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Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists
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Phosphorylated Nuclear Receptor CAR Forms a Homodimer To Repress Its Constitutive Activity for Ligand Activation

Ryota Shizu1, Makoto Osabe1, Lalith Perera2

  • 1Pharmacogenetic Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.

Molecular and Cellular Biology
|March 8, 2017
PubMed

Insights

The nuclear receptor CAR (constitutive androstane receptor) is activated by homodimer-monomer conversion. This process regulates CAR

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Pharmacology

Background:

  • The nuclear receptor CAR (constitutive androstane receptor) is a key regulator of hepatic drug and energy metabolism.
  • CAR's activity is crucial in drug-induced toxicity, diabetes, and tumor development.
  • CAR is constitutively active but can be inactivated by phosphorylation at threonine 38, with protein phosphatase 2A (PP2A) and RACK1 mediating its activation via dephosphorylation.

Purpose of the Study:

  • To elucidate the molecular mechanism regulating CAR activation.
  • To investigate the role of homodimer-monomer conversion in CAR dephosphorylation and activation.

Main Methods:

  • Coexpression of differently tagged CAR proteins in Huh-7 cells, primary hepatocytes, and mouse livers.
  • Coimmunoprecipitation and two-dimensional gel electrophoresis to analyze CAR dimerization.
  • Investigating the effects of epidermal growth factor (EGF), CITCO, and phenobarbital on CAR activation.

Main Results:

  • CAR forms homodimers, burying the PP2A/RACK1 binding site and maintaining an inactive state.
  • Epidermal growth factor (EGF) promotes CAR homodimerization, inhibiting its activity.
  • The ligand CITCO directly binds the CAR homodimer, inducing monomerization and subsequent dephosphorylation.
  • Phenobarbital indirectly activates CAR by reversing EGF-induced dimerization.

Conclusions:

  • CAR homodimer-monomer conversion is the central mechanism controlling its activation.
  • Phosphorylated threonine 38 serves as a common target for both direct and indirect CAR activation pathways.
  • Understanding CAR regulation through dimerization is vital for managing drug toxicity and metabolic diseases.
Keywords:
CITCOcell signalingconstitutive androstane receptorhomodimernuclear receptorsphenobarbitalprotein phosphorylation

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