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At least 19 recordsLinked to original sources
PubMed1997

One-step immunoaffinity purification of recombinant human retinoic acid receptor gamma.

Retinoic acid receptors (RAR) are members of the steroid/thyroid hormone receptor superfamily and serve as ligand-activated transcription factors. In order to facilitate studies of receptor protein, we have generated a monoclonal antibody to the human RAR gamma, and have developed a procedure to purify the full-length receptor expressed in insect cells. The monoclonal antibody (A10) was developed using as antigen a carboxy-terminal fragment of the human RAR gamma expressed as a bacterial fusion protein. The A10 monoclonal antibody binds to both native and denatured forms of the human RAR gamma. This antibody was immobilized on a resin and used to purify full-length, baculovirus-expressed human RAR gamma to near homogeneity. The immunoaffinity-purified receptor is > 90-95% pure as revealed by silver-stained gels. The identity of the single protein band as RAR gamma was verified by immunoblotting using a polyclonal antibody to an epitope distinct from that recognized by the A10 antibody. The pure human RAR gamma is functional with respect to both ligand and DNA binding. Scatchard analysis of 3H-labeled all-trans retinoic acid binding to purified human RAR gamma revealed a single, high-affinity binding site with a Kd of approximately 2 nM. Binding of the pure RAR gamma to a DR5-type retinoic acid response element was also studied. Response element binding by RAR gamma required the presence of the retinoid X receptor, but did not require the presence of additional proteins. Human RAR gamma protein purified in this fashion will be useful in future structural and functional studies.

Animals↗
PubMed1994

Binding of 9-cis-retinoic acid and all-trans-retinoic acid to retinoic acid receptors alpha, beta, and gamma. Retinoic acid receptor gamma binds all-trans-retinoic acid preferentially over 9-cis-retinoic acid.

Both 9-cis-retinoic acid (RA) and all-trans-RA (t-RA) compete for [3H]9-cis-RA binding to RA receptors (RAR alpha, beta, and gamma) in nucleosol fractions from transiently transfected COS-1 cells with IC50 values of approximately 12 and 5 nM, respectively. Curiously, 9-cis-RA competes for [3H]t-RA binding to mouse RAR alpha, beta, and gamma with IC50 values of 31, 8, and 60 nM, respectively, while t-RA itself does not exhibit such differential competition (IC50 values for RARs, 5 nM). A similar pattern is observed with human retinoic acid receptors (RARs). Differential binding of 9-cis-RA to the RAR beta and gamma receptors is also found following in vitro transcription and translation of these receptors. Displacement assays demonstrate that t-RA exhibits similar off-rates for RAR alpha, beta, and gamma. However, 9-cis-RA is 6-fold more rapidly displaced from RAR gamma than from RAR beta. When RAR-transfected COS-1 cells are incubated with [3H]t-RA, [3H]-9-cis-RA or various mixtures of these two radioligands, high performance liquid chromatography analysis demonstrates that the ligands bound in nucleosol fractions from RAR beta-transfected cells reflect the isomer content of the media. However, in identical whole cell assays, nucleosol fractions from RAR gamma-transfected cells preferentially bind t-RA over 9-cis-RA, consistent with the in vitro data. These binding kinetics in vitro and in whole cells suggest that there could be differences in the interactions of the receptor subtypes with the endogenous retinoic acids under physiologic conditions.

Animals↗
PubMed1994

Characterization of cDNAs encoding the chick retinoic acid receptor gamma 2 and preferential distribution of retinoic acid receptor gamma transcripts during chick skin development.

