title: Ezetimibe-sensitive cholesterol uptake by NPC1L1 protein does not require endocytosis.
abstract: Human NPC1L1 protein mediates cholesterol absorption in the intestine and liver and is the target of the drug ezetimibe, which is used to treat hypercholesterolemia. Previous studies concluded that NPC1L1-GFP protein trafficking is regulated by cholesterol binding and that ezetimibe blocks NPC1L1-GFP function by inhibiting its endocytosis. We used cell surface biotinylation to monitor NPC1L1-GFP endocytosis and show that ezetimibe does not alter the rate of NPC1L1-GFP endocytosis in cultured rat hepatocytes grown under normal growth conditions. As expected, NPC1L1-GFP endocytosis depends in part on C-terminal, cytoplasmically oriented sequences, but endocytosis does not require cholesterol binding to NPC1L1's N-terminal domain. In addition, two small- molecule inhibitors of general (and NPC1L1-GFP) endocytosis failed to inhibit the ezetimibe-sensitive uptake of [(3)H]cholesterol from taurocholate micelles. These experiments demonstrate that cholesterol uptake by NPC1L1 does not require endocytosis; moreover, ezetimibe interferes with NPC1L1's cholesterol adsorption activity without blocking NPC1L1 internalization in RH7777 cells.
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307.
authors.forename: Tory A
authors.initials: TA
authors.lastname: Johnson
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307 pfeffer@stanford.edu.
authors.forename: Suzanne R
authors.initials: SR
authors.lastname: Pfeffer
mesh_terms: D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects* / Q000502:physiology; D000069438:Ezetimibe / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006937:Hypercholesterolemia / Q000188:drug therapy / Q000378:metabolism; D007408:Intestinal Absorption / Q000187:drug effects; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport / Q000187:drug effects
chemical_list: D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C410800:NPC1L1 protein, human; D002784:Cholesterol; D000069438:Ezetimibe