Mutational Analysis of Angiotensin II Receptors

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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.