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PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
5
pubmed:dateCreated
2002-4-30
pubmed:abstractText
The retinoid orphan-related receptor-alpha (RORalpha) is a member of the ROR subfamily of orphan receptors and acts as a constitutive activator of transcription in the absence of exogenous ligands. To understand the basis of this activity, we constructed a homology model of RORalpha using the closely related TRbeta as a template. Molecular modeling suggested that bulky hydrophobic side chains occupy the RORalpha ligand cavity leaving a small but distinct cavity that may be involved in receptor stabilization. This model was subject to docking simulation with a receptor-interacting peptide from the steroid receptor coactivator, GR-interacting protein-1, which delineated a coactivator binding surface consisting of the signature motif spanning helices 3-5 and helix 12 [activation function 2 (AF2)]. Probing this surface with scanning alanine mutagenesis showed structural and functional equivalence between homologous residues of RORalpha and TRbeta. This was surprising (given that RORalpha is a ligand-independent activator, whereas TRbeta has an absolute requirement for ligand) and prompted us to use molecular modeling to identify differences between RORalpha and TRbeta in the way that the AF2 helix interacts with the rest of the receptor. Modeling highlighted a nonconserved amino acid in helix 11 of RORalpha (Phe491) and a short-length of 3.10 helix at the N terminus of AF2 which we suggest 1) ensures that AF2 is locked permanently in the holoconformation described for other liganded receptors and thus 2) enables ligand-independent recruitment of coactivators. Consistent with this, mutation of RORalpha Phe491 to either methionine or alanine (methionine is the homologous residue in TRbeta), reduced and ablated transcriptional activation and recruitment of coactivators, respectively. Furthermore, we were able to reconstitute transcriptional activity for both a deletion mutant of RORalpha lacking AF2, and Phe491Met, by overexpression of a GAL-AF2 fusion protein, demonstrating ligand-independent recruitment of AF2 and a role for Phe491 in recruiting AF2.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0888-8809
pubmed:author
pubmed:issnType
Print
pubmed:volume
16
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
998-1012
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed-meshheading:11981035-Alanine, pubmed-meshheading:11981035-Amino Acid Sequence, pubmed-meshheading:11981035-Animals, pubmed-meshheading:11981035-Binding Sites, pubmed-meshheading:11981035-Models, Molecular, pubmed-meshheading:11981035-Molecular Sequence Data, pubmed-meshheading:11981035-Molecular Structure, pubmed-meshheading:11981035-Mutagenesis, Site-Directed, pubmed-meshheading:11981035-Nuclear Receptor Coactivator 2, pubmed-meshheading:11981035-Nuclear Receptor Subfamily 1, Group F, Member 1, pubmed-meshheading:11981035-Protein Structure, Secondary, pubmed-meshheading:11981035-Receptors, Cytoplasmic and Nuclear, pubmed-meshheading:11981035-Receptors, Thyroid Hormone, pubmed-meshheading:11981035-Sequence Alignment, pubmed-meshheading:11981035-Structure-Activity Relationship, pubmed-meshheading:11981035-Trans-Activators, pubmed-meshheading:11981035-Transcription, Genetic, pubmed-meshheading:11981035-Transcription Factors, pubmed-meshheading:11981035-Transfection
pubmed:year
2002
pubmed:articleTitle
Characterization of the retinoid orphan-related receptor-alpha coactivator binding interface: a structural basis for ligand-independent transcription.
pubmed:affiliation
Queensland University of Technology, Centre for Molecular Biotechnology, Brisbane 4001, Queensland, Australia.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't