Lysine 246 of the vitamin D receptor is crucial for ligand-dependent interaction with coactivators and transcriptional activity

Mutant R246A in the predicted helix 3 of the ligand-binding domain, as well as mutants L417S and E420Q in helix 12, which contains the core ligand-dep endent transcriptional activation domain (AF-2), were generated to examine AF-2 activity of the vitamin D receptor (VDR), These mutations abolished vi tamin D-dependent transactivation. In addition, VDR mediates a ligand-depen dent repression of the response of the retinoic acid receptor beta 2 promot er to retinoic acid, and the helix 3 and helix 12 mutants were unable to me diate transrepression. Furthermore, the VDR mutants, but not the native rec eptor, enhanced phorbol ester induction of the activator protein-1-containi ng collagenase promoter. The helix 3 and helix 12 mutations strikingly redu ced the ability of VDR to interact with the coactivators steroid receptor c oactivator-1, ACTR, and the CREB-binding protein. As a consequence, overexp ression of steroid receptor coactivator-1 increased vitamin D-dependent tra nsactivation by VDR but not by the K246A mutant. These results indicate tha t the lysine 246 participates, together with residues in helix 12, in the r ecruitment of coactivators and that AF-2 activity is involved both in ligan d dependent transactivation and in transrepression by VDR.