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首页> 外文期刊>Cellular Physiology and Biochemistry >Na+-H+ exchanger regulatory factor 1 (NHERF1) PDZ scaffold binds an internal binding site in the scavenger receptor megalin
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Na+-H+ exchanger regulatory factor 1 (NHERF1) PDZ scaffold binds an internal binding site in the scavenger receptor megalin

机译：Na + -H +交换子调节因子1（NHERF1）PDZ支架结合清道夫受体巨蛋白中的内部结合位点

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The scavenger receptor megalin binds to albumin in the microvilli of the renal proximal tubule, and transports the ligand to the intravillar cleft for processing by endocytosis. Albumin endocytosis in the proximal tubule is regulated by protein complexes containing a number of transmembrane and accessory proteins including PDZ scaffolds such as NHERF1 and NHERF2. PDZ scaffold proteins bind to class I PDZ binding motifs (S/T-X-?) in the extreme C-terminus of targets. Megalin contains a functional PDZ binding motif (SDV) in its distal terminus, however a potential interaction with the NHERF proteins has not been investigated. As megalin associates with NHE3 in the microvilli and NHE3 is tethered to the intravillar cleft via its interaction with NHERF1, we investigated if there is a direct interaction between megalin and NHERF1 in renal proximal tubule cells. Using confocal microscopy we determined that megalin and NHERF1 co-localise in the apical region in proximal tubule cells. Immunoprecipitation experiments performed using rat kidney lysate indicated that megalin bound NHERF1 in vivo. Using fusion proteins and peptides, we determined that PDZ2 of NHERF1 bound to megalin and that this interaction was via the C-terminus of megalin directly and in the absence of any accessory protein. We next investigated which domain in megalin was regulating this interaction. Using GST fusion proteins we determined that the loss of the most distal C-terminus of megalin containing the PDZ binding motif (SDV) did not alter its ability to bind to NHERF1. Significantly, we then identified an internal NHERF binding domain in the C-terminus of megalin. Using peptide studies we were able to demonstrate that NHERF1 bound to an internal PDZ binding motif in megalin and that a loss of a single threonine residue abolished the interaction between megalin and NHERF1. Finally, in proximal tubule cells, silencing NHERF1 increased megalin expression. Therefore, we have identified a novel protein interaction in proximal tubule cells and specifically identified a new internal PDZ binding motif in the C-terminus of megalin.
机译：清道夫受体巨蛋白结合至肾近端小管的微绒毛中的白蛋白，并将配体转运至绒毛内裂以通过内吞作用进行处理。近端小管中的白蛋白内吞作用受包含许多跨膜和辅助蛋白（包括PDZ支架（如NHERF1和NHERF2））的蛋白复合物调节。 PDZ支架蛋白与目标极端C末端的I类PDZ结合基序（S /T-X-α）结合。巨蛋白在其远端包含一个功能性PDZ结合基序（SDV），但是尚未研究与NHERF蛋白的潜在相互作用。由于巨蛋白与微绒毛中的NHE3缔合，并且NHE3通过其与NHERF1的相互作用而束缚在绒毛内裂上，因此我们研究了巨蛋白和NHERF1在肾小管近端细胞中是否存在直接相互作用。使用共聚焦显微镜，我们确定了巨蛋白和NHERF1共定位在近端小管细胞的根尖区域。使用大鼠肾脏裂解物进行的免疫沉淀实验表明，巨蛋白在体内结合NHERF1。使用融合蛋白和多肽，我们确定NHERF1的PDZ2与巨蛋白结合，并且这种相互作用是通过巨蛋白的C末端直接进行的，并且没有任何辅助蛋白。接下来，我们研究了巨蛋白中的哪个域正在调节这种相互作用。使用GST融合蛋白，我们确定含有PDZ结合基序（SDV）的megalin最远端C末端的丢失不会改变其与NHERF1结合的能力。重要的是，我们随后在巨蛋白的C末端鉴定了一个内部NHERF结合结构域。使用肽研究，我们能够证明NHERF1结合了巨蛋白中的内部PDZ结合基序，单个苏氨酸残基的缺失消除了巨蛋白和NHERF1之间的相互作用。最后，在近端小管细胞中，沉默NHERF1可增加megalin表达。因此，我们已经确定了近端肾小管细胞中的一种新型蛋白质相互作用，并特别地在megalin的C末端确定了一个新的内部PDZ结合基序。

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