scholarly journals Retinoic acid receptor gamma: specific immunodetection and phosphorylation.

1991 ◽  
Vol 115 (2) ◽  
pp. 535-545 ◽  
Author(s):  
C Rochette-Egly ◽  
Y Lutz ◽  
M Saunders ◽  
I Scheuer ◽  
M P Gaub ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Polyclonal Antibodies ◽  
Retinoic Acid Receptor ◽  
Amino Acid Sequences ◽  
Expression Vectors ◽  
Acid Receptor ◽  
F Region ◽  
Nuclear Extracts ◽  
F9 Embryonal Carcinoma Cells ◽  
Human And Mouse

Synthetic peptides corresponding to cDNA-deduced amino acid sequences unique to the human and mouse retinoic acid receptor gamma 1 (hRAR-gamma 1 and mRAR-gamma 1, respectively) were used to generate anti-RAR-gamma 1 antibodies. Four mAbs were selected, which were directed against peptides found in region A1 (Ab1 gamma (A1)), region F (Ab2 gamma (mF) and Ab4 gamma (hF)) and region D2 (Ab5 gamma (D2)). These antibodies specifically immunoprecipitated and recognized by Western blotting RAR-gamma 1 proteins in COS-1 cells transfected with expression vectors containing the RAR-gamma 1 cDNAs. They all reacted with both human and mouse RAR-gamma 1 proteins, except Ab4 gamma (hF) that was specific for hRAR-gamma 1. Rabbit polyclonal antibodies, directed against a peptide from the mRAR-gamma 1 F region were also obtained (RP gamma (mF)) and found to be specific for mouse RAR-gamma 1 protein. Furthermore, in gel retardation/shift assays the antibodies specifically retarded the migration of complexes obtained with a RA response element (RARE). Antibodies raised against regions D2 and F also recognized the RAR-gamma 2 isoform which differs from RAR-gamma 1 only in the A region. On the other hand, antibodies directed against the A1 region of RAR-gamma 1 (Ab1 gamma (A1)) only reacted with the RAR-gamma 1 protein. The antibodies characterized here allowed us to detect the presence of mRAR-gamma 1 and gamma 2 isoforms in mouse embryos and F9 embryonal carcinoma cells nuclear extracts. They were also used to demonstrate that the mRAR-gamma 1 protein can be phosphorylated and that the phosphorylation occurs mainly in the NH2-terminal A/B region.

scholarly journals An RPE cell line as a useful in vitro model for studying retinoic acid receptor β: expression and affinity

2008 ◽  
Vol 28 (6) ◽  
pp. 327-334 ◽  
Author(s):  
Barbara Pavan ◽  
Alessandro Dalpiaz ◽  
Carla Biondi ◽  
Marzia Nieddu ◽  
Antonella De Luca ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Retinoic Acid Receptor ◽  
Retinal Pigment ◽  
Nuclear Extract ◽  
Retinal Pigment Epithelial Cells ◽  
Receptor Subtypes ◽  
Ligand Interaction ◽  
Acid Receptor ◽  
Nuclear Extracts

Retinoids mediate their biological effect by interacting with specific nuclear receptors. Of the several known RAR (retinoic acid receptor) subtypes, RAR-β is of particular interest, since its expression is silenced in many cancers and it is believed to be a tumour suppressor. Specific ligands of RAR-β can potentially be used in anti-cancer therapy. In the present study, we have investigated the feasibility of using HRPE cells (human retinal pigment epithelial cells) as an experimental model for characterizing RAR-β–ligand interaction. RT–PCR (reverse transcription–PCR) and Western blot analyses show that HRPE cells specifically express only RAR-β and none of the other receptor subtypes. In addition, we show that the expression of RAR-β increases with increasing passage number of the cells. Interestingly, the increase in RAR-β expression is not associated with telomere shortening, a typical biomarker of cellular senescence. In the present study, we also describe a protocol for characterizing RAR-β–ligand interactions using nuclear extract from late passage HRPE cells as a source of endogenous RAR-β. Using [3H]CD367 as the ligand, RAR-β in HRPE cells showed an affinity of 9.6±0.6 nM and a Bmax of 780±14 fmol/mg of protein. We have confirmed the feasibility of using this assay to detect the interaction of ligands with RAR-β by investigating the ability of certain flavonoids to inhibit the binding of [3H]CD367 to nuclear extracts from HRPE cells. The inhibition constant of the flavonoids for RAR-β was between approx. 1–30 μM, showing that the flavonoids interact with RAR-β with low affinity.

scholarly journals Positive and negative regulation of the composite octamer motif of the interleukin 2 enhancer by AP-1, Oct-2, and retinoic acid receptor.

1994 ◽  
Vol 180 (4) ◽  
pp. 1485-1497 ◽  
Author(s):  
U de Grazia ◽  
M P Felli ◽  
A Vacca ◽  
A R Farina ◽  
M Maroder ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Retinoic Acid Receptor ◽  
Interleukin 2 ◽  
Expression Vectors ◽  
Regulatory Function ◽  
Acid Receptor ◽  
Cis Element ◽  
Octamer Motif

The differentiating agent retinoic acid (RA) has been previously reported to interfere with 12-O-tetradecanoyl-phorbol-13-acetate (TPA)/Ca2+-induced signals for the regulation of the -96 to -66-bp octamer motif found in the enhancer for the interleukin (IL)-2 gene, which encodes a major T lymphocyte growth factor. The IL-2 octamer motif is a composite cis-element which binds Oct-1 and Oct-2 as well as a TPA/Ca2+-inducible nuclear factor, previously termed octamer-associated protein (OAP40). We show here that Oct-2, despite the presence of an active transcriptional activation domain, requires TPA/Ca2+-induced signals to strongly transactivate the IL-2 octamer motif in Jurkat T cells. This Oct-2-dependent transactivation is inhibited by RA. The presence of an intact COOH-terminal domain of Oct-2 contributes to both TPA/Ca2+-induced transactivation and the RA-mediated repression. We also show that both Fos and Jun components of the AP-1 factors participate in the OAP40 complex. Furthermore, transfected c-jun, jun-B, jun-D, c-fos, or Fos-B expression vectors partially substitute for TPA and Ca2+ and cooperate with Oct-2 for the transactivation of the combined OAP/octamer cis-element. Mutations of the genuine octamer-binding site abrogate both the binding of Oct-1 and Oct-2 and the TPA/Ca2+-induced transactivation of the OAP/octamer motif. OAP confers to Oct-2 responsivity to both TPA/Ca2+ and RA, since specific mutations of the AP-1/OAP-binding site significantly reduce the transactivation by Oct-2 in response to TPA and Ca2+ and abolish the inhibition by RA. Furthermore, retinoic acid receptor (RAR) alpha is able to inhibit in vitro the formation of the complex between the nuclear AP-1/OAP and its specific binding site, resulting in the interference with Oct-2-dependent cis-regulatory function of this AP-1 element. Therefore, we propose that the TPA/calcium-activated AP-1/OAP element is the main target of positive or negative regulatory signals influencing the IL-2 octamer motif, through synergism with Oct-2 and antagonism by RAR.

Contribution of retinoic acid receptor signalling to adrenal cortex morphology and functional zonation through modulation of WNT/[beta]-catenin pathway

2017 ◽  
Author(s):  
Zein Rami El ◽  
Amanda J Rickard ◽  
Golib Dzib Jose Felipe ◽  
Benoit Samson-Couterie ◽  
Angelique Rocha ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Adrenal Cortex ◽  
Retinoic Acid Receptor ◽  
Beta Catenin ◽  
Acid Receptor ◽  
Receptor Signalling
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