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, 94 (15), 7909-14

Activation of the Orphan Receptor RIP14 by Retinoids

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Activation of the Orphan Receptor RIP14 by Retinoids

A M Zavacki et al. Proc Natl Acad Sci U S A.

Abstract

Retinoids are crucial regulators of a wide variety of processes in both developing and adult animals. These effects are thought to be mediated by the retinoic acid (RA) receptors and the retinoid X receptors (RXRs). We have identified an additional retinoid-activated receptor that is neither a retinoic acid receptors nor an RXR. RXR-interacting protein 14 (RIP14), a recently described orphan member of the nuclear receptor superfamily, can be activated by either all-trans-RA (tRA) or the synthetic retinoid TTNPB [[E]-4-[2-(5, 6, 7, 8-tetrahydro-5, 5, 8, 8-tetramethyl-2-naphthalenyl)propen-1-yl]benzoic acid].RIP14 binds to DNA as a heterodimer with RXR. In the presence of either tRA or TTNPB, the addition of 9-cis-RA or the RXR-specific agonist LG1069 [4-[1-(3, 5, 5, 8, 8-pentamethyl-5, 6, 7, 8-tertrahydro-2-naphthyl)ethenyl]benzoic acid] results in additional activation. Mutations of the ligand-dependent transcriptional activation functions indicate that TTNPB activates the RIP14 component of the RIP14-RXR heterodimer, that 9-cis-RA and LG1069 activate RXR, and that tRA activates via both RIP14 and RXR. Despite the very effective activation of RIP14 by tRA or TTNPB, relatively high concentrations of these compounds are required, and no evidence for direct binding of either compound was obtained using several approaches. These results suggest that RIP14 is the receptor for an as-yet-unidentified retinoid metabolite.

Figures

Figure 1
Figure 1
RIP14 is specifically activated by retinoids. (A) RIP14 responds to TTNPB but not farnesol. JEG-3 cells were cotransfected with the indicated expression plasmids, hsp27 EcREx5 Δ mouse mammary tumor virus–luciferase, and a plasmid expressing hGH as an internal control and treated with no ligand, 50 μM farnesol, or 5 μM TTNPB. Luciferase values were normalized to the amount of growth hormone, and the mean ± SD of triplicate wells is graphed. This and all other transfection experiments presented are representative of results obtained in at least three separate experiments. (B) JEG-3 cells were cotransfected as described above and treated with no ligand, 5 μM TTNPB, 100 nM tRA, 1 μM 9-cis-RA, 5 μM TTNPB plus 1 μM 9-cis-RA, or 100 nM tRA plus 1 μM 9-cis-RA. In the same experiment, expression of functional RARβ was confirmed by cotransfection of pRSVRARβ or CDM8 with a β2RARE x3 TK luc (14) reporter plasmid. Normalized luciferase values of 458 ± 44 and 254 ± 14, respectively, were observed in the presence of 5 μM of TTNPB. (C) RIP14–RXR but not RAR–RXR heterodimers bind specifically to the hsp27 EcRE. As indicated, 9.4 fmol (15,000 cpm) of 32P-labeled hsp 27 (lanes 1–13, left to right) or β2RARE (lanes 14–20, left to right) were incubated with: 2 μl of in vitro-translated RIP14 or mock-translated reticulocyte lysate (RRL), 1 μl of E. coli expressed RXR or RARα or a mock preparation of E. coli alone (mock). SP, specific competitor (100 ng of the corresponding unlabeled oligonucleotide); NS, nonspecific competitor [100 ng of the unlabeled Slp HRE-3 GRE (15)]. A nonspecific complex was observed in the presence of reticulocyte lysate; specific complexes are indicated by arrows. The gels shown were run in parallel.
Figure 2
Figure 2
Activation of RIP14 by TTNPB, RXR by LG1069, and both by tRA. (A) Cotransfected RIP14 plus RXR respond to both TTNPB and the RXR-specific agonist LG1069. JEG-3 cells were transfected as in Fig. 1 and treated with no ligand, 5 μM TTNPB, 1 μM LG1069, or 5 μM TTNPB plus 1 μM LG1069. (B) AF-2 regions deleted from RIP14 and RXRα. Amino acids 476–484 of RIP14 and 444–462 of RXRα were deleted to generate RIP14–Δ9C and RXR–Δ19C, respectively. The regions corresponding to the AF-2 motif (36) are identified in bold, and the regions deleted in each receptor are underlined. TRα is included for comparison. (C) AF-2 deletions demonstrate that RIP14 is activated by TTNPB, that RXR is activated by LG1069, and that both receptors respond to tRA. JEG-3 cells were transfected with the indicated expression vectors as in Fig. 1 and treated with either no ligand, 5 μM TTNPB, 10 μM tRA, or 100 nM LG1069.
Figure 3
Figure 3
Dose response of RIP14 activation by retinoids. (A) JEG-3 cells were transfected as in Fig. 1 with RIP14, RXR, and hsp27–EcREx5 Δ mouse mammary tumor virus–luciferase and treated with 0–10 μM TTNPB ± 1 μM 9-cis-RA and 0–10 μM tRA ± 1 μM 9-cis-RA. (B) JEG-3 cells were transfected with CDM8 as a carrier plasmid and the β2RARE x3 TK luc reporter plasmid, and the response of endogenous RAR to either 0–1 μM TTNPB or tRA was determined.

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