: Our broad, long term objective is to understand the antigrowth functions of the AT2 receptor for angiotensin II (Ang II). Accumulating evidences suggests that the AT., receptor regulates cell number during ontogenesis and remodeling of tissues by apoptosis. Abnormality in AT, receptor function is implicated in development of pathological conditions such as CAKUT (congenital abnormality of kidney and urinary tract), flawed ontogenesis of vessels, abnormal post-infarct cardiac remodeling and in neointima formation following vascular injury. Its expression is restricted to a small number of tissues in adults, but during fetal development, tissue repair and remodeling the AT2 receptor is over expressed at sites of remodeling. The AT2 receptor is a novel paradigm for study since it is a 7TM receptor with the unique ability to regulate MAPKS, caspases and protein phosphatases besides G proteins. A complete understanding of the basis of antigrowth functions of the AT2 receptor requires the analysis of structure-function and the identification of molecules involved in signal transduction from this receptor. To undertake a detailed study of the mechanisms involved and to identify the molecules that mediate apoptotic signals from the AT2 receptor, we have established in vitro cell models of apoptosis.
The specific aims for this application are: (1) To define the structure-function relationship of the AT2 receptor in initiating apoptosis. (2) To define the role of a novel adaptor molecule in AT2 receptor-mediated p38 MAPK activation and apoptosis. (3) To isolate novel signaling molecules that interact with the AT, receptor and to characterize their role in its functions. Site-directed mutants defective in AT, receptor-induced apoptosis will be examined for their ability to activate different intracellular signals that co-operatively mount programmed cell death. The activation of various MAPK isoforms, caspases, protein tyrosine phosphatase isoforms, G-protein G-mediated phosphatases; and the down regulation of Cdks and Cdk-inhibitors will be studied in transfected vascular smooth muscle cells. These studies will delineate the cooperative interaction of CD-loops in coupling to distinct signals and in defining the steps that are involved in the AT, receptor-induced death pathway. We will carry out yeast two-hybrid interaction cloning using the AT2 receptor-CD! construct as a bait. Real positive clones will be identified by rigorous tests including genetic screening, protein interaction studies and functional complementation analysis. Full length cDNA will be characterized for their role in mediating AT, receptor-induced apoptosis and expression changes in various pathophysiological states. We will use mutagenesis, protein interaction assays and functional analysis by co-transfection to evaluate the specificity of a candidate adapter protein in AT2-mediated p38 MAPK activation. We anticipate that these studies will shed light on the mechanisms regulating induction of apoptosis by the AT2 receptor in smooth muscle and other types of cells. Furthermore, these studies may lead to an increased understanding of the mechanisms regulating other AT2 receptor-mediated functions such as cardio-protection and fetal development.
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