title: AP2 adaptor complex mediates bile salt export pump internalization and modulates its hepatocanalicular expression and transport function.
abstract: The bile salt export pump (BSEP) mediates the biliary excretion of bile salts and its dysfunction induces intrahepatic cholestasis. Reduced canalicular expression of BSEP resulting from the promotion of its internalization is one of the causes of this disease state. However, the molecular mechanism underlying BSEP internalization from the canalicular membrane (CM) remains unknown. We have shown previously that 4-phenylbutyrate (4PBA), a drug used for ornithine transcarbamylase deficiency (OTCD), inhibited internalization and subsequent degradation of cell-surface-resident BSEP. The current study found that 4PBA treatment decreased significantly the expression of α- and μ2-adaptin, both of which are subunits of the AP2 adaptor complex (AP2) that mediates clathrin-dependent endocytosis, in liver specimens from rats and patients with OTCD, and that BSEP has potential AP2 recognition motifs in its cytosolic region. Based on this, the role of AP2 in BSEP internalization was explored further. In vitro analysis with 3×FLAG-human BSEP-expressing HeLa cells and human sandwich-culture hepatocytes indicates that the impairment of AP2 function by RNA interference targeting of α-adaptin inhibits BSEP internalization from the plasma membrane and increases its cell-surface expression and transport function. Studies using immunostaining, coimmunoprecipitation, glutathione S-transferase pulldown assay, and time-lapse imaging show that AP2 interacts with BSEP at the CM through a tyrosine motif at the carboxyl terminus of BSEP and mediates BSEP internalization from the CM of hepatocytes.
  
  
  CONCLUSIONS
  AP2 mediates the internalization and subsequent degradation of CM-resident BSEP through direct interaction with BSEP and thereby modulates the canalicular expression and transport function of BSEP. This information should be useful for understanding the pathogenesis of severe liver diseases associated with intrahepatic cholestasis.
authors.affiliation: Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan. hayapi@mol.f.u-tokyo.ac.jp
authors.forename: Hisamitsu
authors.initials: H
authors.lastname: Hayashi
authors.forename: Kaori
authors.initials: K
authors.lastname: Inamura
authors.forename: Kensuke
authors.initials: K
authors.lastname: Aida
authors.forename: Sotaro
authors.initials: S
authors.lastname: Naoi
authors.forename: Reiko
authors.initials: R
authors.lastname: Horikawa
authors.forename: Hironori
authors.initials: H
authors.lastname: Nagasaka
authors.forename: Tomozumi
authors.initials: T
authors.lastname: Takatani
authors.forename: Tamio
authors.initials: T
authors.lastname: Fukushima
authors.forename: Asami
authors.initials: A
authors.lastname: Hattori
authors.forename: Takashi
authors.initials: T
authors.lastname: Yabuki
authors.forename: Ikuo
authors.initials: I
authors.lastname: Horii
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Sugiyama
mesh_terms: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D033962:Adaptor Protein Complex 2 / Q000502:physiology*; D033965:Adaptor Protein Complex alpha Subunits / Q000235:genetics / Q000502:physiology; D000818:Animals; D001648:Bile Canaliculi / Q000378:metabolism*; D001692:Biological Transport; D016764:Cell Polarity; D006367:HeLa Cells; D006801:Humans; D008297:Male; D010654:Phenylbutyrates / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D054875:Ubiquitination
chemical_list: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters; C490548:Abcb11 protein, rat; D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D010654:Phenylbutyrates; C075773:4-phenylbutyric acid