It is well recognized that the central actions of angiotensin II (AngII) are importantly involved in the regulation of cardiovascular and body fluid homeostasis. Included amongst the actions of AngII in the brain are behavioral, endocrine, and physiological responses that result from the interaction of the peptide with cell surface receptors. There are two main families of AngII receptors, referred to as AT1 and AT2. Mutagenesis studies and computer modeling have primarily focused on the AT1 receptor, thereby leading to increasing understanding of the molecular mechanisms that define AT1 ligand binding and effector actions. In contrast, limited mutagenesis and no modeling studies have been performed on AT2 subtype. Because the two subtypes share only 34% homology, the extent that AT1 mutagenesis and modeling data are applicable to the AT2 receptor is currently unknown. Accordingly, in the present application, a series of AngII receptor mutants, in deletional mutations, AT1/AT2 chimeras, and point mutations, will be created and analyzed in order to identify the structural elements that define the ligand binding properties of AT2 receptors. Although these experiments focus primarily on the AT2 subtype, later comparison of similarities and differences with the AT1 receptor will provide insight into the ligand binding pocket of the entire AngII receptor family. In addition, because there is growing evidence that receptor dimerization may play an important role in modulating receptor function, the possible formation of AngII receptor dimers will also be studied. Specifically, this proposal will address: (i) identifying ion-pair interactions between AT2 receptor and AngII; (ii) the role of subtype-specific epitopes in the binding of AngII; (iii) the role of cysteine residues in the formation of disulfide bridges and as a source for the differing sensitivities of the two subtypes to the reducing agent dithiothreitol; (iv) identifying the structural elements that are responsible for AT2 subtype selective binding; and (v) investigating possible formation of AngII receptor dimers, either homologous or heterologous.
