title: Phosphorylated Nuclear Receptor CAR Forms a Homodimer To Repress Its Constitutive Activity for Ligand Activation.
abstract: The nuclear receptor CAR (NR1I3) regulates hepatic drug and energy metabolism as well as cell fate. Its activation can be a critical factor in drug-induced toxicity and the development of diseases, including diabetes and tumors. CAR inactivates its constitutive activity by phosphorylation at threonine 38. Utilizing receptor for protein kinase 1 (RACK1) as the regulatory subunit, protein phosphatase 2A (PP2A) dephosphorylates threonine 38 to activate CAR. Here we demonstrate that CAR undergoes homodimer-monomer conversion to regulate this dephosphorylation. By coexpression of two differently tagged CAR proteins in Huh-7 cells, mouse primary hepatocytes, and mouse livers, coimmunoprecipitation and two-dimensional gel electrophoresis revealed that CAR can form a homodimer in a configuration in which the PP2A/RACK1 binding site is buried within its dimer interface. Epidermal growth factor (EGF) was found to stimulate CAR homodimerization, thus constraining CAR in its inactive form. The agonistic ligand CITCO binds directly to the CAR homodimer and dissociates phosphorylated CAR into its monomers, exposing the PP2A/RACK1 binding site for dephosphorylation. Phenobarbital, which is not a CAR ligand, binds the EGF receptor, reversing the EGF signal to monomerize CAR for its indirect activation. Thus, the homodimer-monomer conversion is the underlying molecular mechanism that regulates CAR activation, by placing phosphorylated threonine 38 as the common target for both direct and indirect activation of CAR.
authors.affiliation: Pharmacogenetic Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.
authors.forename: Ryota
authors.initials: R
authors.lastname: Shizu
authors.affiliation: Pharmacogenetic Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.
authors.forename: Makoto
authors.initials: M
authors.lastname: Osabe
authors.affiliation: Computational Chemistry and Molecular Modeling Support Group, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.
authors.forename: Lalith
authors.initials: L
authors.lastname: Perera
authors.affiliation: Pharmacogenetic Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.
authors.forename: Rick
authors.initials: R
authors.lastname: Moore
authors.affiliation: Pharmacogenetic Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sueyoshi
authors.affiliation: Pharmacogenetic Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA negishi@niehs.nih.gov.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Negishi
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D000090702:Constitutive Androstane Receptor; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009479:Neuropeptides / Q000378:metabolism*; D010766:Phosphorylation; D055503:Protein Multimerization; D054648:Protein Phosphatase 2 / Q000378:metabolism*; D000075370:Receptors for Activated C Kinase; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000502:physiology*
chemical_list: D000090702:Constitutive Androstane Receptor; D008024:Ligands; C000716327:NR1I3 protein, human; D009479:Neuropeptides; C000716328:Nr1i3 protein, mouse; C090294:RACK1 protein, mouse; D000075370:Receptors for Activated C Kinase; D018160:Receptors, Cytoplasmic and Nuclear; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D054648:Protein Phosphatase 2
keywords: CITCO; cell signaling; constitutive androstane receptor; homodimer; nuclear receptors; phenobarbital; protein phosphorylation