Identification of HAX-1 as a protein that binds bile salt export protein and regulates its abundance in the apical membrane of Madin-Darby canine kidney cells

J Biol Chem. 2004 Jul 30;279(31):32761-70. doi: 10.1074/jbc.M404337200. Epub 2004 May 24.

Abstract

ATP-binding cassette (ABC)-type proteins are essential for bile formation in vertebrate liver. BSEP, MDR1, MDR2, and MRP2 ABC transporters are targeted to the apical (canalicular) membrane of hepatocytes where they execute ATP-dependent transport of bile acids, drugs, amphipathic cations, phospholipids, and conjugated organic anions, respectively. Changes in activity and abundance of transporters in the canalicular membrane regulate bile flow; however, little is known regarding cellular proteins that bind ABC transporters and regulate their trafficking. A yeast two-hybrid screen identified HAX-1 as a binding partner for BSEP, MDR1, and MDR2. The interactions were validated biochemically by glutathione S-transferase pull-down and co-immunoprecipitation assays. BSEP and HAX-1 were over-represented in rat liver subcellular fractions enriched for canalicular membrane vesicles, microsomes, and clathrin-coated vesicles. HAX-1 was bound to BSEP, MDR1, and MDR2 in canalicular membrane vesicles and co-localized with BSEP and MDR1 in the apical membrane of Madin-Darby canine kidney (MDCK) cells. RNA interference of HAX-1 increased BSEP levels in the apical membrane of MDCK cells by 71%. Pulse-chase studies indicated that HAX-1 depletion did not affect BSEP translation, post-translational modification, delivery to the plasma membrane, or half-life. HAX-1 depletion resulted in an increased peak of metabolically labeled apical membrane BSEP at 4 h and enhanced retention at 6 and 9 h. HAX-1 also interacts with cortactin. Expression of dominant negative cortactin increased steady state levels of BSEP 2-fold in the apical membrane of MDCK cells, as did expression of dominant negative EPS15. These findings suggest that HAX-1 and cortactin participate in BSEP internalization from the apical membrane.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B / metabolism*
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism*
  • ATP Binding Cassette Transporter, Subfamily B, Member 11
  • ATP-Binding Cassette Transporters / metabolism*
  • Adaptor Proteins, Signal Transducing
  • Amino Acid Sequence
  • Animals
  • Bile Acids and Salts / chemistry*
  • Biological Transport
  • Biotinylation
  • Cations
  • Cell Line
  • Cell Membrane / metabolism
  • Cortactin
  • Dogs
  • Escherichia coli / metabolism
  • Genes, Dominant
  • Glutathione Transferase / metabolism
  • Hepatocytes / metabolism
  • Humans
  • Immunoblotting
  • Liver / metabolism
  • Microfilament Proteins / metabolism
  • Microscopy, Fluorescence
  • Models, Biological
  • Molecular Sequence Data
  • Phospholipids / chemistry
  • Plasmids / metabolism
  • Precipitin Tests
  • Protein Binding
  • Protein Biosynthesis
  • Protein Processing, Post-Translational
  • Protein Transport
  • Proteins / chemistry
  • Proteins / physiology*
  • RNA Interference
  • Rats
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Homology, Amino Acid
  • Subcellular Fractions
  • Time Factors
  • Transfection
  • Two-Hybrid System Techniques

Substances

  • ABCB11 protein, human
  • ATP Binding Cassette Transporter, Subfamily B
  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • ATP Binding Cassette Transporter, Subfamily B, Member 11
  • ATP-Binding Cassette Transporters
  • Abcb11 protein, rat
  • Adaptor Proteins, Signal Transducing
  • Bile Acids and Salts
  • CTTN protein, human
  • Cations
  • Cortactin
  • Cttn protein, rat
  • HAX1 protein, human
  • Microfilament Proteins
  • Phospholipids
  • Proteins
  • multidrug resistance protein 3
  • Glutathione Transferase