Extracellular Loop 2 of the Adenosine A1 Receptor Has a Key Role in Orthosteric Ligand Affinity and Agonist Efficacy
Extracellular Loop 2 of the Adenosine A1 Receptor Has a Key Role in Orthosteric Ligand Affinity and Agonist Efficacy
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腺苷 A1 受体的细胞外环 2 在正位配体亲和力和激动剂功效中发挥关键作用
作者：
A. T. Nguyen;Joanne Baltos;Trayder Thomas;Toan D. Nguyen;Laura López Muñoz;K. Gregory;P. White;P. Sexton;A. Christopoulos;L. May
腺苷A1G蛋白偶联受体（A1AR）是涉及广泛的心血管和神经元病症的重要治疗靶标。尽管已经确定A1AR正构位点位于受体的跨膜（TM）束内，但先前的研究已经暗示细胞外环2（ECL 2）在促进正构配体亲和力和各种G蛋白偶联受体（GPCR）的信号传导中具有重要作用。因此，我们进行了广泛的丙氨酸扫描诱变的A1AR-ECL 2，以探讨该域的A1AR正构配体药理学的作用。使用定量分析方法和分子建模，我们确定了直接或间接与正构激动剂和拮抗剂相互作用的ECL 2残基。邻近保守的ECL2-TM3二硫键的离散突变选择性地影响正构配体亲和力，而TM4-ECL2接合处附近的五个残基的簇影响正构激动剂功效。配体对接、分子动力学模拟和诱变结果的组合表明，在进入典型的TM束正构位点之前，正构激动剂5 ′-N-乙基羧酰胺腺苷瞬时结合到由ECL 2和TM 5和TM 7的顶部形成的细胞外前庭。总的来说，这项研究强调了ECL 2在A1AR正构配体结合和受体活化中的关键作用。
The adenosine A1 G protein–coupled receptor (A1AR) is an important therapeutic target implicated in a wide range of cardiovascular and neuronal disorders. Although it is well established that the A1AR orthosteric site is located within the receptor’s transmembrane (TM) bundle, prior studies have implicated extracellular loop 2 (ECL2) as having a significant role in contributing to orthosteric ligand affinity and signaling for various G protein–coupled receptors (GPCRs). We thus performed extensive alanine scanning mutagenesis of A1AR-ECL2 to explore the role of this domain on A1AR orthosteric ligand pharmacology. Using quantitative analytical approaches and molecular modeling, we identified ECL2 residues that interact either directly or indirectly with orthosteric agonists and antagonists. Discrete mutations proximal to a conserved ECL2-TM3 disulfide bond selectively affected orthosteric ligand affinity, whereas a cluster of five residues near the TM4-ECL2 juncture influenced orthosteric agonist efficacy. A combination of ligand docking, molecular dynamics simulations, and mutagenesis results suggested that the orthosteric agonist 5′-N-ethylcarboxamidoadenosine binds transiently to an extracellular vestibule formed by ECL2 and the top of TM5 and TM7, prior to entry into the canonical TM bundle orthosteric site. Collectively, this study highlights a key role for ECL2 in A1AR orthosteric ligand binding and receptor activation.
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人 A(1) 腺苷受体的九个天然半胱氨酸残基的丝氨酸和丙氨酸诱变。
影响因子：
5.8
作者：
Scholl,DJ;Wells,JN
通讯作者：
Wells,JN
影响因子：
3.4
作者：
Mueller, Christa E.;Jacobson, Kenneth A.
通讯作者：
Jacobson, Kenneth A.
影响因子：
4.8
作者：
Lei, S;Liapakis, G;Javitch, JA
通讯作者：
Javitch, JA
激动剂诱导 P2Y2 受体的内化。
影响因子：
3.6
作者：
Sromek,SM;Harden,TK
通讯作者：
Harden,TK
DOI：
10.1073/pnas.2237265100
发表时间：
2004-01-13
影响因子：
11.1
作者：
Shi, L;Javitch, JA
通讯作者：
Javitch, JA