Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 116 Megalin, an Endocytotic Receptor with Signalling Potential MÅRTEN LARSSON ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 UPPSALA ISBN 91-554-6483-1 2006 urn:nbn:se:uu:diva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o Dr. John Pemberton List of original papers This thesis is based on the following papers, referred to in the text by their roman numerals: I Larsson M, Hjälm G, Sakwe AM, Engström Å, Höglund A-S, Larsson E, Robinson RC, Sundberg C and Rask L. (2003) Selec- tive interaction of megalin with postsynaptic density-95 (PSD- 95)-like membrane-associated guanylate kinase (MAGUK) pro- teins. Biochemical Journal 373, 381-391. II Wicher G, Larsson M, Fex Svenningsen Å, Gyllencreutz E, Rask L and Aldskogius H. (2006) Low-density lipoprotein re- ceptor-related protein (LRP)-2/megalin is expressed in oli- godendrocytes in the mouse spinal cord white matter. Journal of Neuroscience Research In press. Published online 3 Feb 2006. III Wicher G, Larsson M, Rask L and Aldskogius H. (2005) Low- density lipoprotein receptor-related protein (LRP)-2/megalin is transiently expressed in a subpopulation of neural progenitors in the embryonic mouse spinal cord. Journal of Comparative Neu- rology 492, 123-131. IV Larsson M, Wicher G, Thuveson M, Hallberg M, Melhus H, Engström Å, Jemth P, Aldskogius H and Rask L. (2006) Low- density lipoprotein receptor-related protein (LRP)-2/megalin plays a role as a receptor for retinol-binding protein on retina pigment epithelial cells of the eye. Manuscript Reprints were made with permissions of the publishers Portland Press Ltd and Wiley (http://www.interscience.Wiley.com). Contents Introduction.....................................................................................................9 LDL-receptor class A ligand-binding domains ...................................13 EGF precursor-like and E-propeller domains ......................................14 Megalin intracellular domain...............................................................15 Megalin ligands ...................................................................................20 Tissue distribution and localization of megalin...................................24 Megalin-deficient mice........................................................................26 Megalin and the brain ..........................................................................27 Aims of the studies........................................................................................30 Present investigations....................................................................................31 Paper I..................................................................................................31 Paper II ................................................................................................32 Paper III ...............................................................................................33 Paper IV...............................................................................................34 Discussion.....................................................................................................35 Concluding remarks......................................................................................38 Acknowledgements.......................................................................................39 References.....................................................................................................40 Abbreviations ApoER2 Apolipoprotein E receptor 2 APP Amyloid precursor protein BBB Blood-brain barrier BMP-4 Bone morphogenic protein 4 CNS Central nervous system CSF Cerebrospinal fluid Dab Disabled DBP Vitamin D-binding protein EGF Epidermal growth factor ER Endoplasmic reticulum EST Expressed sequence tag GAIP GD-interacting protein GIPC GAIP-interacting protein, carboxyterminus GSK-3 Glycogen synthase kinase-3 GST Glutathione S-transferase IGF-1 Insulin-like growth factor-1 LDL Low-density lipoprotein LRP Low-density lipoprotein receptor-related protein MAGUK Membrane-associated guanylate kinase MCTF Megalin carboxyterminal fragment MegBP