Analysis of the ligand-binding domain of human retinoic acid receptor alpha by site-directed mutagenesis.

Three subtypes of retinoic acid receptors (RAR), termed RAR alpha, RAR beta, and RAR gamma, have been described. They are composed of different structural domains, including distinct domains for DNA and ligand binding. RARs specifically bind all-trans-retinoic acid (RA), 9-cis-RA, and retinoid analogs. In this study, we examined the functional role of cysteine and arginine residues in the ligand-binding domain of hRAR alpha (hRAR alpha-LBD, amino acids 154 to 462). All conserved cysteine and arginine residues in this domain were mutated by site-directed mutagenesis, and the mutant proteins were characterized by blocking reactions, ligand-binding experiments, transactivation assays, and protease mapping. Changes of any cysteine residue of the hRAR alpha-LBD had no significant influence on the binding of all-trans RA or 9-cis RA. Interestingly, residue C-235 is specifically important in antagonist binding. With respect to arginine residues, only the two single mutations of R-276 and R-394 to alanine showed a dramatic decrease of agonist and antagonist binding whereas the R272A mutation showed only a slight effect. For all other arginine mutations, no differences in affinity were detectable. The two mutations R217A and R294A caused an increased binding efficiency for antagonists but no change in agonist binding. From these results, we can conclude that electrostatic interactions of retinoids with the RAR alpha-LBD play a significant role in ligand binding. In addition, antagonists show distinctly different requirements for efficient binding, which may contribute to their interference in the ligand-inducible transactivation function of RAR alpha.     

RXR alpha, a promiscuous partner of retinoic acid and thyroid hormone receptors.

Retinoic acid receptor (RAR), thyroid hormone receptor (T3R) and vitamin D3 receptor (VD3R) differ from steroid hormone receptors in that they bind and transactivate through responsive elements organized as direct rather than inverted repeats. We now show that recombinant RAR and T3R are monomers in solution and cannot form stable homodimeric complexes on their responsive elements. Stable binding of the receptors to their responsive elements requires heterodimerization with a nuclear factor. This auxiliary factor is tightly associated with RAR and T3R in the absence of DNA and co-purifies with both receptors. As demonstrated by extensive purification, the same auxiliary factor is required for stable DNA binding of RAR as for that of T3R; the factor also facilitates the formation of a stable VD3R-DNA complex. The auxiliary factor is identical to the retinoid X receptor alpha (RXR alpha) by biochemical and functional criteria. The identification of RXR alpha as a dimerization partner for the RARs, T3Rs and VD3R has important implications as to the function of these receptors and their ligands in development, homeostasis and neoplasia.     

Altered expression of retinoic acid (RA) receptor mRNAs in the fetal mouse secondary palate by all-trans and 13-cis RAs: implications for RA-induced teratogenesis

Retinoic acid (RA) is mandatory for various biological processes and normal embryonic development but is teratogenic at high concentrations. In rodents, one of the major malformations induced by RA is cleft palate (CP). RA mediates its effects by RA receptors (RARs), but the expression patterns of RARs in the developing palate are still unclear. We investigated the normal expression of RAR alpha, beta, and gamma messenger RNAs (mRNAs) in the fetal mouse secondary palate and the effects of all-trans and 13-cis RAs on the expression of RAR mRNAs by Northern blot analysis. RAR alpha (2.8, 3.8 kb), RAR beta (3.3 kb), and RAR gamma (3.7 kb) mRNAs were detected in the fetal palate on gestational days (GD) 12.5-14.5. The expression of RAR alpha and gamma mRNAs did not show apparent sequential changes, but that of RAR beta mRNA increased at GD 13.5. Treatment of pregnant mice with 100 mg/kg all-trans RA induced CP in 94% of the fetuses and elevated the levels of RAR beta and gamma mRNAs in the fetal palate. The up-regulation of RAR beta mRNA by all-trans RA was more marked than that of RAR gamma mRNA. Treatment with 100 mg/kg 13-cis RA induced CP in only 19% of the fetuses. Although 13-cis RA elevated the RAR beta and gamma mRNA levels in fetal palates, its up-regulation was slower and less marked than that induced by all-trans RA. These findings indicate that the induction of RAR beta mRNA in the fetal palate correlates well with the tissue concentration of all-trans RA after RA treatment, and RAR beta may be one of the most influential candidate molecules for RA-induced teratogenesis.     

Retinoic acid receptor isoform RAR gamma 1: an antagonist of the transactivation of the RAR beta RARE in epithelial cell lines and normal human keratinocytes.