Retinoic acid receptors alpha, beta and gamma (RAR alpha, beta and gamma) are ligand-inductible transcriptional activators which belong to the steroid/thyroid hormone receptor superfamily. At least two major isoforms (1 and 2) of each RAR arise by differential use of two promoters and alternative splicing. In mouse, the three RAR genes are expressed in stage- and tissue-specific patterns during embryonic development. In order to understand the role of the different RARs in chick, RAR gamma 2 cDNAs were isolated from an 8.5-day (stage 35 of Hamburger and Hamilton) chick embryo skin library. The deduced chick RAR gamma 2 amino acid sequence displays uncommon features such as 21 specific amino acid replacements, 12 of them being clustered in the amino-terminal region (domains A2 and B), and a truncated acidic carboxy-terminal region (F domain). However, the pattern of RAR gamma expression in chick embryo resembles that reported in mouse, particularly in skin where RAR gamma expression occurs in both the dermal and epidermal layers at the beginning of feather formation, and is subsequently restricted to the differentiating epidermal cells. Northern blot analysis suggests that different RAR gamma isoforms could be successively required during chick development.

Amino Acid Sequence↗
PubMed1992

Patterned distributions of chondroitin sulfate isoforms, retinoic acid receptor gamma and cellular retinoic acid binding proteins in the embryonic mouse incisor.

Odontogenesis is an example of developmental process whereby pluripotential embryonic ectodermal and ectomesenchymal cells give rise to specific, highly ordered, spatial arrangements of overtly differentiated tissues. This process implies the sequential and coordinated expression of several molecules of defined biological activities. The data presented here demonstrate the occurrence, in the embryonic mouse incisor, of a striking correspondence between gradients of mitoses, chondroitin 6-sulfate distribution, retinoic acid receptor gamma and cellular retinoic acid binding protein I transcripts. Although purely phenomenological, these observations provide an important guide to the design of experiments for understanding the mechanisms of pattern formation during tooth development.

Animals↗
PubMed2001

Expression of retinoic acid receptor gamma correlates with retinoic acid sensitivity and metabolism in head and neck squamous cell carcinoma cell lines.

Retinoids, analogues of vitamin A, can reverse premalignant lesions and prevent second primary tumors in patients with head and neck squamous cell carcinoma (HNSCC). The effects of retinoids are mediated by retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which act as ligand-activated transcription factors. The regulation of cell growth, differentiation and retinoid metabolism in normal, premalignant and malignant cells by retinoids is thought to be a result of their effects on gene expression. We investigated mRNA expression of RARs (alpha, beta, and gamma) and RXR-beta by means of RNase protection and related this to retinoic acid (RA)-induced growth inhibition and RA turnover in four HNSCC cell lines (UM-SCC-14C, UM-SCC-22A, UM-SCC-35 and VU-SCC-OE). An RA-resistant subline of UM-SCC-35 was generated by exposure to increasing concentrations of RA for 8 months (designated UM-SCC-35R). RA turnover was determined on the basis of decreasing RA levels in the cells and culture medium after exposure to 1 microM RA. We found that RAR-gamma mRNA expression was strongly correlated with RA-induced growth inhibition (p = 0.016, R = 0.92) and RA turnover (p = 0.041, R = 0.86). RAR-beta transcript levels were reduced in three of five cell lines compared with normal mucosa, and these did not correlate with RA-induced growth inhibition and RA turnover. Expression of RAR-alpha and RXR-beta was not substantially altered in any of the cell lines. These findings suggest that in HNSCC cell lines RAR-gamma is the most important retinoid receptor for regulation of RA turnover rate and RA-induced growth inhibition.

Carcinoma, Squamous Cell↗
PubMed1991

Genomic organization of the retinoic acid receptor gamma gene.

The retinoic acid receptors (RAR) belong to the large family of ligand responsive gene regulatory proteins that includes receptors for steroid and thyroid hormones. These proteins contain two highly conserved domains, involved in determining their DNA and ligand binding activities. Three distinct RARs have been identified (RAR alpha, beta, and gamma) which are encoded by genes on separate chromosomes. Additional isoforms of the three receptors have been described that all differ in the N-terminal regions. To gain insight into the genomic organization and mechanisms of RAR isoform generation, we have analyzed the genomic structure of the RAR gamma gene. The major portion of the RAR gamma protein, including DNA and ligand binding domains, is encoded by seven exons that are identical for all RARg isoforms and are represented by a relatively small portion of the RAR gamma gene. The major portion of this gene encodes separate N-terminal exons for gamma 1 and gamma 2 isoforms and several exons for gamma 1 untranslated regions. We show that RAR gamma 2 transcription is regulated by its own promoter. In comparison with the steroid receptor subfamily, various splice sites of RAR gamma occur at altered positions, suggesting that the RAR subfamily has diverged early during evolution.

