STRUCTURE OF ADENOSINE RECEPTORS

腺苷是一种局部作用于大多数（如果不是全部的话）器官系统的激素。它在细胞低能量水平的压力下被释放，导致氧气需求减少和氧气输送增加。腺苷（AR）受体是G蛋白偶联受体（GPCR）超家族的成员，但关于构成AR四种亚型配体结合袋的氨基酸的生物化学信息很少，并且预计腺苷的独特结构将需要与单胺不同的结合袋。腺苷激动剂和拮抗剂的广泛作用说明了开发AR亚型特异性激动剂和拮抗剂的潜在重要性。该计划的目标是生成可靠的生化数据，可用于开发可靠的AR分子模型，从而促进基于受体的AR亚型特异性激动剂和拮抗剂的设计。目前的研究旨在通过取代半胱氨酸可及性方法（SCAM），A1AR的跨膜跨度（TM）的哪些氨基酸可进入水环境，因此位于A1AR的配体结合缝隙内。这种策略需要用半胱氨酸代替单个氨基酸，并确定半胱氨酸与亲水、疏脂、半胱氨酸特异性试剂的反应性。如果半胱氨酸特异性试剂不可逆地抑制配体结合，并且激动剂和/或拮抗剂的存在延缓了受体的失活速率，则半胱氨酸必须位于配体结合口袋内。活性半胱氨酸的周期性将提供有关TM结构性质的见解。将采用类似的方法来确定激动剂和黄嘌呤型拮抗剂是否占据相同的结合位点，并确定腺苷和黄嘌呤在结合位点内的相对取向。在该项目的第三阶段，研究旨在通过结合A1AR非重叠片段的表达和半胱氨酸扫描诱变与二硫交联来揭示这些接触点，通过确定7个TM之间的一些接触点来描绘A1AR TM的三维排列。这些研究将提供有关通过受体蛋白的信号转导机制的见解，并结合从SCAM研究中产生的数据，将允许区分顺时针和逆时针捆绑的七种TM。因此，这些研究应该提供数据来测试目前可用的GPCR计算模型，并允许基于确定的生化数据建立A1AR的分子模型，这些数据建立了受体跨膜部分的总体三维排列以及从配体结合袋的水环境中可获得的氨基酸。

Adenosine is a hormone with localized effects in most, if not all, organ systems. It is released under stress of low energy levels in a cell, resulting in decreased oxygen demand and increased oxygen delivery. The receptors for adenosine(AR) are members of the G protein-coupled receptor (GPCR) superfamily, but little firm biochemical information is available concerning the amino acids that comprise the ligand-binding pocket of the four subtypes of AR, and it is anticipated the the unique structure of adenosine will require a binding pocket distinct from that for monoamines. The widespread effects of adenosine agonists and antagonists serve to illustrate the potential importance of developing subtype-specific agonists and antagonists of AR. The goal of this program is to generate firm biochemical data that can be utilized to develop reliable molecular models of the AR which will facilitate receptor-based design of subtype specific agonists and antagonists of AR. The present studies aim to establish, by the Substituted Cysteine Accessibility Methods (SCAM), which amino acids of the transmembrane spans (TM) of the A1AR are accessible to the aqueous-milieu and, therefore, are positioned within the ligand-binding crevice of the A1AR. This strategy entails substituting cysteines for individual amino acids and determining the reactivity of the cysteines with hydrophilic, lipophobic, cysteine-specific reagents. If the cysteine-specific reagents irreversibly inhibit ligand binding and the presence of agonists and/or antagonists retard the rate of inactivation of the receptor, the cysteine must be positioned within the ligand binding pocket. The periodicity of reactive cysteines will provide insights concerning the structural nature of the TM. Similar methodology will be employed to determine if agonists and xanthine-type antagonists occupy the same binding site, and determine the relative orientations of adenosine and xanthines within the binding site. In a third phase of this program, studies are designed to delineate the three-dimensional arrangement of the TM of the A1AR by determining some of the contact points between the seven TM by combining expression of non-overlapping fragments of the A1AR and cysteine-scanning mutagenesis with disulfide crosslinking to reveal these contact points. These studies should provide insights concerning the mechanism of signal transduction through the receptor protein and in conjunction with data generated from the SCAM studies will allow discrimination between clockwise and counterclockwise bundling of the seven TM. Thus, these studies should provide data that will test currently available computational models of GPCR and allow the molecular modeling of the A1AR based on firm biochemical data that establishes the gross three-dimensional arrangement of the membrane spanning portions of the receptor and the amino acids accessible from the aqueous environment of the ligand binding pocket.