Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling

Abstract

Cellular responsiveness to retinoic acid and its metabolites is conferred through two structurally and pharmacologically distinct families of receptors: the retinoic acid receptors (RAR) and the retinoid X receptors (RXR). Here we report that the transcriptional activity of RAR and RXR can be reciprocally modulated by direct interactions between the two proteins. RAR and RXR have a high degree of cooperativity in binding to target DNA, consistent with previous reports indicating that the binding of either RAR or RXR to their cognate response elements is enhanced by factors present in nuclear extracts. RXR also interacts directly with and enhances the binding of nuclear receptors conferring responsiveness to vitamin D3 and thyroid hormone T3; the DNA-binding activities of these receptors are also stimulated by the presence of nuclear extracts. Together these data indicate that RXR has a central role in multiple hormonal signalling pathways.

FIG. 1

a, the C terminus of RAR is required for suppression of RXR-dependent transactivation through the CRBPII-RXRE. CV-1 cells were cotransfected in duplicate with the reporter construct ΔSV-CRBPII-CAT, expression plasmid RS-hRXRa, and the control expression vector RS-LUC (no competitor) or the expression plasmids RS-hRARa, RS-Δ81–153, RS-185* and RS-ΔA203–360. Cells (right-hand figure) were then exposed to either ethanol (cross-hatched) or 10 μΜ retinoic acid (filled bars). CAT (chloramphenicol acetyltransferase) activity Is presented as per cent conversion where retinoic acid-induced activation in the presence of RXR is arbitrarily set at 100%. b, RXR enhances RAR-dependent transactivation through an RARE. CV-1 cells were cotransfected in duplicate with the reporter construct tk-DR5.2-LUC, containing two copies of the DR-5 RARE upstream of the thymidine kinase promoter, and expression plasmids RS-CAT (−), RS-RARα, and/or RS-RXRα as Indicated. Cells were then exposed to either ethanol or 10 μΜ retinoic acid (cross-hatched and filled bars respectively). Luciferase activity is presented as per cent normalized response where retinoic acid-induced activation in the presence of RAR is arbitrarily set at 100%. METHODS. CV-1 monkey kidney cell culture, transfections, CAT and luciferase assays were done as previously described^,,. Transfection was on 10-cm plates and, for experiments using CAT, included 1 μg of RS-receptor or RS-LUC, 0.5 μg RS-hRXRα, 1 μg ΔSV-CRBPII-CAT reporter, 5 μg RAS-β- galactosidase reporter and 7.5 μg pGEM4 carrier DNA. Transfections using luciferase included 50 ng RS-RARα and/or 100 ng RXRα (the total amount of RS-expression plasmid was maintained constant in each transfection through the addition of RS-CAT), 0.5 μg tk-DR5.2-LUC reporter, 5 μg RAS-/3- galactosidase reporter and 8.5 μg pGEM4 carrier DNA.

