NRIF3 is a novel coactivator mediating functional specificity of nuclear hormone receptors

Abstract

Many nuclear receptors are capable of recognizing similar DNA elements. The molecular event(s) underlying the functional specificities of these receptors (in regulating the expression of their native target genes) is a very important issue that remains poorly understood. Here we report the cloning and analysis of a novel nuclear receptor coactivator (designated NRIF3) that exhibits a distinct receptor specificity. Fluorescence microscopy shows that NRIF3 localizes to the cell nucleus. The yeast two-hybrid and/or in vitro binding assays indicated that NRIF3 specifically interacts with the thyroid hormone receptor (TR) and retinoid X receptor (RXR) in a ligand-dependent fashion but does not bind to the retinoic acid receptor, vitamin D receptor, progesterone receptor, glucocorticoid receptor, or estrogen receptor. Functional experiments showed that NRIF3 significantly potentiates TR- and RXR-mediated transactivation in vivo but has little effect on other examined nuclear receptors. Domain and mutagenesis analyses indicated that a novel C-terminal domain in NRIF3 plays an essential role in its specific interaction with liganded TR and RXR while the N-terminal LXXLL motif plays a minor role in allowing optimum interaction. Computer modeling and subsequent experimental analysis suggested that the C-terminal domain of NRIF3 directly mediates interaction with liganded receptors through an LXXIL (a variant of the canonical LXXLL) module while the other part of the NRIF3 protein may still play a role in conferring its receptor specificity. Identification of a coactivator with such a unique receptor specificity may provide new insight into the molecular mechanism(s) of receptor-mediated transcriptional activation as well as the functional specificities of nuclear receptors.

FIG. 1

Hormone-dependent interaction of NRIF3 with the LBD of TR. Induction of β-galactosidase activity by thyroid hormone (T3) was measured in the yeast strain EGY48 transformed with a bait vector expressing the LexA-cTRα LBD and a prey plasmid expressing NRIF3 fused to the B42 activation domain (29). The bait LexA alone was used as the negative control. The prey B42–Trip1 was used as the positive control. Hatched bars, without T3; filled bars, with 1 μM T3.

FIG. 2

Nucleotide and deduced amino acid sequences of NRIF3. Only part of the cDNA sequence is shown. A putative nuclear localization signal (KRKK) is underlined. The putative LXXLL motif is shown with a double underline. NRIF3 and EnL have 95% identity. They differ only in their C termini, where the last 16 amino acids (dotted underline) in NRIF3 are replaced with 9 different amino acids (GQPQMSQPL) in EnL. EnS consists of 111 amino acids and is 100% identical to the first 111 amino acids of NRIF3 or EnL.

FIG. 3

NRIF3 is a nuclear protein. HeLa cells were transfected with an expression vector for GFP (left panel) or GFP-NRIF3 (right panel). The cellular location of the expressed proteins was visualized by fluorescence microscopy.

FIG. 4

Characterization of the NRIF3 interaction with nuclear receptors in vitro. A ³⁵S-labeled full-length receptor (cTRα, hRARα, hRXRα, hVDR, hPR, hGR, or hER) was incubated with an affinity-purified GST control or GST-NRIF3 linked to glutathione-agarose beads. The binding was performed in the absence (−) or presence (+) of cognate ligands as described in Materials and Methods. After incubation and washing, the bound receptors were analyzed by sodium dodecyl sulfate–10% polyacrylamide gel electrophoresis and detected by autoradiography. The input lane in each binding assay represents 5% of the total ³⁵S-labeled receptor used in each incubation. GST-RXR was used as a positive control for RAR binding.

FIG. 5

NRIF3 enhances TR-mediated transactivation in vivo. HeLa cells were transfected with a vector expressing cTRα and the IR-ΔMTV-CAT reporter (A) or the GH-TRE-tk-CAT reporter (B) in the presence (filled bars) or the absence (hatched bars) of 1 μM T3. The vector expressing NRIF3 or the empty control vector was cotransfected to examine the effect of NRIF3 on TR-mediated activation. In panel A, the effect of CBP was compared to that of NRIF3. GH, growth hormone.

