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 Thr389, located in the αB-helix and part of the hydrophobic pocket of the PDZ2 domain. Surface plasmon resonance experiments revealed that mutation of SAP97 Thr389 to alanine, as with the other PSD-95-like membrane-associated guanylate kinases, induced binding to megalin.
- membrane-associated guanylate kinase (MAGUK)
- postsynaptic density-95 (PSD-95)
- PSD-95/Drosophila discs-large/zona occludens 1 (PDZ) domain
- synapse-associated protein 97 (SAP97)
↵2 Present address: Department of Molecular Biosciences, Biomedical Centre, Swedish University of Agricultural Sciences, P.O. Box 575, SE-751 23 Uppsala, Sweden.
The nucleotide sequence data reported will appear in DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession number AY265358.
Abbreviations used: Dlg, Drosophila discs-large, FBS, fetal bovine serum; FLAG, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys; GluR, glutamate receptor; GST, glutathione S-transferase; GUK, guanylate kinase-like; LDLR, low-density lipoprotein receptor; MAGUK, membrane-associated guanylate kinase; MALDI–TOF, matrix-assisted laser-desorption ionization–time-of-flight; NHE3, Na+/H+ exchanger 3; NHERF, NHE regulatory factor; PSD, postsynaptic density; PDZ, PSD-95/Dlg/zona occludens 1; RT-PCR, reverse transcription–PCR; RU, resonance units; SAP, synapse-associated protein; SH3, Src-homology 3.
- The Biochemical Society, London ©2003