Linking receptor-mediated endocytosis and cell signaling: evidence for regulated intramembrane proteolysis of megalin in proximal tubule.
Megalin, a member of the low density lipoprotein receptor gene family, is required for efficient protein absorption in the proximal tubule. Recent studies have shown that the low density lipoprotein receptor-related protein, another member of this gene family, is proteolytically processed by gamma-secretase implying a role for low density lipoprotein receptor-related protein in a Notchlike signaling pathway. This pathway has been shown to involve: 1) metalloprotease-mediated ectodomain shedding and gamma-secretase-mediated intramembrane proteolysis of some receptors. Experiments were performed to determine whether megalin undergoes similar processing. By immunocytochemistry, immunoblotting, and a fluorogenic enzyme assay presenilin-1 (required for gamma-secretase activity) and gamma-secretase activity were found in the brush border of proximal kidney tubules where megalin is localized. Using a fluorogenic peptide containing an amyloid precursor protein gamma-secretase cleavage site and Compound E, a specific gamma-secretase inhibitor, we found high levels of gamma-secretase activity in renal brush border membrane vesicles. Immunoblotting analysis of renal microsomes and opossum kidney proximal tubule (OKP) cells using antibodies directed to the cytosolic domain of megalin showed a 35-40-kDa, membrane-associated, carboxyl-terminal fragment of megalin (MCTF). When cells were incubated with 200 nm phorbol 12-myristate 13-acetate, the appearance of the MCTF increased 2.5-fold and was blocked by metalloprotease inhibitors. When the cells were incubated with gamma-secretase inhibitor Compound E, it caused a 2-fold increase in MCTF. Finally, incubating the cells with 1 microm vitamin D-binding protein resulted in a 25% increase in the appearance of the MCTF. In summary, the MCTF is produced by protein kinase C regulated, metalloprotease-mediated ectodomain shedding and is the substrate for gamma-secretase. We postulate that the enzymatic processing of megalin represents part of a novel ligand-dependent signaling pathway in the proximal tubule that links receptor-mediated endocytosis with cell signaling.
Zou
Zhiying
Z
Department of Internal Medicine, School of Medicine, Yale University, New Haven, CT 06520-8029, USA.
Chung
Brian
B
Nguyen
Thao
T
Mentone
Sueann
S
Thomson
Brent
B
Biemesderfer
Daniel
D
D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D016282:Aspartic Acid Endopeptidases; D001665:Binding Sites; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D010450:Endopeptidases / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005982:Glutathione Transferase / Q000378:metabolism; D015151:Immunoblotting; D007150:Immunohistochemistry; D007668:Kidney / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000378:metabolism; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000378:metabolism; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000378:metabolism / Q000502:physiology*; D020782:Matrix Metalloproteinases / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D045726:Metalloproteases / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008861:Microsomes / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D009893:Opossums; D010455:Peptides / Q000737:chemistry; D053764:Presenilin-1; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate; D014809:Vitamin D-Binding Protein / Q000378:metabolism
D000911:Antibodies, Monoclonal; D016895:Culture Media, Serum-Free; D008077:Lipoproteins, LDL; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D008565:Membrane Proteins; D010455:Peptides; D053764:Presenilin-1; D011993:Recombinant Fusion Proteins; D014809:Vitamin D-Binding Protein; D005982:Glutathione Transferase; D011493:Protein Kinase C; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D045726:Metalloproteases; D016282:Aspartic Acid Endopeptidases; C509282:Bace1 protein, mouse; D020782:Matrix Metalloproteinases; D013755:Tetradecanoylphorbol Acetate