Amino Acid Sequence↗
PubMed2002

Down-regulation of the phosphatidylinositol 3-kinase/Akt pathway is involved in retinoic acid-induced phosphorylation, degradation, and transcriptional activity of retinoic acid receptor gamma 2.

Nuclear retinoic acid (RA) receptors (RARs) are phosphorylated at conserved serine residues located in their N-terminal domain. Phosphorylation of RARgamma2 at these residues is increased in response to RA subsequently to the activation of p38MAPK. We show here that this RA-induced phosphorylation of RARgamma2 resulted from the down-regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. By overexpressing Akt and by using agents that activated or inhibited the PI3K/Akt pathway, we also demonstrated that the RA-induced down-regulation of the PI3K/Akt pathway targeted not only the phosphorylation of RARgamma2 but also the turnover and transcriptional activity of the receptor. Altogether these data indicate that the PI3K/Akt pathway plays an important role in retinoic acid signaling.

Animals↗
PubMed2002

Mass-spectrometric analysis of agonist-induced retinoic acid receptor gamma conformational change.

Apo and holo forms of retinoic acid receptors, and other nuclear receptors, display differential sensitivity to proteolytic digestion that likely reflects the distinct conformational states of the free and liganded forms of the receptor. We have developed a method for rapid peptide mapping of holo-retinoic acid receptor gamma that utilizes matrix-assisted laser-desorption-ionization time-of-flight MS to identify peptide fragments that are derived from the partially proteolysed holo-receptor. The peptide maps of retinoic acid receptor gamma bound by four different agonists were identical, suggesting that all four ligands induced a similar conformational change within the ligand-binding domain of the receptor. In all cases, this agonist-induced conformational change promoted the direct association of retinoic acid receptor gamma with the transcriptional co-activator p300 and inhibited interaction of the receptor with the nuclear receptor co-repressor. SR11253, a compound previously reported to exert mixed retinoic acid receptor gamma agonist/antagonist activities in cultured cells, was found to bind directly to, but only weakly altered the protease-sensitivity of, the receptor and failed to promote interaction of the receptor with p300 or induce dissociation of receptor-nuclear receptor co-repressor complexes. This technique should be generally applicable to other members of the nuclear receptor superfamily that undergo an induced structural alteration upon agonist or antagonist binding, DNA binding and/or protein-protein interaction.

Amino Acid Sequence↗
PubMed1990

Specific spatial and temporal distribution of retinoic acid receptor gamma transcripts during mouse embryogenesis.

Retinoic acid (RA), a putative morphogen in vertebrates, has profound effects on development during embryogenesis, chondrogenesis and differentiation of squamous epithelia. The distribution of the transcripts of the retinoic acid receptor gamma (RAR-gamma) gene has been studied here by in situ hybridization during mouse development from days 6.5 to 15.5 post-coïtum (p.c.). RAR-gamma transcripts are detected as early as day 8 p.c. in the presomitic posterior region. Between days 9.5 and 11.5 p.c., the transcripts are uniformly distributed in the mesenchyme of the frontonasal region, pharyngeal arches, limb buds and sclerotomes. At day 12.5 p.c., RAR-gamma transcripts are found in all precartilaginous mesenchymal condensations. From day 13.5 p.c., the transcripts are specifically localized in all cartilages and differentiating squamous keratinizing epithelia, irrespective of their embryological origin. RAR-gamma transcripts are also found in the developing teeth and whisker follicles. The developmental pattern of expression of the RAR-gamma gene suggests that RAR-gamma plays a crucial role for transducing RA signals at the level of gene expression during morphogenesis, chondrogenesis and differentiation of squamous epithelia.

