ADENOSINE RECEPTORS AND ION TRANSPORT IN EPITHELIA

本项目的长期目标是通过以下方式了解这些机制： 腺苷受体调节离子转运。在离子运输中 在上皮细胞中，腺苷起着核心作用，是调节 蜂窝能量需求和可用性。细胞外腺苷， 与细胞工作成比例地释放，作为自体素激活剂 特异性七跨膜（7 TM）G蛋白偶联腺苷 受体。我们已经克隆，测序，并功能性特点， 新的腺苷受体调节氯离子转运（AoAR）似乎 是哺乳动物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 在这些研究中将被利用，包括：（1）双重功能 （与氯化物分泌的刺激和抑制偶联）;（2） 独特的N末端、末端和跨膜残基以及独特的 参与G蛋白偶联的细胞内环;和（3）G蛋白偶联的位置 在某些区域中的碱性氨基酸允许更高水平的蛋白质 受体在E.大肠杆菌中G偶联受体的数量比其它G偶联受体的数量多。通过 利用直肠腺模型系统的优点， 具体目标如下：1）我们将确定特定的残留物 对于激动剂和拮抗剂的独特结合至关重要， AoAR在有限数量的实验中。2)我们将挑战 假设内源性腺苷作用于AoAR， 都能抑制和刺激鲨鱼的跨上皮Cl-分泌 直肠腺我们将研究AoAR与多个G- 在CHO细胞表达系统中，我们获得了高度表达的 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的环中都有碱性氨基酸 赋予了Ao的显著特性， 在E.杆菌使用6 他的净化方案，加上进一步改进， 纯化（离子交换和拮抗剂柱）和重折叠步骤， 我们将纯化足够量的活性Ao腺苷受体 进行这种蛋白质的结构研究。

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.