ADENOSINE RECEPTORS AND ION TRANSPORT IN EPITHELIA

这个项目的长期目标是通过以下方式了解这些机制 哪种腺苷受体调节离子转运。In离子传输 上皮细胞中，腺苷起着中心作用，是调节之间的纽带 蜂窝能量需求和可用性。胞外腺苷， 与细胞功成比例释放，起到金丝桃体激活剂的作用 特异性7种跨膜(7TM)G蛋白偶联腺苷 感受器。我们已经克隆、测序并从功能上描述了 调节氯离子转运的新型腺苷受体(AoAR)似乎 是哺乳动物的腺苷受体的祖先受体 进化了。AoAR具有非凡的功能和结构特征 将在这些研究中利用的内容包括：(1)双重功能 (与刺激和抑制氯分泌相结合)；(2) 唯一的N末端、末端和跨膜残基以及唯一的 参与G蛋白偶联的胞内环；以及(3)G蛋白偶联的定位 某些区域中的碱性氨基酸允许更高水平的蛋白质 该受体在大肠杆菌中的表达高于其他G偶联受体。通过 发挥直肠腺模型系统的优势，我们将追求 具体目标如下：1)我们将确定具体的残留物 对于激动剂和拮抗剂与 AoAR在有限数量的实验中。2)我们将挑战 一种假设是内源性腺苷作用于AoAR以 抑制和刺激鲨鱼跨上皮氯离子的分泌 直肠腺。我们将研究AoAR与多个G-的耦合。 蛋白质在CHO细胞中的表达系统，我们已经获得了高度的 纯化的AoAR具有高特异性结合活性。AoAR的结构 在所有细胞外和细胞内环中都含有碱性氨基酸 赋予了Ao在高海拔地区生产的显著特点 包涵体在大肠杆菌中的高密度密度。使用6 他的净化方案，加上在 纯化(离子交换柱和拮抗柱)和复性步骤， 我们将提纯足够数量的活性Ao腺苷受体 来研究这种蛋白质的结构。

The long term goal of this project is to understand the mechanisms by which adenosine receptors regulate ion transport. In ion transporting epithelia, adenosine plays a central role is a regulatory link between cellular energy demand and availability. Extracellular adenosine, released in proportion to cellular work, acts as an autacoid activator of specific seven transmembrane (7TM) G-protein coupled adenosine receptors. We have cloned, sequenced, and functionally characterized a new adenosine receptor regulating chloride transport (AoAR) appears to be the ancestral receptor from which mammalian adenosine receptors evolved. The AoAR has extraordinary functional and structural features that will be exploited in these studies including: (1) dual function (coupling to both stimulation and inhibition of chloride secretion); (2) unique N terminal, terminal and transmembrane residues and a unique intracellular loop involved in G-protein coupling; and (3) location of basic amino acids in certain domains permitting higher levels of protein expression of the receptor in E. coli than other G coupled receptors. By exploiting advantages of the rectal gland model system we will pursue the following specific aims: 1) We will identify the specific residues that are essential for the unique binding of agonists and antagonist to AoAR in a limited number of experiments. 2) We will challenge the paradigm that a hypothesis is that endogenous adenosine acts at AoAR to both inhibit and stimulate transepithelial Cl- secretion in the shark rectal gland. We will examine the coupling of AoAR to multiple G- proteins both in CHO cells expressing system, we have obtained highly purified AoAR with high specific binding activity. The structure of AoAR in having basic amino acids in all extracellular and intracellular loops confers the remarkable characteristic of Ao to be produced at high densities at high densities in inclusion bodies in E. coli. Using a 6 His purification scheme, coupled with further improvement in purifications (ion exchange and antagonist columns) and refolding steps, we will purify the active Ao adenosine receptor in sufficient amounts for structural studies of this protein.