Interaction of the phosphorylated DNA-binding domain in nuclear receptor CAR with its ligand-binding domain regulates CAR activation

J Biol Chem. 2018 Jan 5;293(1):333-344. doi: 10.1074/jbc.M117.806604. Epub 2017 Nov 13.

Abstract

The nuclear protein constitutive active/androstane receptor (CAR or NR1I3) regulates several liver functions such as drug and energy metabolism and cell growth or death, which are often involved in the development of diseases such as diabetes and hepatocellular carcinoma. CAR undergoes a conversion from inactive homodimers to active heterodimers with retinoid X receptor α (RXRα), and phosphorylation of the DNA-binding domain (DBD) at Thr-38 in CAR regulates this conversion. Here, we uncovered the molecular mechanism by which this phosphorylation regulates the intramolecular interaction between CAR's DBD and ligand-binding domain (LBD), enabling the homodimer-heterodimer conversion. Phosphomimetic substitution of Thr-38 with Asp increased co-immunoprecipitation of the CAR DBD with CAR LBD in Huh-7 cells. Isothermal titration calorimetry assays also revealed that recombinant CAR DBD-T38D, but not nonphosphorylated CAR DBD, bound the CAR LBD peptide. This DBD-LBD interaction masked CAR's dimer interface, preventing CAR homodimer formation. Of note, EGF signaling weakened the interaction of CAR DBD T38D with CAR LBD, converting CAR to the homodimer form. The DBD-T38D-LBD interaction also prevented CAR from forming a heterodimer with RXRα. However, this interaction opened up a CAR surface, allowing interaction with protein phosphatase 2A. Thr-38 dephosphorylation then dissociated the DBD-LBD interaction, allowing CAR heterodimer formation with RXRα. We conclude that the intramolecular interaction of phosphorylated DBD with the LBD enables CAR to adapt a transient monomer configuration that can be converted to either the inactive homodimer or the active heterodimer.

Keywords: CAR; DNA-binding domain; dimerization; heterodimer; homodimer; ligand-binding domain; nuclear receptor; nuclear translocation; phosphorylation; protein-protein interaction.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Cell Line
  • Constitutive Androstane Receptor
  • DNA-Binding Proteins / metabolism
  • Dimerization
  • Humans
  • Ligands
  • Phosphorylation
  • Protein Binding
  • Protein Domains
  • Protein Phosphatase 2 / metabolism
  • Protein Structure, Tertiary
  • Receptors, Cytoplasmic and Nuclear / genetics*
  • Receptors, Cytoplasmic and Nuclear / metabolism*
  • Retinoid X Receptor alpha / metabolism
  • Retinoid X Receptors / metabolism

Substances

  • Constitutive Androstane Receptor
  • DNA-Binding Proteins
  • Ligands
  • NR1I3 protein, human
  • Receptors, Cytoplasmic and Nuclear
  • Retinoid X Receptor alpha
  • Retinoid X Receptors
  • Protein Phosphatase 2