Plasma membrane Ca2+ ATPase isoform 2b interacts preferentially with Na+/H+ exchanger regulatory factor 2 in apical plasma membranes
Plasma membrane Ca2+ ATPase isoform 2b interacts preferentially with Na+/H+ exchanger regulatory factor 2 in apical plasma membranes
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DOI：
10.1074/jbc.m111616200
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发表时间：
2002-03-22
影响因子：
4.8
通讯作者：
Strehler, EE
中科院分区：
文献类型：
--
作者：
DeMarco, SJ;Chicka, MC;Strehler, EE
Ca2+ 信号传导的空间和时间调节需要连接参与 Ca2+ 流入、传感、缓冲和挤出的分子的多蛋白复合物的组装。最近的证据表明，质膜 Ca2+ ATP 酶 (PMCA) 参与局部 Ca2+ 通量的控制，但对特定 PMCA 的多蛋白复合物形成机制知之甚少。使用 PMCA2b COOH 末端尾部作为酵母双杂交筛选中的诱饵，我们鉴定出包含 PSD-95、Dig、ZO-1 (PDZ) 结构域的 Na+/H+ 交换调节因子 2 (NHERF2) 作为相互作用伙伴。使用重组 PMCA2b 和 PMCA4b 以及 NHERF1 和 NHERF2 进行的蛋白质下拉和免疫共沉淀实验表明，PMCA2b 与 NHERF2 的相互作用是特异性和选择性的。 PMCA4b 不与任何一个 NHERF 相互作用，并且 PMCA2b 选择性地偏好 NHERF2 而不是 NHERF1。绿色荧光蛋白标记的 PMCA2b 在 Madin-Darby 犬肾上皮细胞的顶膜上表达，与顶端靶向的 NHERF2 共定位。我们的研究将 NHERF2 确定为 PMCA2b 的第一个特定 PDZ 伴侣，不与 PMCA4b 共享，并证明 PMCA 剪接形式的 COOH 末端残基差异极小，与独特的 PDZ 蛋白相互作用。 NHERF 参与膜蛋白的靶向、保留和调节，包括 β(2)-肾上腺素受体、囊性纤维化跨膜电导调节器和 Trp4 Ca2+ 通道，并且 NHERF2 现在显示也与 PMCA2b 相互作用。这种相互作用可能允许 PMCA2b 在多蛋白 Ca2+ 信号复合物中进行功能组装，促进局部 Ca2+ 流入和流出之间的整合串扰。
Spatial and temporal regulation of Ca2+ signaling require the assembly of multiprotein complexes linking molecules involved in Ca2+ influx, sensing, buffering, and extrusion. Recent evidence indicates that plasma membrane Ca2+ ATPases (PMCAs) participate in the control of local Ca2+ fluxes, but the mechanism of multiprotein complex formation of specific PMCAs is poorly understood. Using the PMCA2b COOH-terminal tail as bait in a yeast two-hybrid screen, we identified the PSD-95, Dig, ZO-1 (PDZ) domain-containing Na+/H+ exchanger regulatory factor-2 (NHERF2) as an interacting partner. Protein pull-down and coimmunoprecipitation experiments using recombinant PMCA2b and PMCA4b as well as NHERF1 and NHERF2 showed that the interaction of PMCA2b with NHERF2 was specific and selective. PMCA4b did not interact with either of the NHERFs, and PMCA2b selectively preferred NHERF2 over NHERF1. Green fluorescent protein-tagged PMCA2b was expressed at the apical membrane in Madin-Darby canine kidney epithelial cells, where it colocalized with apically targeted NHERF2. Our study identifies NHERF2 as the first specific PDZ partner for PMCA2b not shared with PMCA4b, and demonstrates that PMCA splice forms differing only minimally in their COOH-terminal residues interact with unique PDZ proteins. NHERFs have been implicated in the targeting, retention and regulation of membrane proteins including the beta(2)-adrenergic receptor, cystic fibrosis transmembrane conductance regulator, and Trp4 Ca2+ channel, and NHERF2 is now shown to also interact with PMCA2b. This interaction may allow the functional assembly of PMCA2b in a multiprotein Ca2+ signaling complex, facilitating integrated cross-talk between local Ca2+ influx and efflux.