OF CLONED ALPHA2-RECEPTORS

本研究的目的是用分子生物学的方法检测 方法，不同功能特性的结构基础 α2肾上腺素能受体。我们计划克隆猪的编码基因 对于我们已经纯化成同质的α2-肾上腺素能受体。 我们将利用从结构域映射和生化中获得的见解 对纯化的受体进行分析以描绘受体的那些区域 我们可以通过缺失和定点突变来进一步分析 了解配基识别涉及哪些结构成分， 变构对Na~+、H~+和5-氨基取代的肾上腺素能结合的影响 阿米洛利类似物，受体-GTP结合蛋白相互作用， 受体促进的Na+/H+交换和受体介导的抑制 腺苷环化酶。此外，我们计划研究不同的角色 受体的功能域在整个生理过程中发挥作用 受α2肾上腺素能受体影响的功能，如抑制 神经递质的释放，通过表达突变的与野生型受体 在适当的目标单元格中确定离散删除或 位点突变对特定的α2受体功能有影响。我们期待着 拟议的研究将提供对结构性问题的新见解 α2受体的许多功能特性的基础，可能 通过模仿或阻断基因提示药物开发的新基因座 α2-肾上腺素能药在特定生理过程中的作用。

The present studies are aimed at examining, using a molecular biological approach, the structural basis for the diverse functional properties of alpha2-adrenergic receptors. We plan to clone the porcine gene that codes for the alpha2-adrenergic receptor that we have purified to homogeneity. We will exploit insights gained from domain mapping and biochemical analysis of the purified receptor to delineate those areas of the receptor that we can analyze further by deletion and site-directed mutagenesis to learn what structural components are involved in ligand recognition, allosteric effects on adrenergic binding by Na+, H+ and 5-amino-substituted analogs of amiloride, receptor-GTP binding protein interactions, receptor-accelerated Na+/H+ exchange and receptor mediated inhibition of adenylate cyclase. Furthermore, we plan to examine what role different functional domains of the receptor play in the overall physiological functions influenced by alpha2-adrenergic receptors, such as suppression of neurotransmitter release, by expressing mutated versus wild type receptors in appropriate target cells to determine what effect discrete deletion or site mutations have on particular alpha2-receptor functions. We anticipate that the proposed studies will provide new insights into the structural basis for many of the functional properties of alpha2-receptors and perhaps suggest novel loci for drug development in mimicking, or blocking, the effects of alpha2-adrenergic agents in particular physiological processes.