The diversity of isoforms of retinoic acid (RA) receptors (RARs) and of DNA sequences of retinoic acid-responsive elements (RAREs) suggests the existence of selectivities in the RAR/RARE recognition or in the subsequent gene modulation. Such selectivities might be particularly important for RAREs involved in positive feedback, eg. the RAR beta RARE. In the present work we found that in several epithelial cell lines, reporter constructs containing the RAR beta RARE linked to the HSV-tk promoter were transactivated in the presence of RA by endogenous RARs and co-transfected RAR alpha 1 and RAR beta 2 isoforms, but not by RAR gamam 1. On the contrary, this latter isoform behaved towards the RAR beta RARE as an inhibitor of the transactivation produced by endogenous RARs and by cotransfected RAR alpha 1 and RAR beta 2. RAR gamma 1 also behaved as an antagonist of the transactivation produced by cotransfected RXR alpha. The natural RAR beta gene promoter or RAR beta RARE tk constructs were not activated by the endogenous receptors of normal human keratinocytes (NHK), which are known to contain predominantly RAR gamma 1. It was, however, possible to activate to a certain extent RAR beta RARE-reporter constructs in NHK by co-transfecting RAR alpha 1, RAR beta 2 or RXR alpha. The antagonist behavior of RAR gamma 1 towards the RAR beta RARE may explain why in certain cell types such as keratinocytes, RAR beta is neither expressed nor induced by RA.     

Targeted disruption of retinoic acid receptor alpha (RAR alpha) and RAR gamma results in receptor-specific alterations in retinoic acid-mediated differentiation and retinoic acid metabolism.

F9 embryonic teratocarcinoma stem cells differentiate into an epithelial cell type called extraembryonic endoderm when treated with retinoic acid (RA), a derivative of retinol (vitamin A). This differentiation is presumably mediated through the actions of retinoid receptors, the RARs and RXRs. To delineate the functions of each of the different retinoid receptors in this model system, we have generated F9 cell lines in which both copies of either the RAR alpha gene or the RAR gamma gene are disrupted by homologous recombination. The absence of RAR alpha is associated with a reduction in the RA-induced expression of both the CRABP-II and Hoxb-1 (formerly 2.9) genes. The absence of RAR gamma is associated with a loss of the RA-inducible expression of the Hoxa-1 (formerly Hox-1.6), Hoxa-3 (formerly Hox-1.5), laminin B1, collagen IV (alpha 1), GATA-4, and BMP-2 genes. Furthermore, the loss of RAR gamma is associated with a reduction in the metabolism of all-trans-RA to more polar derivatives, while the loss of RAR alpha is associated with an increase in metabolism of RA relative to wild-type F9 cells. Thus, each of these RARs exhibits some specificity with respect to the regulation of differentiation-specific gene expression. These results provide an explanation for the expression of multiple RAR types within one cell type and suggest that each RAR has specific functions.     

Murine RARbeta4 displays reduced transactivation activity, lower affinity for retinoic acid, and no anti-AP1 activity

The biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARalpha, beta, and gamma) and retinoid X receptors (RXR alpha, beta, and gamma). Each of the RARs is expressed as four to seven different isoforms. Four isoforms of RAR beta (beta1, beta2, beta3, and beta4), which differ only in their N-terminal sequence (A domain) have been described. These RARbeta isoforms display a specific pattern of expression in developing and adult animals and are highly evolutionarily conserved suggesting that they mediate distinct cellular effects of vitamin A. Experiments were performed to examine directly the RA-binding activity, transactivation activity, and anti-AP1 activity of each of these four RARbeta isoforms. The results demonstrate that RARbeta1, beta2, and beta3 bind RA with a similar K(d) value, have a similar EC(50) value in RA-dependent transactivation assays and inhibit AP1 activity to a similar level. By contrast, RARbeta4 has an elevated K(d) for RA, an increased EC(50) value in RA-dependent transactivation assays and does not display the ability to inhibit AP1 activity. This provides additional evidence that at least one RAR isoform, RARbeta4, may mediate distinct activities within a cell. Furthermore, these data suggest that the presence of an A domain in RARbeta is important for modulating these activities of RARs.     

Unliganded T3R, but not its oncogenic variant, v-erbA, suppresses RAR-dependent transactivation by titrating out RXR.

