Weinman Edward J, Wang Yu, Wang Fengying, Greer Charmaine, Steplock Deborah, Shenolikar Shirish
Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Biochemistry. 2003 Nov 4;42(43):12662-8. doi: 10.1021/bi035244l.
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.
NHERF-1是一种含有两个串联PDZ结构域的蛋白质衔接子，最初被鉴定为细胞内cAMP升高时NHE3活性磷酸化和下调所必需的辅助因子。NHERF-1包含多个蛋白质相互作用结构域，但其与NHE3结合的机制尚不清楚。酵母双杂交分析表明，NHE3的C末端序列STHM构成了与其与NHERF-1结合至关重要的PDZ基序。在该实验中，当NHE3以分离结构域形式呈现时，它能与PDZ-I和PDZ-II结合，但全长NHERF-1中单个PDZ结构域的突变表明NHE3对PDZ-II结构域有明显偏好。为了研究细胞中NHERF-1/NHE3的结合情况，使用镍-NTA-琼脂糖从表达六组氨酸标签的NHERF-1和NHE3的PS120细胞中分离NHERF-1复合物。在这些实验中，突变C末端PDZ基序仍能使NHE3与NHERF-1结合，这表明存在其他机制或成分来稳定细胞内的NHE3/NHERF-1复合物。然而，PS120细胞中的转运实验表明，NHE3中的C末端PDZ基序和NHERF-1中的功能性PDZ-II结构域是对福斯可林和8-溴-cAMP反应时最大程度抑制钠氢交换所必需的。总之，数据表明NHE3 C末端与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作用。