FIG. 2

Direct Interactions between RAR and RXR in the absence or presence of DNA. a, RAR and RXR form a complex In solution. Immunoprecipltation reactions were done using in vitro synthesized, ³⁵S-methionlne-labelled RARα (lanes 1 and 2), the ligand binding domain of RARα (LBD) (amino acids 155–462) (lanes 3 and 4), or the GR (glucocorticoid receptor) (lanes 5 and 6) In either the absence (lanes 1,3,5) or presence (lanes 2,4,6) of bacterlally expressed RXR (bRXR). Polyclonal antisera prepared against RXRa was used, in vitro synthesized RAR, LBD, and GR proteins not subjected to Immunoprecipitatlon are shown in lanes 7–9. b, RAR and RXR bind cooperatively to the CRBPII-RXRE. Gel mobility shift assays were done using in vitro synthesized RAR and/or RXR as indicated and ³²P-labelled CRBPII-RXRE oligonucleotide. Polyclonal antisera prepared against either RAR (RARab) (lane 5) or RXR (RXRab) (lane 6) or prelmmune serum (PI) (lane 7) were included in the reactions as indicated, c, Binding specificity of the RAR-RXR complex. Gel mobility shift competition reactions were done using ³²P-labelled CRBPII-RXRE oligonucleotide and either a 10-fold (10 ×) or 40-fold (40 ×) excess of unlabelled competitor oligonucleotide encoding either the CRBPII-RXRE (lanes 2 and 3), the βRARE (lanes 4 and 5), or a palindromic GRE (lanes 6 and 7). d, RAR and RXR bind cooperatively to the βRARE. Gel mobility shift assays were done using in vitro synthesized RAR and/or RXR as indicated and ³²P-labelled βRARE oligonucleotide. Polyclonal antisera prepared against either RAR (RARab) (lane 5) or RXR (RXRab) (lane 6) or preimmune serum (PI) (lane 7) were Included in the reactions as Indicated, e, RAR and RXR overexpressed in COS cells bind cooperatively to the βRARE. Gel mobility shift assays were done using ³²P-labelled βRARE and whole cell extracts prepared from COS cells in which RAR (lane 2), RXR (lane 3) or RAR and RXR (lanes 4–9) were overexpressed. Prelmmune serum (3 μl) (lane 5) or 0.2 μl or 1 μl RAR-specific antiserum (RARab) (lanes 6 and 7, respectively) or 1 μl or 3 μl RXR-specific antiserum (RXRab) (lanes 8 and 9, respectively) were included in the reactions as Indicated. The position of the RAR-RXR-βRARE complex is Indicted by an arrowhead. METHODS. The LBD expression vector was generated through insertion of an Xhol-BamHI fragment of Δ81–153, including amino acids 155–462 of RARα, Into the pCMX expression vector containing a synthetic translation start site sequence^,. RARα, LBD, and GR RNA was prepared and subsequently translated in rabbit reticulocyte lysates as directed by the supplier (Promega). RXR was expressed in bacteria as a fusion with glutathlone-S-transferase using the pGEX-2T expression vector (Pharmacia) and purified as previously described. Immunoprecipltation reactions (20 μl) included 5 μl ³⁵S-methionine-labelled receptor protein and 150 ng of either purified GST-RXR or GST alone in 20 mM Tris, pH 8.0. Proteins were incubated 20 min on Ice before the addition of 5 μl polyclonal antisera prepared against an RXRα peptide (amino acids 214–229). Antigen-antibody complexes were collected by the addition of Protein A-Sepharose (Pharmacia) and the Immunocomplexes washed three times with 400 μl RIPA buffer (10 mM Tris (pH 8.0), 150 mM NaCI, 1% Triton X-100,1% sodium deoxycholate). Immunopreclpl- tated complexes were resolved by SDS-PAGE on 10% gels which were then fixed In 30% methanol, 10% acetic acid, dried and autoradiographed. Gel mobility shift assays (20 μl) contained 10 mM Tris (pH 8.0), 40 mM KCI, 0.05% Nonidet P-40,6% glycerol, 1 mM DTT, 0.2 pg of poly(dl-dC) and 2.5 μl each of in vitro synthesized RAR and RXR proteins. The total amount of reticulocyte lysate was maintained constant in each reaction (5 μl) through the addition of unprogrammed lysate. Where indicated, prelmmune serum or polyclonal rabbit antisera prepared against bacterlally expressed RARa or an RXRa peptide (amino acids 214–229) were included. After a 10 min Incubation on ice 1 ng ³²P-labelled oligonucleotide was added and the incubation continued for an additional 10 min. DNA-protein complexes were resolved on a 4% polyacrylamide gel in 0.5× TBE (lx TBE is 90mM Tris, 90mM boric acid, 2 mM EDTA). Gels were dried and autoradiographed at −70°. Gel mobility shift assays using COS cell-expressed receptors were as described, using 5 μg whole cell extracts prepared from COS cells transfected with 10 μg of either pCMX-hRARα, pCMX-hRXRα, or both expression plasmids.

FIG. 3

Direct interactions between RXR and TR or VDR in the absence or presence of DNA. a, RXR forms complexes with either TR or VDR in solution. Immunoprecipitation reactions were done with RXR-specific antiserum and in vitro synthesized, ³⁵S-methionine-labelled TRβ (lanes 1 and 2) or VDR (lanes 3 and 4) in either the absence (lanes 1, 3) or presence (lanes 2, 4) of bacterially expressed RXR (bRXR). Vitamin D₃ (1 ×10⁻⁷M) was included in reactions containing the VDR. In vitro synthesized TR and VDR proteins not subjected to immunoprecipitation are shown in lanes 5 and 6. b, RXR interacts cooperatively with TR and VDR in DNA binding. Gel mobility shift assays were done using in vitro synthesized RXR, TR, VDR and GR as indicated and ³²P-labelled oligonucleotides encoding Moloney leukaemia virus LTR TRE (lanes 1–4), the mouse osteopontin VDRE (lanes 5–8), or the palindromic GRE (lanes 9–12). METHODS. TRβ and VDR RNA was prepared and subsequently translated in rabbit reticulocyte lysates as directed by the supplier (Promega). Immunoprecipitation and gel mobility shift assays are described in Fig. 2 legend.