Identification of an apical sorting determinant in the cytoplasmic tail of megalin

Megalin is the main endocytic receptor of the proximal tubule and is responsible for reabsorption of many filtered proteins. In contrast to other members of the low-density lipoprotein (LDL) receptor gene family, it is expressed on the apical plasma membrane ( PM) of polarized epithelial cells. To identify megalin's apical sorting signal, we generated deletion mutants and chimeric mini-receptors composed of complementary regions of megalin and LDL receptor-related protein (LRP) and assessed the distribution of the mutants in Madin-Darby canine kidney (MDCK) cells by immunofluorescence and cell surface biotinylation. Megalin and LRP minireceptors are correctly targeted to the apical and basolateral PM, respectively, of MDCK cells. We found that the information that directs apical sorting is present in the cytoplasmic tail (CT) of megalin, which contains three NPXY motifs, YXXO, SH3, and dileucine motifs, and a PDZ-binding motif at its COOH terminus. Deletion analysis established that amino acids 107 136 of the megalin-CT containing the second NPXY-like motif are critical for apical sorting and targeting, whereas the regions containing the first and third NPXY motifs are required for efficient endocytosis. We conclude that the megalin-CT contains a novel apical sorting determinant and that cytoplasmic sorting machinery exists in MDCK cells for some apical transmembrane proteins.