Abstract
The silencing mediator for retinoid and thyroid hormone receptors (SMRT) serves as a platform for transcriptional repression elicited by several steroid/nuclear receptors and transcription factors. SMRT exists in two major splicing isoforms, α and τ, with SMRTα containing only an extra 46-amino acid sequence inserted immediately downstream from the C-terminal corepressor motif. Little is known about potential functional differences between these two isoforms. Here we show that the pregnane X receptor (PXR) interacts more strongly with SMRTα than with SMRTτ both in vitro and in vivo. It is interesting that the PXR-SMRTα interaction is also resistant to PXR ligand-induced dissociation, in contrast to the PXR-SMRTτ interaction. SMRTα consistently inhibits PXR activity more efficiently than does SMRTτ in transfection assays, although they possess comparable intrinsic repression activity and association with histone deacetylase. We further show that the mechanism for the enhanced PXR-SMRTα interaction involves both the 46-amino acid insert and the C-terminal corepressor motif. In particular, the first five amino acids of the SMRTα insert are essential and sufficient for the enhanced binding of SMRTα to PXR. Furthermore, we demonstrate that Tyr2354 and Asp2355 residues of the SMRTα insert are most critical for the enhanced interaction. In addition, expression data show that SMRTα is more abundantly expressed in most human tissues and cancer cell lines, and together these data suggest that SMRTα may play a more important role than SMRTτ in the negative regulation of PXR.
Footnotes
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This work was supported by the National Institutes of Health [Grant DK52542].
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ABBREVIATIONS: NR, nuclear receptor; PXR, pregnane X receptor; SMRT, silencing mediator for retinoid and thyroid hormone receptors; N-CoR, nuclear receptor corepressor; HDAC, histone deacetylase; ID, interacting domain; RXR, retinoid X receptor; CAR, constitutive androstane receptor; TR, thyroid hormone receptor; PCN, pregnenolone-16α-carbonitrile; GST, glutathione transferase; Rif, rifampicin; CTZ, clotrimazole; HA, hemagglutinin; aa, amino acid(s); PCR, polymerase chain reaction; AD, activation domain; DMSO, dimethyl sulfoxide; PAGE, polyacrylamide gel electrophoresis; HEK, human embryonic kidney; RAR, retinoic acid receptor; VDR, vitamin D receptor; ROR, retinoid-related orphan receptor; EGFP, enhanced green fluorescent protein.
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↵1 Current affiliation: MD Anderson Cancer Center, University of Texas, Houston, Texas.
- Received April 10, 2008.
- Accepted October 30, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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