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.