STRUCTURE OF ADENOSINE RECEPTORS

腺苷是一种激素，在大多数器官系统(如果不是全部的话)都有局部作用。它在细胞内低能量水平的压力下释放，导致氧气需求减少和氧气输送增加。腺苷受体(AR)是G蛋白偶联受体(GPCR超家族)的成员，但关于构成AR四种亚型的配体结合口袋的氨基酸的确切生化信息很少，预计腺苷的独特结构将需要一个不同于单胺的结合口袋。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。该计划的目标是产生可靠的生化数据，可用于开发可靠的AR分子模型，这将促进基于受体的AR亚型特定激动剂和拮抗剂的设计。本研究旨在通过替代半胱氨酸可及性方法(SCAM)建立A1AR跨膜跨膜(TM)的哪些氨基酸是水环境可及的，从而定位于A1AR的配体结合缝隙内。这一策略需要用半胱氨酸取代单个氨基酸，并确定半胱氨酸与亲水性、疏脂性、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，而激动剂和/或拮抗剂的存在减缓了受体的失活速度，则半胱氨酸必须位于配体结合口袋内。反应性半胱氨酸的周期性将提供关于TM结构性质的见解。类似的方法将被用来确定激动剂和黄嘌呤类拮抗剂是否占据相同的结合部位，并确定腺苷和黄嘌呤在结合部位内的相对取向。在该计划的第三阶段，研究旨在描绘A1AR的TM的三维排列，方法是通过结合A1AR的非重叠片段的表达和半胱氨酸扫描突变与二硫键交联来揭示这些接触点，从而确定七个TM之间的一些接触点。这些研究应该提供关于通过受体蛋白进行信号转导的机制的见解，并结合从SCAM研究产生的数据，将允许区分七个TM的顺时针和逆时针捆绑。因此，这些研究应该提供数据，测试目前可用的GPCR计算模型，并允许基于可靠的生化数据对A1AR进行分子建模，该数据建立了跨越受体部分的膜和可从配体结合口袋的水环境中获得的氨基酸的总体三维排列。

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.