Abstract
In most mammalian cells, the retinoic acid receptor (RAR) is nuclear rather than cytoplasmic, regardless of its cognate ligand, retinoic acid (RA). In testis Sertoli cells, however, RAR is retained in the cytoplasm and moves to the nucleus only when RA is supplied. This led us to identify a protein that regulates the translocation of RAR. From yeast two-hybrid screening, we identified a novel RAR-interacting protein called CART1 (cytoplasmic adaptor for RAR and TR). Systematic interaction assays using deletion mutants showed that the C-terminal CoRNR box of CART1 was responsible for the interaction with the NCoR binding region of RAR and TR. Such interaction was impaired in the presence of ligand RA, as further determined by GST pulldown assays in vitro and immunoprecipitation assays in vivo. Fluorescence microscopy showed that unliganded RAR was captured by CART1 in the cytoplasm, whereas liganded RAR was liberated and moved to the nucleus. Overexpression of CART1 blocked the transcriptional repressing activity of unliganded apoRAR, mediated by corepressor NCoR in the nucleus. CART1 siRNA treatment in a mouse Sertoli cell line, TM4, allowed RAR to move to the nucleus and blocked the derepressing function of CART1, suggesting that CART1 might be a cytoplasmic, testis-specific derepressor of RAR.
Highlights
Nuclear receptors (NRs)3 are ligand-dependent transcription factors that control diverse aspects of development and homeostasis by regulating expression of their target genes [1, 2]
Our findings suggest that CART1 may be a cytoplasmic, testis-specific derepressor of retinoic acid receptor (RAR), which could be an additional checkpoint in the fine control of gene expression
Some studies have demonstrated that RAR and TR are retained in the cytoplasm and move to the nucleus when ligands are supplied [13,14,15,16]
Summary
Nuclear receptors (NRs)3 are ligand-dependent transcription factors that control diverse aspects of development and homeostasis by regulating expression of their target genes [1, 2]. To determine the effect of CART1 on the subcellular localization of RAR in living cells, we whereas helix H12 (or AF-2 AD) is dispensable for the interac- appended the GFP to human CART1 and the HcRed to mouse tion but essential for ligand-dependent disruption of the inter- RAR for fluorescence microscopy.
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