Slattery Craig, Jenkin Kayte A, Lee Aven, Simcocks Anna C, McAinch Andrew J, Poronnik Philip, Hryciw Deanne H
UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, Ireland.
Cell Physiol Biochem. 2011;27(2):171-8. doi: 10.1159/000325219. Epub 2011 Feb 11.
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-Φ) 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.
清道夫受体巨蛋白在肾近端小管的微绒毛中与白蛋白结合，并将配体转运至绒毛内裂隙进行内吞作用处理。近端小管中的白蛋白内吞作用受包含多种跨膜蛋白和辅助蛋白的蛋白质复合物调控，这些蛋白包括PDZ支架蛋白，如NHERF1和NHERF2。PDZ支架蛋白与靶标极端C末端的I类PDZ结合基序（S/T-X-Φ）结合。巨蛋白在其远端末端含有一个功能性PDZ结合基序（SDV），然而尚未研究其与NHERF蛋白的潜在相互作用。由于巨蛋白在微绒毛中与NHE3相关联，且NHE3通过与NHERF1的相互作用被拴系在绒毛内裂隙，我们研究了肾近端小管细胞中巨蛋白与NHERF1之间是否存在直接相互作用。使用共聚焦显微镜，我们确定巨蛋白和NHERF1在近端小管细胞的顶端区域共定位。使用大鼠肾裂解物进行的免疫沉淀实验表明，巨蛋白在体内与NHERF1结合。使用融合蛋白和肽，我们确定NHERF1的PDZ2与巨蛋白结合，且这种相互作用直接通过巨蛋白的C末端，且无需任何辅助蛋白。接下来，我们研究了巨蛋白中的哪个结构域调节这种相互作用。使用GST融合蛋白，我们确定巨蛋白最远端含PDZ结合基序（SDV）的C末端缺失并不改变其与NHERF1结合的能力。重要的是，我们随后在巨蛋白的C末端鉴定出一个内部NHERF结合结构域。使用肽研究，我们能够证明NHERF1与巨蛋白中的一个内部PDZ结合基序结合，且单个苏氨酸残基的缺失消除了巨蛋白与NHERF1之间的相互作用。最后，在近端小管细胞中，沉默NHERF1可增加巨蛋白的表达。因此，我们在近端小管细胞中鉴定出一种新型蛋白质相互作用，并特别在巨蛋白的C末端鉴定出一个新的内部PDZ结合基序。
