Weinman E J, Steplock D, Donowitz M, Shenolikar S
Department of Medicine, University of Maryland School of Medicine and Medical Service, Department of Veterans Affairs Medical Center, Baltimore, Maryland 21201, USA.
Biochemistry. 2000 May 23;39(20):6123-9. doi: 10.1021/bi000064m.
The sodium-hydrogen exchanger regulatory factor (NHERF) is an essential cofactor for cAMP-mediated inhibition of the Na(+)/H(+) exchanger isoform, NHE3, in renal brush border membranes. NHERF is also an ezrin-binding protein. To define the functional importance of ezrin binding for NHERF's function as a NHE3 regulator, we transfected stable PS120 cells expressing NHE3 with plasmids encoding WT and truncated mouse NHERF proteins. Co-immunoprecipitation established that in PS120 cells, NHE3 bound to full-length NHERF(1-355), the C-terminal domain, NHERF(147-355), and NHERF(1-325), which lacks the proposed ezrin-binding domain. The N-terminal domain, NHERF(1-146), failed to bind the antiporter. Ezrin was also co-immunoprecipitated with NHERF(1-355) but not with NHERF(1-325). 8Br-cAMP inhibited NHE3 activity in cells that expressed NHERF(1-355) or NHERF(147-355) but had no effect on the formation of NHE3-NHERF or NHERF-ezrin complexes. Na(+)/H(+) exchange was unaffected by 8Br-cAMP in cells that expressed NHERF(1-146) or NHERF(1-325). NHE3 phosphorylation in vivo was enhanced by 8Br-cAMP only in cells where NHERF bound to both NHE3 and ezrin. The data suggest that NHERF functions as a scaffold to link NHE3 with ezrin and that this multiprotein complex is essential for cAMP-mediated phosphorylation of NHE3 and the inhibition of Na(+)/H(+) exchange.
钠氢交换调节因子（NHERF）是环磷酸腺苷（cAMP）介导抑制肾刷状缘膜中钠（+）/氢（+）交换异构体NHE3所必需的辅助因子。NHERF也是一种埃兹蛋白结合蛋白。为了确定埃兹蛋白结合对NHERF作为NHE3调节因子功能的重要性，我们用编码野生型和截短型小鼠NHERF蛋白的质粒转染表达NHE3的稳定PS120细胞。免疫共沉淀表明，在PS120细胞中，NHE3与全长NHERF（1-355）、C末端结构域NHERF（147-355）以及缺乏假定埃兹蛋白结合结构域的NHERF（1-325）结合。N末端结构域NHERF（1-146）未能与该反向转运体结合。埃兹蛋白也与NHERF（1-355）进行了免疫共沉淀，但未与NHERF（1-325）共沉淀。8-溴环磷酸腺苷（8Br-cAMP）抑制表达NHERF（1-355）或NHERF（147-355）的细胞中的NHE3活性，但对NHE3-NHERF或NHERF-埃兹蛋白复合物的形成没有影响。在表达NHERF（1-146）或NHERF（1-325）的细胞中，8Br-cAMP对钠（+）/氢（+）交换没有影响。仅在NHERF与NHE3和埃兹蛋白都结合的细胞中，8Br-cAMP增强了体内NHE3的磷酸化。数据表明，NHERF作为一种支架将NHE3与埃兹蛋白连接起来，并且这种多蛋白复合物对于cAMP介导的NHE3磷酸化和钠（+）/氢（+）交换的抑制至关重要。