V-erbA is thought to be an antagonist of thyroid hormone receptor (T3R) function. Here we show that unliganded T3R, but not v-erbA, suppresses retinoic acid (RA)-dependent induction of the RAR-beta 2 promoter by competing for the common dimerization partner, the retinoid X receptor (RXR). Firstly, T3R suppression can be alleviated by co-transfection of RXR. Secondly, T3R, but not v-erbA, competes with RAR for RXR and causes the dissociation of a preformed RAR/RXR-RARE ternary complex in vitro. A single point mutation located in the dimerization interface of v-erbA (Pro349 to Ser) abolishes the transdominant phenotype when introduced at the respective position in T3R. The hypertransforming v-erbA variant r12, in which this mutation is reversed (Ser349 to Pro) suppresses RA-induced differentiation in chicken erythroid progenitors, while v-erbA does not. Our data thus suggest that unliganded T3R and v-erbA act as dominant suppressors through mechanistically distinct pathways.     

Retinoid receptors involved in the effects of retinoic acid on rat testis development

We have previously shown that retinoic acid (RA) is able to act on the development of Leydig, Sertoli, and germ cells in the testis in culture (Livera et al., Biol Reprod 2000; 62:1303-1314). To identify which receptors mediate these effects, we have now added selective agonists and antagonists of retinoic acid receptors (RARs) or retinoid X receptors (RXRs) in the same organotypic culture system. The RAR alpha agonist mimicked most of the effects of RA on the cultured fetal or neonatal testis, whereas the RAR beta, gamma, and pan RXR agonists did not. The RAR alpha agonist decreased the testosterone production, the number of gonocytes, and the cAMP response to FSH of fetal testis explanted at 14.5 days postconception (dpc). The RAR alpha agonist disorganized the cords of the 14.5-dpc cultured testis and increased the cord diameter in cultured 3-days-postpartum (dpp) testis in the same way as RA. All these RA effects could be reversed by an RAR alpha antagonist and were unchanged by an RAR beta/gamma antagonist. The RAR beta agonist, however, increased Sertoli cell proliferation in the 3-dpp testis in the same way as RA, and this effect was blocked by an RAR beta antagonist. The RAR gamma and the pan RXR agonists had no selective effect. These results suggest that all the effects of RA on development of the fetal and neonatal testis are mediated via RAR alpha, except for its effect on Sertoli cell proliferation, which involves RAR beta.     

Ligand specificities of recombinant retinoic acid receptors RAR alpha and RAR beta.

Binding of retinoic acid (RA) to specific RA receptors alpha and beta (RAR alpha and RAR beta) was studied. Receptors were obtained in two ways: (1) full-length receptors were produced by transient expression of the respective human cDNAs in COS 1 cells; and (2) the ligand-binding domains of RAR alpha and RAR beta were produced in Escherichia coli. RA binding to the wild-type and truncated forms of the receptor was identical for both RAR alpha and RAR beta, indicating that the ligand-binding domains have retained the binding characteristics of the intact receptors. Furthermore, RA bound with the same affinity to both RAR alpha and RAR beta. Only retinoid analogues with an acidic end-group were able to actively bind to both receptors. On measuring the binding of various retinoids, we have found that the properties of the ligand-binding sites of RAR alpha and RAR beta were rather similar. Two retinoid analogues were capable of binding preferentially to either RAR alpha or RAR beta, suggesting that it may be possible to synthesize specific ligands for RAR alpha and RAR beta.     

Distinct stages in adipogenesis revealed by retinoid inhibition of differentiation after induction of PPARgamma.

Retinoic acid (RA) inhibits adipocyte differentiation of 3T3-L1 preadipocytes but is effective only early in adipogenesis. RA prevented induction of the adipogenic factors PPARgamma and C/EBPalpha. Using receptor-specific ligands, we determined that the effects of RA were mediated by liganded RA receptors (RARs) rather than retinoid X receptors. Preadipocytes expressed primarily RARalpha and RARgamma; during adipocyte differentiation, RARalpha gene expression was nearly constant, whereas RARgamma1 mRNA and protein levels dramatically decreased. Ectopic expression of RARgamma1 extended the period of effectiveness of RA by 24 to 48h; RARalpha expression had a similar effect, suggesting functional redundancy of RAR subtypes. Remarkably, RA inhibited differentiation when added after PPARgamma1 and PPARgamma2 proteins had already been expressed and resulted in the loss of PPARgamma proteins from cells. By 72 to 96 h after the induction of differentiation, RA failed to prevent differentiation of even ectopic-RAR-expressing cells. Thus, the unresponsiveness of 3T3-L1 preadipocytes to RA after the induction of differentiation is initially due to the reduction in cellular RAR concentration rather than to the induction of PPARgamma. At later times cells continue along the differentiation pathway in a manner which is RA and RAR independent.