Coactivator-Vitamin D Receptor Interactions Mediate Inhibition of the Atrial Natriuretic Peptide Promoter

Songcang Chen,Junfeng Cui,Karl Nakamura,Ralff C.J Ribeiro,Brian L West,David G Gardner

doi:10.1074/jbc.275.20.15039

Songcang Chen, Junfeng Cui + Show 4 more

Open Access

https://doi.org/10.1074/jbc.275.20.15039

Copy DOI

Abstract

We have discovered a role for coactivators binding to the AF-2 surface of the 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 predicted 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 transfected DR3 TK luciferase reporter) and inhibition (assessed with an hANPCAT reporter) functions of the receptor when tested in cultured neonatal rat atrial myocytes and HeLa cells. Collectively, these mutations also suppressed association of VDR with the coactivators GRIP1 and steroid receptor coactivator 1 in vitro but had little or no effect on ligand binding, heterodimerization with the retinoid X receptor, or association with a VDR-specific DNA recognition element. Co-transfection with GRIP1 or steroid receptor coactivator 1 amplified both the positive and negative responses to wild type VDR but had little or no effect on the functionally impaired mutants described above. The interaction between VDR and GRIP1 proved to be heavily dependent upon the integrity of nuclear box III in the latter protein. Mutations in this region of GRIP1 impaired its ability to associate with VDR in vitro and to amplify VDR activity in intact cells. These studies establish a role for coactivators recruited to the same receptor surface in both the activating and inhibitory activity of the liganded receptor.

Highlights

We have discovered a role for coactivators binding to the AF-2 surface of the vitamin D receptor (VDR) in its negative effects on gene transcription
The canonical sequence preferred by the VDR is composed of a direct repeat of the sequence AGGTCA spaced by three nucleotides (DR3) [7], which the VDR occupies as a heterodimeric complex with the retinoid X receptor (RXR)
We carried out a sequence alignment to identify probable homologues of those residues recently identified in hTR␤1 [12] as being critically involved in the activation function of that receptor (Fig. 1)

Summary

Introduction

We have discovered a role for coactivators binding to the AF-2 surface of the vitamin D receptor (VDR) in its negative effects on gene transcription. Mutation of six of these residues in VDR resulted in loss of both the activation (assessed with a transfected DR3 TK luciferase reporter) and inhibition (assessed with an hANPCAT reporter) functions of the receptor when tested in cultured neonatal rat atrial myocytes and HeLa cells These mutations suppressed association of VDR with the coactivators GRIP1 and steroid receptor coactivator 1 in vitro but had little or no effect on ligand binding, heterodimerization with the retinoid X receptor, or association with a VDR-specific DNA recognition element. Feng et al [12] used a scanning surface mutagenesis approach based on x-ray crystallographic data [13] to define the surface residues of the thyroid hormone receptor (TR) LBD that are required for association with coactivator proteins and for the hormone-dependent stimulation of transcription They identified a collection of 6 amino acid residues on the surface which are essential for both coactivator binding and the activation function [12]. Subsequent studies of Darimont et al [14] with TR␤1, Shiau et al [15] with ER␣, and Nolte et al [16] with the peroxisome proliferator-activated receptor ␥ confirmed this prediction with structural analyses demonstrating the alignment of specific regions of the coactivator, the so-called nuclear receptor or NR boxes (see below), within the hydrophobic cleft of these receptors

Methods

Results

Conclusion