Signaling Mechanisms of the Angll Receptor AT2

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DESCRIPTION (provided by applicant): Angiotensin II (Ang II) is well known for its involvement in the regulation of blood pressure and hydromineral balance, as well as in the pathogenesis of hypertension, congestive heart failure and a number of other clinical conditions. We have demonstrated that co-expression of the Angll receptors AT1 and AT2 in Xenopus oocytes resulted in AT2-mediated inhibition of the AT1-mediated IP3 production, and that the region of the 3rd ICL of AT2 that spans amino acids 240-256 is sufficient for this function. We have also shown that AT2 directly interacts with the ErbB3 receptor and has proposed that this could be one of the mechanisms by which AT2 exerts its anti-growth effects. The general goal of this research proposal is to characterize the signaling mechanisms of the Ang II receptor AT2, and elucidate the role of direct protein-protein interaction in this function. With the help of funding from AREA grant R15HL60241-01, we generated a large number of the AT2 mutants and have demonstrated two new AT2-mediated signaling mechanisms in Xenopus oocytes:a) the AT2-mediated inhibition of the ATl-mediated IP3 production, and b) AT2-mediated cGMP reduction.We have also shown that AT2 directly interacts with the ErbB3 receptor and have proposed that this could be a mechanism by which AT2 exerts its anti-growth effects. The specific aim 1 of this proposal is to identify the region of the AT2 involved in reduction of basal levels of cGMP in Xenopus oocytes, and determine whether the AT2 exerts a negative regulatory effect on the AT1 mediated increase of the cGMP in these cells. The specific aim 2 is to identify the region of the AT2 responsible for the inhibition of tyrosine phosphorylation of the insulin receptor (IR) beta chain in Chinese Hamster Ovary (CHO) cells, and determine whether this AT2-mediated inhibition of IR phosphorylation is via direct protein-protein interaction. We propose that a direct interaction between the AT2 and IR beta chain, similar to what we have seen in the case of AT2 and ErbB3 interaction, may be involved in AT2-mediated inhibition of IR signaling. We will use the AT2 mutants to determine which region of AT2 is involved in the inhibition of IR signaling in CHO cells. Thus, funding of this proposal will not only help in elucidating newly identified signaling mechanisms of Ang II, but also allow us to continue the training we have been giving to a large number of graduate and undergraduate students in cellular and molecular research at BGSU.