Signaling Mechanisms of the Angll Receptor AT2

说明(申请人提供)：血管紧张素II(Ang II)因其参与调节血压和水矿平衡以及高血压、充血性心力衰竭和其他一些临床疾病的发病机制而广为人知。我们已经证明，在非洲爪哇卵母细胞中，Ang11受体AT1和AT2的共表达导致了AT2介导的抑制AT1介导的IP3的产生，并且AT2的第三ICL区域跨越240-256个氨基酸，足以实现这一功能。我们还证明了AT2与ErbB3受体直接相互作用，并提出这可能是AT2发挥其抗生长作用的机制之一。本研究的总体目标是研究血管紧张素Ⅱ受体AT2的信号机制，并阐明蛋白质与蛋白质的直接相互作用在这一功能中的作用。在区域基金R15HL60241-01的资助下，我们在非洲爪哇卵母细胞中产生了大量的AT2突变体，并证明了AT2介导的两种新的信号机制：a)AT2介导的抑制AT1介导的IP3的产生，b)AT2介导的cGMP减少。我们还证明了AT2直接与ErbB3受体相互作用，并提出这可能是AT2发挥其抗生长作用的机制。本研究的具体目的1是确定非洲爪哇卵母细胞中与cGMP基础水平降低有关的AT2区域，并确定AT2是否对AT1介导的cGMP在这些细胞中的升高起到负调控作用。本研究的目的2是确定中国仓鼠卵巢(CHO)细胞胰岛素受体(IR)β链酪氨酸磷酸化抑制的AT2区域，并确定AT2是否通过直接的蛋白质相互作用来抑制IR的磷酸化。我们认为，AT2和IRβ链之间的直接相互作用，类似于我们在AT2和ErbB3相互作用中看到的情况，可能参与了AT2介导的IR信号的抑制。我们将使用AT2突变体来确定AT2的哪个区域参与抑制CHO细胞的IR信号。因此，这项建议的资助不仅有助于阐明新发现的Ang II的信号机制，还将使我们能够继续为北京国立大学细胞和分子研究方面的大量研究生和本科生提供培训。

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.