The adaptor protein ARH escorts megalin to and through endosomes

Megalin is an endocytic receptor that binds multiple ligands and is essential for many physiological processes such as brain development and uptake of proteins by the kidney tubule, yolk sac, and thyroid. The cytoplasmic tail of megalin contains two FXNPXY motifs. Autosomal recessive hypercholesterolemia (ARH) is an adaptor protein that binds to the FXNPXY motif of the low-density lipoprotein receptor as well as clathrin and AP-2. We found that ARH also binds to the first FXNPXY motif of megalin in two-hybrid, pull-down and coimmunoprecipitation assays. ARH colocalizes with megalin in clathrin coated pits and in recycling endosomes in the Golgi region. When cells are treated with nocodazole, the recycling endosomes containing megalin and ARH disperse. On internalization of megalin, ARH and megalin are first seen in clathrin coated pits followed by sequential localization in early endosomes and tubular recycling endosomes in the pericentriolar region followed by their reappearance at the cell surface. Expression of ARH in Madin-Darby canine kidney cells expressing megalin mini-receptors enhances megalin-mediated uptake of I-125-lactoferrin, a megalin ligand. These results show that ARH facilitates endocytosis of megalin, escorts megalin along its endocytic route and raise the possibility that transport through the endosomal system is selective and requires interaction with specific adaptor proteins.