Ezrin binding domain-deficient NHERF attenuates cAMP-mediated inhibition of Na(+)/H(+) exchange in OK cells.
Ezrin binding domain-deficient NHERF attenuates cAMP-mediated inhibition of Na(+)/H(+) exchange in OK cells.
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Ezrin 结合域缺陷的 NHERF 减弱了 OK 细胞中 cAMP 介导的 Na( )/H( ) 交换抑制。
DOI：
10.1152/ajprenal.2001.281.2.f374
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Shenolikar,S
中科院分区：
文献类型：
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作者：
Weinman,EJ;Steplock,D;Wade,JB;Shenolikar,S
Na+/H+交换调节因子（NHERF）是cAMP介导的Na+/H+交换转运蛋白3（NHE 3）抑制中的一种重要蛋白质辅因子，可促进包括NHE 3、NHERF和ezrin在内的蛋白质信号复合物的形成。这种NHE 3调节模型是在成纤维细胞中开发的，其对上皮细胞的适用性仍有待建立。用空载体（对照）、全长小鼠（m）NHERF（1-355）或截短的mNHERF（1- 325）转染负鼠肾（OK）细胞，所述截短的mNHERF（1-325）缺乏ezrin结合，并且已在成纤维细胞中证明结合NHE 3但不介导其cAMP相关抑制。8-10− 4 M的溴腺苷3′，5 ′-环一磷酸（8-BrcAMP）对表达野生型mNHERF（1-355）的对照细胞和OK细胞的Na+/H+交换活性的抑制率>60%，但对表达mNHERF（1-325）的细胞的抑制率<10%。NHE 3与mNHERF共免疫沉淀（1-325），但在表达mNHERF的细胞中NHE 3的cAMP磷酸化受损（1-325）。高渗对NHE 3活性和3-O-甲基-D-葡萄糖摄取的抑制作用在所有三种细胞系中是相同的。在任何细胞系中，cAMP均未改变NHE 3的细胞表面表达。这些数据表明，NHERF-埃兹蛋白信号复合物的破坏减弱了cAMP对OK细胞中NHE 3活性的抑制作用，并提供了支持所提出的上皮细胞中NHE 3的蛋白激酶A调节模型的证据。
Na+/H+exchanger regulatory factor (NHERF), an essential protein cofactor in cAMP-mediated inhibition of Na+/H+exchange transporter 3 (NHE3), facilitates the formation of a signal complex of proteins that includes NHE3, NHERF, and ezrin. This model for NHE3 regulation was developed in fibroblasts and its applicability to epithelial cells remains to be established. Opossum kidney(OK) cells were transfected with either empty vector (control), full-length mouse (m) NHERF(1–355), or a truncated mNHERF(1–325) that lacked ezrin binding and had been demonstrated in fibroblasts to bind NHE3 but not mediate its cAMP-associated inhibition. 8-Bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP) at 10−4M inhibited Na+/H+exchange activity in control and OK cells expressing wild-type mNHERF(1–355) by >60% but by <10% in cells expressing mNHERF(1–325). NHE3 coimmunoprecipitated with mNHERF(1–325), but cAMP phosphorylation of NHE3 was impaired in cells expressing mNHERF(1–325). The inhibitory effect of hyperosmolality on NHE3 activity and the uptake of 3-O-methyl-d-glucose was the same in all three cell lines. Cell surface expression of NHE3 was not changed by cAMP in any of the cells lines. These data indicate that disruption of the NHERF-ezrin signal complex attenuates the inhibitory effect of cAMP on NHE3 activity in OK cells and provides evidence supporting the proposed model of protein kinase A regulation of NHE3 in epithelial cells.