Yun C H, Lamprecht G, Forster D V, Sidor A
Department of Medicine, Gastroenterology Division, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
J Biol Chem. 1998 Oct 2;273(40):25856-63. doi: 10.1074/jbc.273.40.25856.
Cyclic AMP is a major second messenger that inhibits the brush border Na+/H+ exchanger NHE3. We have previously shown that either of two related regulatory proteins, E3KARP or NHERF, is necessary for the cAMP-dependent inhibition of NHE3. In the present study, we characterized the interaction between NHE3 and E3KARP using in vitro binding assays. We found that NHE3 directly binds to E3KARP and that the entirety of the second PSD-95/Dlg/ZO-1 (PDZ) domain plus the carboxyl-terminal domain of E3KARP are required to bind NHE3. E3KARP binds an internal region within the NHE3 C-terminal cytoplasmic tail, defining a new mode of PDZ domain interaction. Analyses of cellular distribution of NHE3 and E3KARP expressed in PS120 fibroblasts show that NHE3 and E3KARP are co-localized on the plasma membrane, but not in a distinct juxtanuclear compartment in which NHE3 is predominantly expressed. The distributions of NHE3 and E3KARP were not affected by treatment with 8-bromo-cAMP. As shown earlier for the human homolog of NHERF, we also found that the cytoskeletal protein ezrin binds to the carboxyl-terminal domain of E3KARP. These results are consistent with the possibility that E3KARP and NHERF may function as scaffold proteins that bind to both NHE3 and ezrin. Since ezrin is a protein kinase A anchoring protein, we suggest that the scaffolding function of E3KARP binding to both ezrin and NHE3 localizes cAMP-dependent protein kinase in the vicinity of the cytoplasmic domain of NHE3, which is phosphorylated by elevated cAMP.
环磷酸腺苷（cAMP）是一种主要的第二信使，可抑制刷状缘钠/氢交换体NHE3。我们之前已经表明，两种相关的调节蛋白E3KARP或NHERF中的任何一种对于cAMP依赖性抑制NHE3都是必需的。在本研究中，我们使用体外结合试验对NHE3与E3KARP之间的相互作用进行了表征。我们发现NHE3直接与E3KARP结合，并且E3KARP的整个第二个PSD-95/Dlg/ZO-1（PDZ）结构域加上羧基末端结构域是结合NHE3所必需的。E3KARP结合NHE3 C末端细胞质尾巴内的一个内部区域，确定了一种新的PDZ结构域相互作用模式。对PS120成纤维细胞中表达的NHE3和E3KARP的细胞分布分析表明，NHE3和E3KARP共定位于质膜上，但不存在于NHE3主要表达的明显的近核区室中。NHE3和E3KARP的分布不受8-溴-cAMP处理的影响。正如之前对NHERF的人类同源物所显示的那样，我们还发现细胞骨架蛋白埃兹蛋白（ezrin）与E3KARP的羧基末端结构域结合。这些结果与E3KARP和NHERF可能作为与NHE3和埃兹蛋白都结合的支架蛋白发挥作用的可能性一致。由于埃兹蛋白是一种蛋白激酶A锚定蛋白，我们认为E3KARP与埃兹蛋白和NHE3结合的支架功能将cAMP依赖性蛋白激酶定位在NHE3细胞质结构域附近，该结构域会被升高的cAMP磷酸化。