FIG. 6

NRIF3 functions as a coactivator for RXR but not RAR. (A) NRIF3 potentiates the activity of the endogenous RXR(s) but not the RAR(s). HeLa cells were transfected with the IR-ΔMTV-CAT reporter (without any receptor expression vector) to examine the activation by endogenous retinoid receptors. The NRIF3 expression vector or the empty control vector was cotransfected to examine the effect of NRIF3 on the activity of the endogenous RXR(s) or RAR(s). Relative CAT activity was determined in the presence (filled bars) or absence (hatched bars) of the indicated ligands (1 μM). (B and C) NRIF3 potentiates the activity of the exogenously expressed RXR. A vector expressing hRXRα was cotransfected into HeLa cells with the IR-ΔMTV-CAT reporter (B) or the DR1-ΔMTV-CAT reporter (C) in the presence (filled bars) or absence (hatched bars) of the indicated ligands (1 μM). The effect of NRIF3 on RXR-mediated transactivation was examined as described for panel A. TTNPB, a synthetic ligand for RAR.

FIG. 7

NRIF3 does not potentiate the activity of GR, PR, ER, or VDR. HeLa cells were transfected with the following CAT reporters and appropriate receptor expression vectors: GRE/PRE-tk-CAT and rGR or hPR (A), ERE-ΔMTV-CAT and hER (B), and VDRE-ΔMTV-CAT and hVDR (C). Cells were incubated in the presence (filled bars) or absence (hatched bars) of 100 nM dexamethasone for GR, progesterone for PR, estradiol for ER, and 1,25-(OH)₂-vitamin D₃ for VDR. Cotransfection of NRIF3 was found to have little effect on the activities of these receptors.

FIG. 8

The NCD is essential for the interaction with liganded TR or RXR. (A) Schematic comparison of NRIF3 with EnS and EnL. EnS is 100% identical to the first 111 amino acids of NRIF3 and EnL (open boxes). The regions from amino acids 112 to 161 in NRIF3 and EnL (stippled boxes) are 100% identical. NRIF3 and EnL differ in their C termini (16 amino acids in NRIF3 [hatched box] and 9 amino acids in EnL [filled box]). The positions of the LXXLL motif and a putative nuclear localization signal (KRKK) are also indicated. (B) NRIF3 (N), EnS (S), or EnL (L) was examined for interaction with LexA-TR or LexA-RXR in a yeast two-hybrid assay as described in Materials and Methods. The assays were performed in the absence (hatched bars) or the presence (filled bars) of 1 μM T3 (for TR) or 9-cis RA (for RXR).

FIG. 9

The LXXLL motif of NRIF3 is required for optimum interaction with TR and RXR. Wild-type NRIF3 (WT) or the L9A NRIF3 mutant (L9A) was examined for interaction with LexA-TR or LexA-RXR in a yeast two-hybrid assay as described in Materials and Methods. β-Galactosidase activities were determined in the absence (filled bars) or presence (stippled bars) of cognate ligands (1 μM T3 for TR; 1 μM 9-cis RA for RXR).

FIG. 10

Hypothetical model of the interaction of the NCD and the liganded LBD. The docking of the C-terminal helix of NRIF3, which contains an LXXIL module, to the ligand-bound LBDs was carried out as described in Materials and Methods. The NCD-TR LBD model is shown here as an example. The side chains of the two leucines (green) and one isoleucine (cyan) of the LXXIL core fit within a hydrophobic groove (salmon) on the surface of the liganded LBD (80). A similar modeling procedure was carried out with an LXXLL box of SRC-1 (result not shown). Putative binding energies (−21 kcal/mol for the NCD and −18 kcal/mol for the LXXLL box of SRC-1) were calculated as described in Materials and Methods. See the text for details.

FIG. 11

Interaction of the NCD with the receptor LBDs and the role of the LXXIL motif. The wild-type NCD (WT) or the NCD mutant form (Mut) in which the three core hydrophobic residues of the LXXIL motif (two leucines and one isoleucine) are changed into alanines was examined for interaction with the LBDs of TR, RXR, and RAR in a yeast two-hybrid assay as described in Materials and Methods. β-Galactosidase activities were determined in the absence (open bars) or presence (stippled bars) of cognate ligands. The prey expressing B42 alone was used as a negative control.