Larsson Mårten, Hjälm Göran, Sakwe Amos M, Engström Ake, Höglund Anna-Stina, Larsson Erik, Robinson Robert C, Sundberg Christian, Rask Lars
Department of Medical Biochemistry and Microbiology, Uppsala University, PO Box 582, SE-751 23 Uppsala, Sweden.
Biochem J. 2003 Jul 15;373(Pt 2):381-91. doi: 10.1042/BJ20021958.
Megalin is an integral membrane receptor belonging to the low-density lipoprotein receptor family. In addition to its role as an endocytotic receptor, megalin has also been proposed to have signalling functions. Using interaction cloning in yeast, we identified the membrane-associated guanylate kinase family member postsynaptic density-95 (PSD-95) as an interaction partner for megalin. PSD-95 and a truncated version of megalin were co-immunoprecipitated from HEK-293 cell lysates overexpressing the two proteins, which confirmed the interaction. The two proteins were found to be co-localized in these cells by confocal microscopy. Immunocytochemical studies showed that cells in the parathyroid, proximal tubuli of the kidney and placenta express both megalin and PSD-95. We found that the interaction between the two proteins is mediated by the binding of the C-terminus of megalin, which has a type I PSD-95/ Drosophila discs-large/zona occludens 1 (PDZ)-binding motif, to the PDZ2 domain of PSD-95. The PSD-95-like membrane-associated guanylate kinase ('MAGUK') family contains three additional members: PSD-93, synapse-associated protein 97 (SAP97) and SAP102. We detected these proteins, apart from SAP102, in parathyroid chief cells, a cell type having a marked expression of megalin. The PDZ2 domains of PSD-93 and SAP102 were also shown to interact with megalin, whereas no interaction was detected for SAP97. The SAP97 PDZ2 domain differed at four positions from the other members of the PSD-95 subfamily. One of these residues was Thr(389), located in the alphaB-helix and part of the hydrophobic pocket of the PDZ2 domain. Surface plasmon resonance experiments revealed that mutation of SAP97 Thr(389) to alanine, as with the other PSD-95-like membrane-associated guanylate kinases, induced binding to megalin.
巨膜蛋白是一种属于低密度脂蛋白受体家族的整合膜受体。除了作为内吞受体的作用外，巨膜蛋白还被认为具有信号传导功能。利用酵母中的相互作用克隆技术，我们鉴定出膜相关鸟苷酸激酶家族成员突触后密度蛋白95（PSD - 95）是巨膜蛋白的一个相互作用伙伴。PSD - 95和巨膜蛋白的一个截短版本从过表达这两种蛋白的HEK - 293细胞裂解物中被共免疫沉淀，这证实了两者之间的相互作用。通过共聚焦显微镜发现这两种蛋白在这些细胞中共定位。免疫细胞化学研究表明，甲状旁腺、肾近端小管和胎盘的细胞同时表达巨膜蛋白和PSD - 95。我们发现这两种蛋白之间的相互作用是由巨膜蛋白的C末端介导的，该末端具有I型PSD - 95/果蝇盘大蛋白/紧密连接蛋白1（PDZ）结合基序，与PSD - 95的PDZ2结构域结合。PSD - 95样膜相关鸟苷酸激酶（“MAGUK”）家族还包含另外三个成员：PSD - 93、突触相关蛋白97（SAP97）和SAP102。我们在甲状旁腺主细胞（一种巨膜蛋白有显著表达的细胞类型）中检测到了除SAP102之外的这些蛋白。PSD - 93和SAP102的PDZ2结构域也被证明与巨膜蛋白相互作用，而未检测到SAP97与巨膜蛋白的相互作用。SAP97的PDZ2结构域在四个位置上与PSD - 95亚家族的其他成员不同。其中一个残基是苏氨酸（Thr）389，位于αB螺旋中，是PDZ2结构域疏水口袋 的一部分。表面等离子体共振实验表明，将SAP97的苏氨酸（Thr）389突变为丙氨酸后，与其他PSD - 95样膜相关鸟苷酸激酶一样，诱导其与巨膜蛋白结合。
