Regulation of NHE3 by glucocorticoids, SGK1 and NHERF2

描述（由申请人提供）：二十多年来，人们已经知道糖皮质激素对维持体液和电解质平衡的刺激作用。药理剂量的甲基强的松龙刺激小动物的Na+吸收。然而，这种激活的分子机制仍然难以捉摸。小肠和肾脏刷状缘膜上的Na+/H+交换体NHE 3在Na+的跨上皮吸收中起主要作用。我们以前证明，甲基强的松龙在兔特异性刺激回肠NHE 3 mRNA，而不影响NHE 1 mRNA水平。其他研究表明，糖皮质激素特异性激活回肠、近端结肠和肾近端小管中的NHE 3 mRNA。这些结果表明，糖皮质激素通过NHE 3的基因表达激活NHE 3活性。然而，我们最近发现使用Caco-2细胞，地塞米松激活NHE 3转运而不影响NHE 3 mRNA表达。地塞米松对NHE 3的这种非转录激活在OK细胞中得到证实，表明NHE 3的转录激活可能不是糖皮质激素刺激NHE 3的唯一决定因素。我们发现地塞米松仅在NHE 3调节蛋白NHERF 2存在下增强NHE 3活性。我们确定了血清和糖皮质激素诱导的蛋白激酶1，SGK 1，作为一种蛋白质与PDZ结构域的NHERF 2。我们证明了SGK 1激活NHE 3活性，OK细胞中“激酶死亡”SGK 1的表达显著阻断了地塞米松的作用，证明了SGKI的重要性。我们还表明，SGK 1直接磷酸化NHE 3在体外表明磷酸化依赖性调节NHE 3。在本申请中，我们拟研究：（1）SGK 1和NHERF 2在糖皮质激素激活NHE 3中的作用;（2）SGK 1和NHERF 2响应糖皮质激素激活NHE 3的分子机制;以及（3）SGK 1，NHERF 2和NHE 3之间的相互作用。这一建议将解决糖皮质激素调节NHE 3的复杂性，并增强我们对膜转运蛋白调节中蛋白质-蛋白质相互作用的理解。

DESCRIPTION (provided by applicant): The stimulative effect of glucocorticoids on the maintenance of fluid and electrolyte balance has been known for more than two decades. Pharmacological doses of methylprednisolone stimulated Na+ absorption in small animals. However, molecular mechanisms underlying this activation remain elusive. Na+/H+ exchanger NHE3 in the brush border membrane of intestine and kidney plays a major role in transepithelial Na+ absorption. We previously demonstrated that methylprednisolone in rabbits specifically stimulated NHE3 mRNA in ileum without affecting NHE1 mRNA levels. Others have shown that glucocorticoids specifically activated NHE3 mRNA in ileum, proximal colon and renal proximal tubules. These results suggest that glucocorticoids activate NHE3 activity by gene expression of NHE3. However, we recently found using Caco-2 cells that dexamethasone activates NHE3 transport without affecting NHE3 mRNA expression. This non-transcriptional activation of NHE3 by dexamethasone was demonstrated in OK cells, suggesting that transcriptional activation of NHE3 may not be the only determining factor in glucocorticoid-stimulation of NHE3. We found that dexamethasone enhanced NHE3 activity only in the presence of a NHE3 regulatory protein, NHERF2. We identified serum- and glucocorticoids-induced protein kinase 1, SGK1, as a protein interacting with PDZ domains of NHERF2. We demonstrated that SGK1 activated NHE3 activity and expression of "kinase-dead" SGK1 in OK cells markedly blocked the dexamethasone effect, demonstrating the importance of SGKI. We also showed that SGK1 directly phosphorylated NHE3 in vitro suggesting phosphorylation-dependent regulation of NHE3. In this application, we propose to investigate: (1) the roles of SGK1 and NHERF2 in activation of NHE3 by glucocorticoids; (2) molecular mechanisms underlying the activation of NHE3 by SGK1 and NHERF2 in response to glucocorticoid; and (3) the interaction between SGK1, NHERF2 and NHE3. This proposal will resolve complexity in NHE3 regulation by glucocorticoids, and enhance our understanding of protein-protein interaction in regulation of membrane transport proteins.