STRUCTURE OF ADENOSINE RECEPTORS

腺苷是一种激素，在大多数（如果不是全部）器官系统中具有局部效应。 它在细胞中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（adenosine，AR）受体属于G蛋白偶联受体（G protein-coupled receptor，GPCR）超家族，但目前关于AR四种亚型的配体结合口袋的氨基酸组成的生物化学信息很少，而且腺苷的独特结构需要一个与单胺不同的结合口袋。 腺苷激动剂和拮抗剂的广泛作用用于说明开发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用于开发AR的可靠分子模型的可靠生化数据，这将有助于AR亚型特异性激动剂和拮抗剂的基于受体的设计。 本研究旨在通过取代半胱氨酸可及性方法（SCAM）确定A1 AR跨膜跨度（TM）的哪些氨基酸可接近水环境，因此位于A1 AR的配体结合缝隙内。 该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并且激动剂和/或拮抗剂的存在延迟受体的失活速率，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类拮抗剂是否占据相同的结合位点，并确定结合位点内腺苷和黄嘌呤的相对取向。 在该计划的第三阶段，研究的目的是描绘的A1 AR的TM的三维排列，通过确定之间的一些接触点的七个TM结合表达的A1 AR和半胱氨酸扫描诱变与二硫键交联的非重叠片段，以揭示这些接触点。 这些研究应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研究产生的数据将允许区分顺时针和逆时针捆绑的七个TM。 因此，这些研究应该提供的数据，将测试目前可用的计算模型的GPCR和允许的A1 AR的分子建模的基础上，建立了总的三维排列的跨膜部分的受体和氨基酸的配体结合口袋的水环境中访问的坚实的生化数据。

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.