Mutational Analysis of Angiotensin II Receptors

描述（由申请人提供）：众所周知，肽激素血管紧张素II （AngII）在调节心血管和体液稳态中起重要作用。由于这种肽在生理、内分泌和行为方面的重要作用，对AngII有反应的受体被积极研究，作为治疗与内皮损伤、动脉粥样硬化、高血压和心力衰竭相关的血管病变的潜在靶点。有两个主要的AngII受体家族，被称为1型（AT0）和2型（AT2）亚型。这些亚型的克隆表明，这两种受体都符合g蛋白偶联受体的七螺旋结构基序。受体诱变是阐明g蛋白偶联受体在配体结合、受体激活和g蛋白偶联方面的结构/功能关系的宝贵方法。对于AngII受体，诱变研究主要集中在AT1受体上，并提出了几种该亚型的计算机模型。由于两种亚型之间的同源性较低（仅34%），再加上许多关键的AT1受体残基在AT2受体上并不保守，因此将目前的AT1受体模型外推到AT2受体上一直是模棱两可的。该实验室和其他实验室已经开始定义重要的AT2受体残基，这些残基赋予了该亚型的结合和功能特性。我们已经展示了AT1和AT2受体在AngII结合和受体激活方面的一些惊人的共性以及一些不同之处。在目前的应用中，我们建议使用受体诱变来继续定义两种AnglI受体亚型的结构/功能关系。比较和对比AT1和AT2受体之间的结构数据将继续为整个AngII受体家族的结构设计提供更多的见解。具体而言，该提案将解决：(i)识别at2特异性配体的结合表位，即CGP42112A和PD123319；（ii）在跨膜结构域3和7中定位维持AT1受体药物原生、非活性状态的特定残基；（iii）确定AT1受体激活驱动有丝分裂原激活蛋白激酶活性的机制；（iv）识别控制ATz受体亚型受体激活的结构元件；(v)研究可能形成的AngII受体二聚体，无论是同源的还是异源的。

DESCRIPTION (provided by applicant): It is well recognized that the peptide hormone angiotensin II (AngII) is importantly involved in the regulation of cardiovascular and body fluid homeostasis. Due to the important physiological, endocrine, and behavioral effects of this pepfide, the receptors that respond to AngII are actively studied as targets for potential therapies against vascular lesions associated with endothelial damage, atherosclerosis, hypertension, and cardiac failure. There are two main families of AngII receptors, referred to as the Type 1 (AT0 and the Type 2 (AT2) subtypes. Cloning of these subtypes have revealed that both receptors conform to the heptahelical structural motif of G-protein coupled receptors. Use of receptor mutagenesis has been an invaluable approach to elucidate the structure/functional relationships of G-protein coupled receptors with respect to ligand binding, receptor activation, and G-protein coupling. For AngII receptors, mutagenesis studies have focused primarily on the AT1 receptor and have led to several proposed computer models of this subtype. Due to the low homology (only 34 percent) shared between the two subtypes coupled with findings that many of the key AT1 receptor residues are not conserved on AT2 receptors, extrapolating current AT1 receptor models to AT2 receptors has been equivocal. Efforts by this laboratory and others have begun to define important AT2 receptor residues that confer this subtype's binding and functional properties. We have shown some surprising commonalities as well as some dissimilarities between AT1 and AT2 receptors with respect to AngII binding and receptor activation. In the present application, we propose to use receptor mutagenesis to continue defining the structure/function relationships for both AnglI receptor subtypes. Comparing and contrasting the structural data between AT1 and AT2 receptors will continue to provide additional insights towards the structural design of the entire AngII receptor family. Specifically, this proposal will address: (i) identifying binding epitopes for AT2-specific ligands, i.e. CGP42112A and PD123319; (ii) locating specific residues in transmembrane domains 3 and 7 that maintain the AT1 receptor's drug-native, inactive state; (iii) determining the mechanisms of AT1 receptor activation that drive mitogen activating protein kinase activity; (iv) identifying structural elements that control receptor activation for the ATz receptor subtype; and (v) investigating possible formation of AngII receptor dimers, either homologous or heterologous.