The octapeptide angiotensin II (Ang II) has long been recognized as a key component in the neuroendocrine control of cardiovascular function. Ang II also plays an important role in the development of oocytes in both amphibians and mammals. Angiotensin II exerts its effects by binding to its high affinity receptors AT1 and AT2. Both of these receptors share seven-transmembrane domain topology and 34% amino acid identity. The responses induced by the AT2 receptor are sensitive to pertussis toxin suggesting that this receptor is a Gi-protein-coupled receptor. However, this receptor does not demonstrate the GTP-gamma induced shift to a low affinity form that is characteristic of the G-protein-linked receptors. Therefore it was suggested that this receptor mediates its effects through a non-classical, Gi type G-protein. The long-term objectives of the research in this laboratory are: a) to understand how the ligand-induced responses of a cell are regulated, and b) to elucidate how the receptor-mediated signal transduction regulates the molecular switch that determines the fate (growth or apoptosis) of a cell. The primary objective of this proposal is to analyze the structure-function relationship of rat AT2 receptor and to elucidate what are the intracellular signaling cascades that are activated in response to the binding of Ang II to this receptor when expressed in Xenopus oocytes. Our recent findings indicate that activation of rat AT2 receptor expressed in Xenopus oocytes causes an elevation in the intracellular IP3 levels. However, Ang II binding to rat AT2 receptors expressed in Xenopus oocytes causes an elevation in the intracellular IP3 levels. However, Ang II binding to rat AT2 receptors expressed in Xenopus oocytes is not sensitive to GTPgammaS. Therefore, the PI hypothesizes that the rat AT2 receptor mediated activation of the phospholipase C pathway in Xenopus oocytes is also effected through a non-classical Gi-protein endogenous to these cells. Experiments are directed to identify and characterize the nature of this endogenous mediator that couples rat AT2 receptor to PLC pathway. To do this, the PI proposes to study the structure-function relationship of AT2 receptor by generating site-directed mutants and AT2:AT1 chimeric proteins. The PI also proposes to analyze what other signaling mechanisms are activated by rat AT2 receptor in Xenopus oocytes. The PI hopes these studies will provide new insights into the signaling by AT2 receptor and its possible role in oocyte development.
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