Animals↗
PubMed1996

Implication of retinoic acid receptor gamma in squamous differentiation and response to retinoic acid in head and neck SqCC/Y1 squamous carcinoma cells.

Nuclear retinoic acid receptors are considered to be the mediators of most of the effects of retinoic acid (RA) on gene expression. To explore the role of RA receptor gamma (RARgamma) in the growth and differentiation of SqCC/Y1 head and neck squamous carcinoma cells, they were transfected with RARgamma sense and antisense expression vectors and stable clones in which RARgamma expression was either increased or blocked were isolated. The growth inhibitory effect of RA in monolayer culture was enhanced in the sense transfectants and decreased in the antisense ones. The ability to form colonies in semisolid medium was abolished by RA in the sense transfectants, while the antisense transfected clones exhibited heterogeneous responses. The expression the squamous differentiation markers cytokeratin K1 transglutaminase type I, and involucrin was increased in the absence of exogenous retinoid in a sense transfected clone and decreased in an antisense transfected clone. RA suppressed squamous differentiation in both types of transfectant. The expression of epidermal growth factor receptor (EGFR) was higher in the antisense and lower in the sense transfectant than in the parental cells and RA decreased EGFR mRNA level in the parental and the sense transfectant but not in the antisense transfectant. In addition activator protein-1 (AP-1) binding activity was decreased by the RA treatment in the sense clones, but not in the antisense ones. These results suggest that RARgamma mediates the effects of RA on the cell growth both in monolayer culture and in semisolid medium possibly through AP-1 suppression.

Base Sequence↗
PubMed1993

Function of retinoic acid receptor gamma in the mouse.

Null mutant mice for retinoic acid receptor gamma 2 (RAR gamma 2) or all RAR gamma isoforms were generated. RAR gamma 2 mutants appeared normal, whereas RAR gamma mutants exhibited growth deficiency, early lethality, and male sterility due to squamous metaplasia of the seminal vesicles and prostate. These defects were previously observed in vitamin A-deficient animals and could be prevented by RA administration, demonstrating that RAR gamma mediates some of the retinoid signal in vivo. Congenital defects included Harderian gland agenesis, tracheal cartilage malformations, and homeotic transformations along the rostral axial skeleton, establishing a direct link between RA and patterning of the axial skeleton. We also show that in utero RA-induced lumbosacral truncations are mediated by RAR gamma. The observed RAR gamma null phenotype suggests a high degree of functional redundancy among the RARs. The variable penetrance of some of the observed defects is discussed in light of this redundancy and stochastic variation of gene activity.

Abnormalities, Multiple↗
PubMed1998

Arg278, but not Lys229 or Lys236, plays an important role in the binding of retinoic acid by retinoic acid receptor gamma.

The diverse 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). Although the ligand-binding domains of RARs share the same novel folding pattern, many RAR subtype-specific retinoids have been synthesized indicating that the ligand-binding pocket of each RAR subtype has unique features. Previously we have demonstrated the importance for RA binding and RA-dependent transactivation of Arg276 of RARalpha alone and in RARbeta Arg269 in conjunction with Lys220. In this study, we have examined the role of the homologous amino acid residues (Lys229 and Arg278) in RARgamma for these activities. Like RARalpha but dissimilar to RARbeta, Arg278 in RARgamma alone was found to play an important role in RA binding and RA-dependent transactivation. Since Lys236 in RARgamma was suggested from the crystal structure of holo-RARgamma to interact with RA, we also examined its role and that of its homologs in RARalpha and RARbeta. Despite the suggestion from the crystal structure, neither Lys236 nor its homologs in RARalpha and RARbeta play a role in the binding of RA or RA-dependent transactivation. It is likely that Lys236 in RARgamma and its homologs in RARalpha and RARbeta are solvent exposed rather than pointing into the RA-binding pocket.

