A C-terminal PDZ motif in NHE3 binds NHERF-1 and enhances cAMP inhibition of sodium-hydrogen exchange.
A C-terminal PDZ motif in NHE3 binds NHERF-1 and enhances cAMP inhibition of sodium-hydrogen exchange.
复制标题
NHE3 中的 C 端 PDZ 基序结合 NHERF-1 并增强 cAMP 对钠氢交换的抑制。
DOI：
10.1021/bi035244l
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发表时间：
2003
期刊：
影响因子：
2.9
通讯作者：
Shenolikar,Shirish
中科院分区：
文献类型：
--
作者：
Weinman,EdwardJ;Wang,Yu;Wang,Fengying;Greer,Charmaine;Steplock,Deborah;Shenolikar,Shirish
NHERF-1是一种包含两个串联PDZ结构域的蛋白质适配器，首次被确定为响应细胞内cAMP升高的NHE3活性磷酸化和下调所需的重要辅助因子。NHERF-1包含多个蛋白相互作用域，但其结合NHE3的机制尚不清楚。酵母双杂交分析表明，NHE3的c端序列STHM构成了与NHERF-1关联的PDZ基序。在该实验中，NHE3作为分离结构域结合PDZ- 1和PDZ- ii，但全长NHERF-1中单个PDZ结构域的突变表明NHE3明显倾向于PDZ- ii结构域。为了研究NHERF-1/NHE3在细胞中的相关性，利用镍- nta -琼脂糖从表达六组氨酸标记的NHERF-1和NHE3的PS120细胞中分离出NHERF-1复合物。在这些实验中，突变c端PDZ基序仍然允许NHE3与NHERF-1结合，这表明存在其他稳定细胞NHE3/NHERF-1复合物的机制或成分。然而，在PS120细胞中的转运实验表明，NHE3中的c端PDZ基序和NHERF-1中的功能PDZ- ii结构域是最大限度地抑制forskolin和8-Br-cAMP对钠-氢交换的响应所必需的。综上所述，这些数据表明NHE3 c端与NHERF-1 PDZ结构域之间的PDZ相互作用增强了camp升高激素对钠-氢交换的调节。
NHERF-1, a protein adapter containing two tandem PDZ domains, was first identified as an essential cofactor required for the phosphorylation and downregulation of NHE3 activity in response to elevated intracellular cAMP. NHERF-1 contains multiple protein interaction domains, but the mechanism by which it binds NHE3 remains unknown. Yeast two-hybrid analyses demonstrated that the C-terminal sequence, STHM, of NHE3 constitutes a PDZ motif critical for its association with NHERF-1. In this assay, NHE3 bound both PDZ-I and PDZ-II when presented as isolated domains, but mutations of the individual PDZ domains in the full-length NHERF-1 suggested a significant preference of NHE3 for the PDZ-II domain. To investigate NHERF-1/NHE3 association in cells, NHERF-1 complexes were isolated from PS120 cells expressing hexahistidine-tagged NHERF-1 and NHE3 using nickel-NTA−agarose. In these experiments, mutating the C-terminal PDZ motif still allowed NHE3 binding to NHERF-1, suggesting the presence of additional mechanisms or components that stabilized a cellular NHE3/NHERF-1 complex. Transport assays in PS120 cells, however, showed that the C-terminal PDZ motif in NHE3 and a functional PDZ-II domain in NHERF-1 were required for maximal inhibition of sodium−hydrogen exchange in response to forskolin and 8-Br-cAMP. Together, the data suggested that the PDZ interaction between the NHE3 C-terminus and a NHERF-1 PDZ domain enhanced the regulation of sodium−hydrogen exchange by cAMP-elevating hormones.