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Selective interaction of megalin with postsynaptic density-95 (PSD-95)-like membrane-associated guanylate kinase (MAGUK) proteins
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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2003 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 373, no 2, 381-391 p.Article in journal (Refereed) Published
Description
Abstract [en]

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.

Place, publisher, year, edition, pages
2003. Vol. 373, no 2, 381-391 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-94090DOI: 10.1042/BJ20021958OAI: oai:DiVA.org:uu-94090DiVA: diva2:167824
Available from: 2006-03-10 Created: 2006-03-10 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Megalin, an Endocytotic Receptor with Signalling Potential
Open this publication in new window or tab >>Megalin, an Endocytotic Receptor with Signalling Potential
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Megalin is an endocytotic receptor belonging to the low-density lipoprotein family. It has often been viewed only as merely a scavenger receptor of absorptive and secretory epithelia. Recent work has revealed that the megalin intracellular domain contains several motifs potentially binding proteins involved in signal transduction.

To find potential intracellular proteins binding to megalin, a yeast two-hybrid screening was initiated with the intracellular tail of megalin as the bait. A partial clone encoding the scaffolding protein postsynaptic protein 95 (PSD-95) was found to bind to megalin with its second PDZ-domain. Co-localization experiments in HEK-293 cells and kidney, placenta and parathyroid tissue confirmed this interaction. The PSD-95 related proteins PSD-93 and SAP102 were also confirmed to bind megalin with their PDZ2-domains, but the corresponding domain from SAP97 did not bind. Mutation analysis revealed that an amino acid residue change Ala to Thr was the cause of this.

Megalin has within the central nervous system (CNS) been shown to be expressed only in the ependymal cells and choroid plexus. Nothing has been known about megalin expression in the spinal cord. To study spatio-temporal expression of megalin in the spinal cord, extensive staining of prenatal and postnatal mouse spinal cord was undertaken. Megalin expression was found in the dorsal part of the embryonic spinal cord. Most of these cells also expressed vimentin, suggesting that megalin has a role in the normal development of astrocytes. In the postnatal mouse, megalin seems to be expressed in oligodendrocytes only in the spinal cord white matter, and co-incident with myelination. This suggests that megalin is involved in the formation and maintenance of myelin along long spinal pathways. Megalin staining was clearly seen in the nucleus of these cells, indicating that megalin works in a notch-like signalling pathway.

Uptake of retinol to the retina pigment epithelium (RPE) has long been thought to be a diffusion process. Staining for megalin in RPE revealed strong expression, and uptake experiments with 3H-retinol bound to retinol-binding protein and blocking with the LDL-receptor family specific antagonist receptor-associated protein (RAP) showed that megalin is a receptor for uptake of retinol to the RPE.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 60 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 116
Keyword
Biochemistry, Megalin, LRP-2, Postsynaptic density-95, Retinol-binding protein, Oligodendrocytes, Central nervous system, Biokemi
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-6585 (URN)91-554-6483-1 (ISBN)
Public defence
2006-03-31, B41, BMC, Uppsala, 13:15
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Available from: 2006-03-10 Created: 2006-03-10 Last updated: 2013-05-23Bibliographically approved

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Hjälm, GöranEngström, ÅkeLarsson, ErikSundberg, ChristianRask, Lars

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Larsson, MårtenHjälm, GöranEngström, ÅkeLarsson, ErikSundberg, ChristianRask, Lars
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