Amino Acid Substitution↗
PubMed2000

TFIIH interacts with the retinoic acid receptor gamma and phosphorylates its AF-1-activating domain through cdk7.

Retinoic acid receptor gamma (RARgamma) is phosphorylated in COS-1 cells at two conserved serine residues located in the N-terminal region (serines 77 and 79 in RARgamma1 and serines 66 and 68 in RARgamma2) that contains the activation function AF-1. These serines are phosphorylated in vitro by cdk7, a cyclin-dependent kinase associated to cyclin H and MAT1 in the CAK complex (cdk7.cyclin H. MAT1), that is found either free or as a component of the transcription/DNA repair factor TFIIH. RARgamma is more efficiently phosphorylated by TFIIH than by CAK and interacts not only with cdk7 but also with several additional subunits of TFIIH. RARgamma phosphorylation and interaction with TFIIH occur in a ligand-independent manner. Our data demonstrate also that phosphorylation of the AF-1 function modulates RARgamma transcriptional activity in a response gene-dependent manner.

Animals↗
PubMed1995

Two isoforms of Xenopus retinoic acid receptor gamma 2 (B) exhibit differential expression and sensitivity to retinoic acid during embryogenesis.

We report the isolation of two retinoic acid receptor isoforms (RAR gamma), which differ only in the 5'untranslated and putative N-terminus A regions. The two isoforms appear to serve as early markers for the presumptive neural axis; however, their expression patterns differ. RAR-gamma 2.1 is first expressed at gastrulation at the dorsal lip and subsequently along the presumptive neural axis. RAR- gamma 2.2 represents the full-length sequence of a receptor cDNA already partially characterized and present as a maternal transcript [Ellinger-Ziegelbauer and Dreyer (1991); Genes Dev 5:94-104, (1993): Mech Dev 41:31-46; Pfeffer and DeRobertis, (1994) Mech Dev: 45:147-153]. Unlike RAR-gamma 2.2, the 2.1 variant is not expressed either in pre-somitic mesoderm or notochord. RAR-gamma 2.1 is strongly expressed in branchial arches and to a lesser extent in the neural floor plate. The two isoforms also exhibit differential sensitivity to retinoic acid. Constitutive expression of RAR gamma 2.2 following neurulation appears to be depressed by treatment with retinoic acid, but domains of highest expression, namely, the head and tail, remain relatively unaffected, as do patterns of expression prior to late neurulation. By contrast, RAR-gamma 2.1 is not transcribed in retinoid-inhibited structures. Using microinjection techniques, we show that changes of RAR-gamma 2.1 expression in presumptive head structures occur as an early and local consequence of retinoic acid administration. Since RAR-gamma 2.1 expression is inhibited by retinoic acid, we tested to see if other treatments that perturb axis formation had any effect. Surprisingly, UV irradiation did not suppress that its inhibition by retinoic acid is not due solely to inhibition of anterior neural development. These experiments demonstrate a new subdivision of isoforms that undergo differential expression during development and that exhibit differential sensitivity to retinoic acid and to UV. This sensitivity and the presence of this isoform variant in regions that are known to exhibit polarizing activity strengthen the hypothesis that these receptors play a primary role during morphogenesis.

Amino Acid Sequence↗
PubMed1997

Contribution of retinoic acid receptor gamma to retinoid-induced craniofacial and axial defects.

