title: Sorting nexin 27 interacts with multidrug resistance-associated protein 4 (MRP4) and mediates internalization of MRP4.
abstract: Multidrug resistance-associated protein 4 (MRP4/ABCC4) makes a vital contribution to the bodily distribution of drugs and endogenous compounds because of its cellular efflux abilities. However, little is known about the mechanism regulating its cell surface expression. MRP4 has a PDZ-binding motif, which is a potential sequence that modulates the membrane expression of MRP4 via interaction with PDZ adaptor proteins. To investigate this possible relationship, we performed GST pull-down assays and subsequent analysis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. This method identified sorting nexin 27 (SNX27) as the interacting PDZ adaptor protein with a PDZ-binding motif of MRP4. Its interaction was confirmed by a coimmunoprecipitation study using HEK293 cells. Knockdown of SNX27 by siRNA in HEK293 cells raised MRP4 expression on the plasma membrane, increased the extrusion of 6-[(14)C]mercaptopurine, an MRP4 substrate, and conferred resistance against 6-[(14)C]mercaptopurine. Cell surface biotinylation studies indicated that the inhibition of MRP4 internalization was responsible for these results. Immunocytochemistry and cell surface biotinylation studies using COS-1 cells showed that SNX27 localized to both the early endosome and the plasma membrane. These data suggest that SNX27 interacts with MRP4 near the plasma membrane and promotes endocytosis of MRP4 and thereby negatively regulates its cell surface expression and transport function.
authors.affiliation: Laboratory of Molecular Pharmacokinetics, Department of Medical Pharmaceutics, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. hayapi@mol.f.u-tokyo.ac.jp
authors.forename: Hisamitsu
authors.initials: H
authors.lastname: Hayashi
authors.forename: Sotaro
authors.initials: S
authors.lastname: Naoi
authors.forename: Takayuki
authors.initials: T
authors.lastname: Nakagawa
authors.forename: Toru
authors.initials: T
authors.lastname: Nishikawa
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Fukuda
authors.forename: Shinobu
authors.initials: S
authors.lastname: Imajoh-Ohmi
authors.forename: Ayano
authors.initials: A
authors.lastname: Kondo
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Kubo
authors.forename: Takashi
authors.initials: T
authors.lastname: Yabuki
authors.forename: Asami
authors.initials: A
authors.lastname: Hattori
authors.forename: Masakazu
authors.initials: M
authors.lastname: Hirouchi
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Sugiyama
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001693:Biological Transport, Active / Q000502:physiology; D019556:COS Cells; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000096:biosynthesis* / Q000235:genetics; D054731:PDZ Domains; D011485:Protein Binding; D034741:RNA, Small Interfering; D058305:Sorting Nexins / Q000235:genetics / Q000378:metabolism*
chemical_list: D027425:ATP-Binding Cassette, Sub-Family C Proteins; D034741:RNA, Small Interfering; D058305:Sorting Nexins; C073492:ABCC4 protein, human