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Isolation and characterization of a novel coactivator protein, NCoA-62, involved in vitamin D-mediated transcription
T A Baudino1, D M Kraichely, S C Jefcoat
1Department of Pharmacological and Physiological Science, Program in Cell and Molecular Biology, St. Louis University Health Sciences Center, St. Louis, Missouri 63104, USA.
Abstract:
The vitamin D receptor (VDR) forms a heterodimeric complex with retinoid X receptor (RXR) and binds to vitamin D-responsive promoter elements to regulate the transcription of specific genes or gene networks. The precise mechanism of transcriptional regulation by the VDR.RXR heterodimer is not well understood, but it may involve interactions of VDR.RXR with transcriptional coactivator or corepressor proteins. Here, a yeast two-hybrid strategy was used to isolate proteins that selectively interacted with VDR and other nuclear receptors. One cDNA clone designated NCoA-62, encoded a 62, 000-Da protein that is highly related to BX42, a Drosophila melanogaster nuclear protein involved in ecdysone-stimulated gene expression. Yeast two-hybrid studies and in vitro protein-protein interaction assays using glutathione S-transferase fusion proteins demonstrated that NCoA-62 formed a direct protein-protein contact with the ligand binding domain of VDR. Coexpression of NCoA-62 in a vitamin D-responsive transient gene expression system augmented 1, 25-dihydroxyvitamin D3-activated transcription, but it had little or no effect on basal transcription or gal4-VP16-activated transcription. NCoA-62 also interacted with retinoid receptors, and its expression enhanced retinoic acid-, estrogen-, and glucocorticoid-mediated gene expression. These data indicate that NCoA-62 may be classified into an emerging set of transcriptional coactivator proteins that function to facilitate vitamin D- and other nuclear receptor-mediated transcriptional pathways.
Insights
NCoA-62 is a novel coactivator protein that directly interacts with the vitamin D receptor (VDR). This interaction enhances vitamin D-activated gene transcription, suggesting a key role for NCoA-62 in nuclear receptor signaling pathways.
Area of Science:
- Molecular Biology
- Genetics
- Endocrinology
Background:
- The vitamin D receptor (VDR) forms a heterodimer with the retinoid X receptor (RXR) to regulate gene transcription.
- The exact mechanisms of VDR.RXR heterodimer transcriptional regulation are not fully understood.
- Interactions with coactivator or corepressor proteins are hypothesized to be involved.
Purpose of the Study:
- To identify proteins that interact with VDR using a yeast two-hybrid system.
- To characterize the function of a novel interacting protein, NCoA-62, in nuclear receptor-mediated transcription.
Main Methods:
- Yeast two-hybrid screening to identify VDR-interacting proteins.
- In vitro protein-protein interaction assays using glutathione S-transferase fusion proteins.
- Transient gene expression assays to assess transcriptional activity.
Main Results:
- A cDNA clone, NCoA-62, was isolated, encoding a protein related to Drosophila BX42.
- NCoA-62 directly binds to the ligand-binding domain of VDR.
- Coexpression of NCoA-62 enhanced 1,25-dihydroxyvitamin D3-activated transcription.
- NCoA-62 also interacted with retinoid, estrogen, and glucocorticoid receptors, enhancing their mediated gene expression.
Conclusions:
- NCoA-62 functions as a transcriptional coactivator.
- It facilitates transcriptional pathways mediated by VDR and other nuclear receptors.
- NCoA-62 represents a new class of coactivator proteins involved in hormone signaling.