Huang Peng, Steplock Deborah, Weinman Edward J, Hall Randy A, Ding Zhe, Li Jianguo, Wang Yulin, Liu-Chen Lee-Yuan
Department of Pharmacology and Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
J Biol Chem. 2004 Jun 11;279(24):25002-9. doi: 10.1074/jbc.M313366200. Epub 2004 Apr 7.
We previously showed that Na(+)/H(+)-exchanger regulatory factor-1/Ezrin-radixin-moesin-binding phosphoprotein-50 (NHERF-1/EBP50) co-immunoprecipitated with the human kappa opioid receptor (hKOR) and that its overexpression blocked the kappa agonist U50,488H-induced hKOR down-regulation by enhancing recycling. Here, we show that glutathione S-transferase (GST)-hKOR C-tail interacted with purified NHERF-1/EBP50, whereas GST or GST-C-tails of micro or delta opioid receptors did not. GST-hKOR C-tail, but not GST, bound HA-NHERF-1/EBP50 transfected into Chinese hamster ovary cells and endogenous NHERF-1/EBP50 in opossum kidney proximal tubule epithelial cells (OK cells). The PDZ domain I, but not II, of NHERF-1/EBP50 was involved in the interaction. Association of NHERF-1/EBP50 with hKOR C-tail enhanced oligomerization of NHERF-1/EBP50. NHERF-1/EBP50 was previously shown to regulate Na(+)/H(+)-exchanger 3 (NHE3) activities in OK cells. We found stimulation of OK cells with U50,488H significantly enhanced Na(+)/H(+) exchange, which was blocked by naloxone but not by pertussis toxin pretreatment, indicating it is mediated by KORs but independent of G(i)/G(o) proteins. In OKH cells, a subclone of OK cells expressing a much lower level of NHERF-1/EBP50, U50,488H had no effect on Na(+)/H(+) exchange, although it enhanced p44/42 mitogen-activated protein kinase phosphorylation via G(i)/G(o) proteins similar to that in OK cells. Stable transfection of NHERF-1/EBP50 into OKH cells restored the stimulatory effect of U50,488H upon Na(+)/H(+) exchange. Thus, NHERF-1/EBP50 binds directly to KOR, and this association plays an important role in accelerating Na(+)/H(+) exchange. We hypothesize that binding of the KOR to NHERF-1/EBP50 facilitates oligomerization of NHERF-1/EBP50, leading to stimulation of NHE3. This study provides the first direct evidence that a G protein-coupled receptor through association with NHERF-1/EBP-50 stimulates NHE3.
我们之前的研究表明，钠氢交换调节因子-1/埃兹蛋白-根蛋白-膜突蛋白结合磷蛋白-50（NHERF-1/EBP50）与人κ阿片受体（hKOR）共免疫沉淀，并且其过表达通过增强循环来阻断κ激动剂U50,488H诱导的hKOR下调。在此，我们发现谷胱甘肽S-转移酶（GST）-hKOR C末端与纯化的NHERF-1/EBP50相互作用，而GST或μ或δ阿片受体的GST-C末端则无此作用。GST-hKOR C末端，但不是GST，能结合转染到中国仓鼠卵巢细胞中的HA-NHERF-1/EBP50以及负鼠肾近端小管上皮细胞（OK细胞）中的内源性NHERF-1/EBP50。NHERF-1/EBP50的PDZ结构域I而非II参与了这种相互作用。NHERF-1/EBP50与hKOR C末端的结合增强了NHERF-1/EBP50的寡聚化。之前的研究表明NHERF-1/EBP50可调节OK细胞中钠氢交换体3（NHE3）的活性。我们发现用U50,488H刺激OK细胞可显著增强钠氢交换，这被纳洛酮阻断，但百日咳毒素预处理则无此作用，表明这是由KOR介导的，但不依赖于G(i)/G(o)蛋白。在OKH细胞（OK细胞的一个亚克隆，表达水平低得多的NHERF-1/EBP50）中，U50,488H对钠氢交换无影响，尽管它通过G(i)/G(o)蛋白增强了p44/42丝裂原活化蛋白激酶的磷酸化，与OK细胞中的情况类似。将NHERF-1/EBP50稳定转染到OKH细胞中可恢复U50,488H对钠氢交换的刺激作用。因此，NHERF-1/EBP50直接与KOR结合，这种结合在加速钠氢交换中起重要作用。我们推测KOR与NHERF-1/EBP50的结合促进了NHERF-1/EBP50的寡聚化，从而导致NHE3的激活。本研究首次提供了直接证据，表明一种G蛋白偶联受体通过与NHERF-1/EBP-50结合来刺激NHE3。
