EVIDENCE FOR LIGAND-DEPENDENT INTRAMOLECULAR FOLDING OF THE AF-2 DOMAIN IN VITAMIN-D RECEPTOR-ACTIVATED TRANSCRIPTION AND COACTIVATOR INTERACTION

A ligand-dependent transcriptional activation domain (AF-2) exists in region E of the nuclear receptors. This highly conserved domain may co ntact several coactivators that are putatively involved in nuclear rec eptor-mediated transcription. In this study, a panel of vitamin D rece ptor (VDR) AF-2 mutants was created to examine the importance of sever al 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 interactio n, but they were transcriptionally inactive in a VDR-responsive report er gene assay. All AF-2 mutations that abolished VDR-mediated transact ivation also eliminated interactions between VDR and several putative coactivator proteins including suppressor of gall (SUG1), steroid horm one receptor coactivator-1 (SRC-1), or receptor interacting protein (R IP140), suggesting that coactivator interaction is important for AF-5- mediated transcription. In support of this concept, the minimal AF-2 d omain [VDR(408- 427] fused to the gal4 DNA binding domain was sufficie nt to mediate transactivation as well as interaction with putative coa ctivators. 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 int eracted with an AF-2 deletion mutant of the VDR in a 1,25-(OH)(2)D-3-d ependent manner, suggesting a ligand-induced intramolecular folding of the VDR AF-2 domain. The L417S mutant of this domain disrupted the in teraction with VDR ligand-binding domain, while the E420Q mutant did n ot affect this interaction. These studies suggest that the conserved A F-2 motif may mediate transactivation through ligand-dependent intermo lecular interaction with coactivators and through ligand-induced intra molecular contacts with the VDR ligand-binding domain itself.