Signaling Mechanisms of the Angll Receptor AT2

描述（由申请人提供）：众所周知，血管紧张素II（Ang II）参与血压和水矿物质平衡的调节，以及参与高血压、充血性心力衰竭和许多其他临床病症的发病机制。我们已经证明，AngII受体AT 1和AT 2在爪蟾卵母细胞中的共表达导致AT 1介导的IP 3产生的AT 2介导的抑制，并且跨越氨基酸240-256的AT 2的第三ICL的区域足以实现该功能。我们还表明，AT 2直接与ErbB 3受体相互作用，并提出这可能是AT 2发挥其抗生长作用的机制之一。本研究的总体目标是表征血管紧张素II受体AT 2的信号传导机制，并阐明直接蛋白质-蛋白质相互作用在此功能中的作用。在AREA基金R15 HL 60241 -01的资助下，我们产生了大量的AT 2突变体，并在非洲爪蟾卵母细胞中证明了两种新的AT 2介导的信号传导机制：a）AT 2介导的对AT 1介导的IP 3产生的抑制，和B）AT 2介导的cGMP减少。我们还表明AT 2直接与ErbB 3受体相互作用，并提出这可能是AT 2发挥其抗生长作用的机制。本提案的具体目的1是鉴定参与爪蟾卵母细胞中cGMP基础水平降低的AT 2区域，并确定AT 2是否对这些细胞中AT 1介导的cGMP增加发挥负调节作用。具体目的2是鉴定负责抑制中国人卵巢（CHO）细胞中胰岛素受体（IR）β链酪氨酸磷酸化的AT 2区域，并确定AT 2介导的IR磷酸化抑制是否通过直接蛋白质-蛋白质相互作用。我们提出，AT 2和IR β链之间的直接相互作用，类似于我们在AT 2和ErbB 3相互作用的情况下所看到的，可能参与AT 2介导的IR信号抑制。我们将使用AT 2突变体来确定AT 2的哪个区域参与抑制CHO细胞中的IR信号传导。因此，该提案的资助不仅有助于阐明新发现的Ang II信号传导机制，而且还使我们能够继续对BGSU细胞和分子研究的大量研究生和本科生进行培训。

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.