Megalin-binding protein NHE3 Na+/H+ exchanger isoform 3 PDZ PSD-95/Drosophila discs-large/zona occludens 1 PI3-kinase Phosphatidylinositol 3-kinase PKC Protein kinase C PSD-95 Postsynaptic density 95 RAP Receptor-associated protein RBP Retinol-binding protein RIP Regulated intramembrane proteolysis RPE Retina pigment epithelium RT-PCR Reverse transcriptase-polymerase chain reaction SAP Synapse-associated protein SH2/SH3 Src homology 2/Src homology 3 SHBG Sex hormone-binding globulin SHH Sonic hedgehog VLDL Very low-density lipoprotein Introduction Epithelial cells are lining all cavities in the living body. These epithelial cells are organized into a polarized cytoarchitecture, with an apical and a baso- lateral surface with distinctly different protein- and lipid compositions. The main function of polarized epithelial cells is to carry out vectorial transport from one compartment to another. This is for instance done by the uptake of nutrients from the intestine to the blood or from the primary urine to the blood in the kidney. Receptor-mediated endocytosis (Fig. 1) is one of several important systems the cell has developed to take up important nutrients, hormones, vitamins and co-factors [1]. The term receptor-mediated endocy- tosis was first established by Michael S. Brown and Joseph L. Goldstein in 1986 [2] by their work on the low-density lipoprotein (LDL)-receptor and cholesterol, a work which rendered them the Nobel Prize in Physiology or Medicine in 1985. Simplified, receptor-mediated endocytosis starts with a ligand binding to a transmembrane receptor, which is located in a clathrin- coated pit on the cell surface. The ligand-receptor complex is internalized into sorting endosomes, which are acidified by ATP-dependent proton pumps. The acidification causes the ligand to be released from the receptor. Most receptors rapidly exit the sorting endosomes and return to the cell membrane directly, or via the endocytic recycling compartment. The sorting endosomes mature into late endosomes and further to lysosomes where the ligands are degraded. In the case of polarized epithelial cells, ligands can in many cases also be transported from the apical surface, across the cell, to the basolateral surface in a process called transcytosis. Megalin (also known as LRP2, gp330 or gp600) is a type 1 transmem- brane receptor expressed mainly on the apical side of absorptive and secre- tory epithelial cells. It belongs to the LDL-receptor family, a family consist- ing of seven core members in mammals (Fig. 2A). The family is, apart from the archetypal LDL-receptor and megalin, also made up by the very low- density lipoprotein (VLDL)-receptor, apolipoprotein E receptor 2 (ApoER2)/LRP8/LR7, Multiple EGF-domain 7 (MEGF7)/LRP4, LDL- receptor related protein 1 (LRP1)/D2-macroglobulin receptor/CD91 and LRP1B (Fig. 2A). Three receptors are more distantly related, namely the LRP5/LRP7/LR3, LRP6 and LR11/sorting protein related receptor (SorLA) (Fig. 2B). Even more distantly related receptors (LRP3, LRP9/LRP10 and ST7/LRP12) are referred to as members of the LDL-receptor family due to their content of LDL-receptor class A motifs (see below). 9 LDL LDL D D YWTD WT Y YWT Y W Y T W YWTD D TD Extracellular space LDL D D YWTD WT D Y D YWT YWTD T YWT W Y Y WT YW YWTD YW D T YW D T YWTD Cytosol D TD Clathrin- coated pit D D YWTD WT T Y W Y YW Y T WT YWTD D D Sorting endosome D D YWTD YWT YWT YW YWT T YWTD pH 5.9-6.0 D D LDL D D YWTD WT Y WT Y Y W YW T YWTD D TD Late endosome pH 5.0-6.0 LDL Endocytic recycling compartment pH 6.4-6.5 Lysosome Trans-Golgi LDL pH 5.0-5.5 pH 6.4-6.5 YWTD YWTD YWTD YW Y T WTD YWTD D Medial-Golgi RAP pH 6.4-6.5 D D YWTD RAP YWT YWT YW YW T YWTD Boca D T D Boca Figure 1. Endocytotic recycling pathway of the LDL-receptor. pH of different intra- cellular compartments is noted. 10 The core members of the LDL-receptor family (Fig. 2A) are characterized by containing the same structural motifs in the same arrangement: cysteine- rich ligand-binding LDL-receptor class A (also referred to as complement type repeats) motifs followed by E-propeller (YWTD-containing) domains, which are flanked by epidermal growth factor (EGF) precursor-like