Interactions of ANG II AT2 receptor signaling with ANF

心房利钠因子（ANF）和血管紧张素（Ang II）对电解质平衡和血压的调节有重要作用。ANF和angii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蛋白酪氨酸磷酸酶抑制ANF刺激的鸟苷酸环化酶活性。PC12和兔肾小管上皮细胞已被证明具有AT2受体亚型。使用这些细胞，我们发现Ang II抑制anf刺激的鸟苷酸环化酶活性和刺激蛋白酪氨酸磷酸酶活性，并且转染丝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）抑制anf刺激的鸟苷酸环化酶活性。因此，MKP-1或SHP-1可能是介导Ang II对ANF信号传导抑制作用的蛋白酪氨酸磷酸酶。我们的研究表明，ANF的结合诱导GCRP/GCRP-2（鸟苷酸环化酶调节蛋白）与鸟苷酸环化酶的关联，导致酶激活。最近，道格拉斯博士的实验室发现，AT2受体通过G蛋白β - γ亚基释放花生四烯酸，然后激活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以诱导MKP-1的表达。因此，G蛋白β - γ亚基/花生四烯酸或EGF受体/ERK级联可能介导Ang II对MKP-1诱导或MKP-1/SHP-1激活的影响。基于这些发现，我们假设：1)Ang II通过G蛋白β - γ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随后与鸟苷酸环化酶或GCRP/GCRP-2结合并使其去磷酸化，从而抑制anf刺激的鸟苷酸环化酶活性。为了验证这些假设，我们使用兔血管紧张素II AT1和AT2受体敲除小鼠的PC12和肾小管上皮细胞，我们提出：1)检测MKP-1/SHP-1是否在Ang II激活后的G蛋白β -酰胺亚基/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）结合并使其去磷酸化，从而抑制anf刺激的鸟苷酸环化酶活性。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.