Coactivator-vitamin D receptor interactions mediate inhibition of the atrial natriuretic peptide promoter

We have discovered a role for coactivators binding to the AF-2 surface of t he vitamin D receptor (VDR) in its negative effects on gene transcription. We tested nine amino acid residues (Ser(235), Ile(242), Lys(246), Asp(253), Ile(260), Leu(263), Leu(417), Leu(419), and Glu(420)) in, human VDR which, based on homology to the human thyroid hormone receptor, would be predicte d to lie in or near the coactivator-binding site. Mutation of six of these residues in VDR resulted in loss of both the activation (assessed with a tr ansfected DR3 TK luciferase reporter) and inhibition (assessed with an hANP CAT reporter) functions of the receptor when tested in cultured neonatal ra t atrial myocytes and HeLa cells. Collectively, these mutations also suppre ssed association of VDR with the coactivators GRIP1 and steroid receptor co activator 1 in vitro but had little or no effect on ligand binding, heterod imerization with the retinoid X receptor, or association with a VDR-specifi c DNA recognition element. Co-transfection with GRIP1 or steroid receptor c oactivator 1 amplified both the positive and negative responses to wild typ e VDR but had little or no effect on the functionally impaired mutants desc ribed above. The interaction between VDR and GRIP1 proved to be heavily dep endent upon the integrity of nuclear box III in the latter protein. Mutatio ns in this region of GRIP1 impaired its ability to associate with VDR in vi tro and to amplify VDR activity in intact cells. These studies establish a role for coactivators recruited to the same receptor surface in both the ac tivating and inhibitory activity of the liganded receptor.