Interactions of ANG II AT2 receptor signaling with ANF

心钠素(ANF)和血管紧张素Ⅱ(Ang II)在调节电解质平衡和血压方面发挥着重要作用。ANF和Ang II通常相互对抗。然而，Ang II和ANF相互作用的分子事件仍有待确定。Ang II AT2受体已被证明通过一种未知的蛋白质酪氨酸磷酸酶抑制ANF刺激的鸟苷环化酶活性。PC12和兔肾小管上皮细胞具有AT2受体亚型。利用这些细胞，我们发现Ang II抑制ANF刺激的鸟苷环化酶的活性，并刺激蛋白酪氨酸磷酸酶的活性，而有丝分裂原激活的蛋白激酶磷酸酶(MKP-1)和蛋白酪氨酸磷酸酶SHP-1的转基因则抑制ANF刺激的鸟氨酸环化酶的活性。因此，MKP-1或SHP-1可能是介导Ang II抑制ANF信号转导的蛋白酪氨酸磷酸酶。我们的研究人员指出，ANF的结合诱导了GCRP/GCRP-2(鸟苷环化酶调节蛋白)与鸟苷环化酶的关联，从而导致酶的激活。最近，道格拉斯博士的实验室发现，AT2受体通过G蛋白β淀粉亚基释放花生四烯酸，花生四烯酸随后激活EGF受体/ERK(丝裂原激活蛋白激酶)级联反应。我们发现花生四烯酸可诱导MKP-1的表达。因此，G蛋白β亚基/花生四烯酸或EGF受体/ERK级联通路可能介导Ang II对MKP-1的诱导或MKP-1/SHP-1的激活。根据这些发现，我们假设：1)Ang II通过G蛋白β-亚基/花生四烯酸或EGF受体/ERK级联激活或诱导MKP-1/SHP-1；2)MKP-1或SHP-1与鸟苷环化酶或GCRP/GCRP-2结合并去磷酸化，从而抑制ANF刺激的鸟苷环化酶活性。为了验证这些假说，我们利用兔血管紧张素II AT1和AT2受体敲除小鼠的PC12和肾小管上皮细胞，建议：1)检测MKP-1/SHP-1是否位于G蛋白β亚基/花生四烯酸或EGF受体/ERK级联反应的下游；3)检测MKP-1/SHP-1是否与鸟苷酸环化酶或GCRP(或GCRP-2)相关并使其去磷酸化，从而抑制ANF刺激的鸟巢晚期环化酶活性。这些研究产生的信息将有助于在分子水平上理解Ang II和ANF信号的相互作用。

Atrial natriuretic factor (ANF) and angiotensin (Ang II) exert potent effects on the regulation of electrolyte balance and blood pressure. ANF and Ang II normally antagonize each other. However, the molecular event underlying the interplay of Ang II and ANF remains to be established. The Ang II AT2 receptor has been shown to inhibit ANF- stimulated guanylate cyclase activity through an unknown protein tyrosine phosphatase. PC12 and rabbit renal tubular epithelial cells have been shown to possess an AT2 receptor subtype. Using these cells, we have found that Ang II inhibits ANF-stimulated guanylate cyclase activity and stimulates protein tyrosine phosphatase activities, and that transfection of the mitogen-activated protein kinase phosphatase (MKP- 1) and protein tyrosine phosphatase, SHP-1, inhibits ANF-stimulated guanylate cyclase activity. Therefore, MKP-1 or SHP-1 may be the protein tyrosine phosphatase that mediates the inhibitory effect of Ang II on ANF signaling. Our studiers indicate that binding of ANF induces the association of GCRP/GCRP-2 (guanylate cyclase regulatory proteins) with guanylate cyclase leading to enzyme activation. Recently, Dr. Douglas's laboratory has found that the AT2 receptor, through G protein betagamma subunits, releases arachidonic acid which then activates EGF receptor/ERK (mitogen-activate protein kinase) cascade. We have found that arachidonic acid induces the expression of MKP-1. Therefore, G protein betagamma subunits/arachidonic acid or EGF receptor/ERK cascade may mediate the effect of Ang II on MKP-1 induction or MKP-1/SHP-1 activation. Based on these findings, we hypothesize that: 1) Ang II activates or induces MKP-1/SHP-1 through G protein betagamma subunits/arachidonic acid or EGF receptor/ERK cascade; and 2) MKP-1 or SHP-1 then associates with and dephosphorylates either guanylate cyclase or GCRP/GCRP-2 leading to the inhibition of ANF-stimulated guanylate cyclase activity. To evaluate these hypotheses, using PC12 and renal tubular epithelial cells from rabbit angiotensin II AT1 and AT2 receptor knock out mice, we propose: 1) to examine whether MKP-1/SHP-1 is downstream of G protein betagamma subunits/arachidonic acid or EGF receptor/ERK cascade following Ang II activation; and 3) to examine whether MKP- 1/SHP-1 associate with and dephosphorylates either guanylate cyclase or GCRP (or GCRP-2) leading to the inhibition of ANF-stimulated guanylate cyclase activity. The information generated from these studies will help to understand the interplay of Ang II and ANF signaling in the molecular level.