The N-terminus of the WD5 repeat of human RACK1 binds to airway epithelial NHERF1.
The N-terminus of the WD5 repeat of human RACK1 binds to airway epithelial NHERF1.
复制标题
人 RACK1 的 WD5 重复序列的 N 末端与气道上皮 NHERF1 结合。
DOI：
10.1021/bi0607249
复制
发表时间：
2006
期刊：
影响因子：
2.9
通讯作者：
Wang,Xiangyun
中科院分区：
文献类型：
--
作者：
Liedtke,CaroleM;Wang,Xiangyun
CFTR Cl通道功能的调节涉及与RACK 1结合的活化蛋白激酶C ε（PKC ε）和与人气道上皮细胞中的人Na +/H+交换调节因子（NHERF 1）结合的RACK 1的蛋白复合物，RACK 1是活化C激酶的受体。NHERF1与RACK1的结合通过NHERF1 − PDZ1结构域介导。本研究的目的是鉴定RACK 1上人NHERF 1的结合基序。我们使用编码人RACK 1的7个WD 40重复单位的肽检查了NHERF 1在RACK 1上的结合位点。WD重复肽WD 5直接结合NHERF 1和PDZ 1结构域，具有相似的EC 50值，阻断重组RACK 1和NHERF 1的结合，并以剂量依赖性方式从Calu-3细胞裂解物中拉下内源性RACK 1。剩余的WD重复肽不阻断RACK1 − NHERF1结合。编码PDZ 1结构域上的位点的11个氨基酸的肽阻断WD 5重复肽与PDZ 1结构域的结合。WD 5重复肽的N末端12个氨基酸片段（包含四条反向平行β链中的第一条）与NHERF 1的PDZ 1结构域剂量依赖性结合，并阻断PDZ 1结构域与RACK 1的结合。这些结果表明，结合位点可能形成具有足以结合NHERF1的拓扑结构的β转角。我们的研究结果还表明NHERF在WD 5重复序列处与RACK 1结合，这与RACK 1的WD 6重复序列上的PKC ε结合位点不同。
Regulation of the CFTR Cl channel function involves a protein complex of activated protein kinase Cε (PKCε) bound to RACK1, a receptor for activated C kinase, and RACK1 bound to the human Na+/H+exchanger regulatory factor (NHERF1) in human airway epithelial cells. Binding of NHERF1 to RACK1 is mediated via a NHERF1−PDZ1 domain. The goal of this study was to identify the binding motif for human NHERF1 on RACK1. We examined the site of binding of NHERF1 on RACK1 using peptides encoding the seven WD40 repeat units of human RACK1. One WD repeat peptide, WD5, directly binds NHERF1 and the PDZ1 domain with similar EC50values, blocks binding of recombinant RACK1 and NHERF1, and pulls down endogenous RACK1 from Calu-3 cell lysate in a dose-dependent manner. The remaining WD repeat peptides did not block RACK1−NHERF1 binding. An 11-amino acid peptide encoding a site on the PDZ1 domain blocks binding of the WD5 repeat peptide with the PDZ1 domain. An N-terminal 12-amino acid segment of the WD5 repeat peptide, which comprises the first of four antiparallel β-strands, dose-dependently binds to the PDZ1 domain of NHERF1 and blocks binding of the PDZ1 domain to RACK1. These results suggest that the binding site might form a β-turn with topology sufficient for binding of NHERF1. Our results also demonstrate binding of NHERF to RACK1 at the WD5 repeat, which is distinct from the PKCε binding site on the WD6 repeat of RACK1.