title: Evidence for ligand-dependent intramolecular folding of the AF-2 domain in vitamin D receptor-activated transcription and coactivator interaction.
abstract: A ligand-dependent transcriptional activation domain (AF-2) exists in region E of the nuclear receptors. This highly conserved domain may contact several coactivators that are putatively involved in nuclear receptor-mediated transcription. In this study, a panel of vitamin D receptor (VDR) AF-2 mutants was created to examine the importance of several conserved residues in VDR-activated transcription. Two AF-2 mutants (L417S and E420Q) exhibited normal ligand binding, heterodimerization with retinoid X receptor, and vitamin D-responsive element interaction, but they were transcriptionally inactive in a VDR-responsive reporter gene assay. All AF-2 mutations that abolished VDR-mediated transactivation also eliminated interactions between VDR and several putative coactivator proteins including suppressor of gal1 (SUG1), steroid hormone receptor coactivator-1 (SRC-1), or receptor interacting protein (RIP140), suggesting that coactivator interaction is important for AF-2-mediated transcription. In support of this concept, the minimal AF-2 domain [VDR(408-427)] fused to the gal4 DNA binding domain was sufficient to mediate transactivation as well as interaction with putative coactivators. Introducing the L417S and E420Q mutations into the minimal AF-2 domain abolished this autonomous transactivation and coactivator interactions. Finally, we demonstrate that the minimal AF-2 domain interacted with an AF-2 deletion mutant of the VDR in a 1,25-(OH)2D3-dependent manner, suggesting a ligand-induced intramolecular folding of the VDR AF-2 domain. The L417S mutant of this domain disrupted the interaction with VDR ligand-binding domain, while the E420Q mutant did not affect this interaction. These studies suggest that the conserved AF-2 motif may mediate transactivation through ligand-dependent intermolecular interaction with coactivators and through ligand-induced intramolecular contacts with the VDR ligand-binding domain itself.
authors.affiliation: Department of Pharmacological and Physiological Science, Saint Louis University Health Science Center, Missouri 63104, USA.
authors.forename: H
authors.initials: H
authors.lastname: Masuyama
authors.forename: C M
authors.initials: CM
authors.lastname: Brownfield
authors.forename: R
authors.initials: R
authors.lastname: St-Arnaud
authors.forename: P N
authors.initials: PN
authors.lastname: MacDonald
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D000078723:Nuclear Receptor Interacting Protein 1; D017510:Protein Folding; D018167:Receptors, Calcitriol / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D015533:Transcriptional Activation; D014807:Vitamin D / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D008024:Ligands; D009687:Nuclear Proteins; D000078723:Nuclear Receptor Interacting Protein 1; D018167:Receptors, Calcitriol; D014807:Vitamin D