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）重要地参与心血管和体液稳态的调节。 由于这种肽的重要生理、内分泌和行为作用，对AngII有反应的受体被积极研究作为对抗与内皮损伤、动脉粥样硬化、高血压和心力衰竭相关的血管病变的潜在疗法的靶点。 有两个主要的AngII受体家族，称为1型（AT 0）和2型（AT 2）亚型。 这些亚型的克隆揭示了这两种受体符合G蛋白偶联受体的七螺旋结构基序。 使用受体诱变已经是一种非常有价值的方法来阐明G-蛋白偶联受体在配体结合、受体活化和G-蛋白偶联方面的结构/功能关系。 对于AngII受体，诱变研究主要集中在AT 1受体，并导致了几个建议的计算机模型，这种亚型。 由于两种亚型之间的同源性较低（仅34%），再加上许多关键的AT 1受体残基在AT 2受体上不保守的发现，将当前的AT 1受体模型外推到AT 2受体是不确定的。 该实验室和其他实验室的努力已经开始定义赋予该亚型结合和功能特性的重要AT 2受体残基。 我们已经显示了一些令人惊讶的共性，以及一些AT 1和AT 2受体之间的差异，就血管紧张素Ⅱ结合和受体活化。 在本申请中，我们提出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AT 1和AT 2受体之间的结构数据将继续提供对整个AngII受体家族的结构设计的额外见解。 具体地，该提议将解决：（i）鉴定AT 2特异性配体（即CGP 42112 A和PD 123319）的结合表位;（ii）定位维持AT 1受体的药物天然、非活性状态的跨膜结构域3和7中的特异性残基;（iii）确定驱动促分裂原活化蛋白激酶活性的AT 1受体活化机制;（iv）鉴定控制ATz受体亚型的受体活化的结构元件;和（v）研究同源或异源AngII受体二聚体的可能形成。

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.