The Na+/H+ exchanger isoform 1 (NHE1) is a novel member of the calmodulin-binding proteins. Identification and characterization of calmodulin-binding sites.
The Na+/H+ exchanger isoform 1 (NHE1) is a novel member of the calmodulin-binding proteins. Identification and characterization of calmodulin-binding sites.
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DOI：
10.1016/s0021-9258(17)36887-4
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发表时间：
1994-05
期刊：
影响因子：
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通讯作者：
B. Bertrand;Shigeo Wakabayashi;T. Ikeda;Jacques Pouysségur;M. Shigekawa
中科院分区：
文献类型：
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作者：
B. Bertrand;Shigeo Wakabayashi;T. Ikeda;Jacques Pouysségur;M. Shigekawa
Na+/H+ 交换活性（NHE1 人类异构体）响应生长因子和高渗应激而迅速激活。为了深入了解 NHE1 激活的机制，我们研究了普遍存在的 Ca(2+) 依赖性调节因子钙调蛋白 (CaM) 与 NHE1 的直接相互作用。 CaM-Sepharose 的结合实验以及丹磺酰化 CaM 的荧光测量表明，NHE1 胞质结构域以 Ca(2+) 依赖性方式强烈结合 CaM。缺失突变体的融合蛋白分析提供了位于 NHE1 邻近区域（氨基酸 636-656 和 657-700）的高（Kd 约 20 nM）和中（Kd 约 350 nm）亲和力 CaM 结合位点的证据。为了评估 CaM 结合位点的调节作用，生成了几种在高亲和力位点具有缺失和点突变的 cDNA，并在交换器缺陷型成纤维细胞系 PS120 中表达。高亲和力 CaM 结合位点的带正电残基的缺失和点突变导致生长因子（α-凝血酶等）和 100 mM 蔗糖引起的细胞质碱化分别减少高达 50% 和 80%。在这些突变体中，碱化的降低显然与 CaM 结合能力的降低成正比。这些结果表明，Ca2+/CaM 与高亲和力位点的结合至少部分参与了 NHE1 响应不同细胞外信号的激活。
The Na+/H+ exchange activity (NHE1 human isoform) is rapidly activated in response to growth factors and hyperosmotic stress. To get insight into the mechanism of NHE1 activation, we studied the direct interaction of a ubiquitous Ca(2+)-dependent regulatory factor, calmodulin (CaM) with NHE1. Binding experiments with CaM-Sepharose, as well as fluorescence measurements with dansylated CaM, revealed that the NHE1 cytoplasmic domain strongly binds CaM in a Ca(2+)-dependent manner. Fusion protein analysis with deletion mutants provided evidence for high (Kd approximately 20 nM) and intermediate (Kd approximately 350 nm) affinity CaM-binding sites located in neighboring regions of NHE1 (amino acids 636-656 and 657-700). To assess a regulatory role of CaM-binding sites, several cDNAs having deletion and point mutations in the high affinity site were generated and expressed in the exchanger-deficient fibroblast cell line PS120. Deletion and point mutations of positively charged residues of the high affinity CaM-binding site resulted in up to 50 and 80% reductions of cytoplasmic alkalinization caused by growth factors (alpha-thrombin, etc.) and 100 mM sucrose, respectively. In these mutants, the reduction in alkalinization was apparently in proportion to that of the CaM-binding ability. These results suggest that binding of Ca2+/CaM to the high affinity site is involved at least partly in the activation of NHE1 in response to different extracellular signals.