Exogenous retinoic acid (RA) administered during mouse embryogenesis can alter the pattern of the axial skeleton during two developmental periods: an early window (7 to 8.5 days post-coitum; dpc) and a late window (9.5 to 11.5 dpc). Treatment during the early window results in vertebral homeotic transformations (predominantly posteriorizations) concomitant with rostral shifts in Hox gene expression, while treatment at the later window results in similar transformations without detectable alterations in Hox gene expression patterns. Mice null for retinoic acid receptor gamma (RAR gamma) exhibit axial defects, including homeosis of several vertebrae, therefore establishing a role for this receptor in normal axial specification RAR gamma null mutants are also completely resistant to RA-induced spina bifida, which occurs in wildtype embryos treated at 8.5-9.0 dpc, suggesting that this receptor specifically transduces at least a subset of the teratogenic effects of retinoids. To further investigate the role of RAR gamma in RA-induced defects during the early and late windows of retinoid-sensitive vertebral patterning, RAR gamma heterozygotes were intercrossed, pregnant females treated with vehicle or RA at 7.3, 10.5 or 11.5 dpc and full-term fetuses assessed for skeletal defects. Relative to wildtype littermates, RAR gamma null mutants treated at 7.3 dpc were markedly resistant to RA-induced embryolethality, craniofacial malformations, and neural tube defects. Furthermore, while RAR gamma null mutants were modestly resistant to certain vertebral malformations elicited by RA treatment at 7.3, they exhibited more pronounced resistance following treatment at 10.5 and 11.5 dpc. Moreover, several of the vertebral defects inherent to the RAR gamma null phenotype were abolished by RA treatment specifically at 10.5 dpc, suggesting that RAR alpha and/or RAR beta isoforms may substitute for certain RAR gamma functions, and that RAR gamma may elicit its normal effects on vertebral morphogenesis at this developmental stage.

Animals↗
PubMed1993

Retinoic acid receptor gamma 2 gene expression is up-regulated by retinoic acid in 3T3-L1 preadipocytes.

Retinoids, especially all-trans retinoic acid (RA), have been shown to inhibit the differentiation of preadipose cells. In the present study, the expression of retinoic acid receptors (RAR alpha, beta and gamma) and retinoid X receptors (RXR alpha, beta and gamma) was examined by Northern blot analysis in rat adipose tissue and mouse 3T3-L1 adipose cells. The adipose tissue and/or 3T3-L1 cells expressed mRNAs for a number of nuclear retinoid receptors, including RAR alpha, beta and gamma, and RXR alpha, beta and gamma. RAR alpha, RAR gamma, RXR alpha and RXR beta mRNAs were abundant in adipose tissue and 3T3-L1 cells. RXR gamma mRNA was detected in adipose tissue but not in 3T3-L1 cells. Treatment of 3T3-L1 cells with 1 microM RA led to a 4-5-fold increase in the RAR gamma mRNA level, but only a trace amount of RAR beta mRNA was detected. RAR gamma mRNA expression was rapidly (within 2 h) induced by physiological concentrations of RA in a dose-dependent manner. The response of RAR gamma mRNA expression to RA was reversible; rapid disappearance of RAR gamma mRNA occurred on RA removal. In addition, the induction of RAR gamma expression did not require de novo protein synthesis, but was completely abolished by an inhibitor of RNA synthesis. Using RAR gamma 1 and gamma 2 isoform-specific probes, the patterns of RAR gamma 1 and gamma 2 mRNA expression in 3T3-L1 cells in the presence and absence of RA were examined. RAR gamma 1 mRNA was detected in 3T3-L1 cells but was not affected by RA treatment; however, RAR gamma 2 mRNA was strongly induced by RA.

3T3 Cells↗
PubMed1990

Identification of a novel isoform of the retinoic acid receptor gamma expressed in the mouse embryo.

Retinoic acid is known to have profound effects on developmental processes. It has been implicated as a putative morphogen in the developing chick limb bud and regenerating amphibian limb blastema and has been demonstrated to have powerful teratogenic effects in mammals, including humans. Recently, three specific retinoic acid receptors (RARs), RAR alpha, -beta, and -gamma, were identified and shown to be members of the steroid receptor superfamily. We report the identification of a novel RAR gamma isoform, mRAR gamma B, which differs from the previously described mouse RAR gamma at its amino terminus. In addition, we show that both RAR gamma isoforms are expressed maximally at midgestation in structures known to be affected adversely by retinoic acid administration to pregnant mice. Multiple RAR isoforms, each of which may play a unique or combinatorial role as a regulator of mammalian development, are thus expressed in the mouse embryo.

Amino Acid Sequence↗