Physicochemical Parameters Affecting the Charcoal Adsorption Assay for Quantitative Retinoid-Binding Measurement

Sablonnière, Bernard; Dallery, Nathalie; Grillier, I; Formstecher, Pierre; Dautrevaux, M

doi:10.1006/abio.1994.1090

Cited by 9 publications

(5 citation statements)

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“…The specific binding observed for [ 3 H]9-cis retinoic acid was 133 + − 9 fmol/mg of protein (results not shown). Taking into account that CD367 is a relatively stable ligand and more specific to the RAR subtype than [ 3 H]9-cis-retinoic acid [13], we have therefore chosen [ 3 H]CD367 to characterize the binding properties of nuclear RARs. 9-Cis-retinoic acid is known to be a high-affinity ligand of both RAR and RXR subtypes [32,33]; however RT-PCR and Western blotting results reported above show that HRPE cells selectively express the RAR-β subtype.…”

Section: In Vitro Binding Assaymentioning

confidence: 99%

“…Similar observations were made by Lee et al [12], in aging fibroblasts with decreased proliferative capacity, who observed a selective up-regulation of RAR-β expression in response to retinoic acid and its derivatives in these cells. However, the accuracy and reproducibility of different methods for studying the binding affinities of specific compounds to RARs can be compromised by several factors, which can be summarized as: (i) a low abundance of RARs in natural cells and tissues, resulting in a low ratio of specific compared with non-specific binding sites; (ii) the chemical instability of the natural ligand, retinoic acid; and (iii) the difficulty of designing a perfect method to separate free from bound ligand [13]. Indeed, RAR-β expression is up-regulated with serial passage in senescent normal mammary epithelial cells, whereas it is down-regulated in mammary cancer cell lines [1].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2008

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Retinoids mediate their biological effect by interacting with specific nuclear receptors. Of the several known RAR (retinoic acid receptor) subtypes, RAR-beta 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-beta 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-beta-ligand interaction. RT-PCR (reverse transcription-PCR) and Western blot analyses show that HRPE cells specifically express only RAR-beta and none of the other receptor subtypes. In addition, we show that the expression of RAR-beta increases with increasing passage number of the cells. Interestingly, the increase in RAR-beta 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-beta-ligand interactions using nuclear extract from late passage HRPE cells as a source of endogenous RAR-beta. Using [(3)H]CD367 as the ligand, RAR-beta in HRPE cells showed an affinity of 9.6 +/- 0.6 nM and a B(max) of 780 +/- 14 fmol/mg of protein. We have confirmed the feasibility of using this assay to detect the interaction of ligands with RAR-beta by investigating the ability of certain flavonoids to inhibit the binding of [(3)H]CD367 to nuclear extracts from HRPE cells. The inhibition constant of the flavonoids for RAR-beta was between approx. 1-30 microM, showing that the flavonoids interact with RAR-beta with low affinity.

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Section: In Vitro Binding Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2008

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“…Nonspecific binding was determined in the presence of 100-fold molar excess unlabeled RA. Bound RA was separated from free by charcoal-dextran adsorption as described (36). Specific RA binding was determined by subtracting the nonspecific binding from the total binding.…”

Section: Methodsmentioning

confidence: 99%

Critical Role of Tyrosine 277 in the Ligand-Binding and Transactivating Properties of Retinoic Acid Receptor α

et al. 2000

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Retinoic acid receptors specifically bind all-trans-retinoic acid (RA) and function as RA-inducible transcriptional regulatory factors. Binding of RA to RARalpha, beta, and gamma is sensitive to nitration with tetranitromethane, a tyrosine-specific modifying reagent. To identify tyrosine residue(s) that are important for RA binding, we carried out chemical modification experiments with purified RARalpha ligand-binding domain (RARalpha-LBD) subjected to partial acid hydrolysis and selective proteolysis. The chemically modified peptides containing each of the three Tyr residues present in the RARalpha-LBD sequence were then analyzed and identified by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC/ESI-MS). We found that RA binding to RARalpha-LBD protected Tyr(277)-containing peptides from nitration. Protection of Tyr(277) could result either from direct masking by the bound ligand or from ligand-induced changes in receptor conformation and tyrosine accessibility. The role of Tyr residues was further documented by site directed mutagenesis using three site-specific RARalpha mutants: Y208A, Y277A, and Y362A. The affinity for RA of these mutant receptors was in the range of that of the wild-type protein, except for the Y277A receptor mutant, which displays a 15-20-fold reduction in affinity and transactivation activity for RA. Whereas mutation of Tyr(277) into alanine had a variable effect on different agonists and antagonists binding, it caused a dramatic decrease of retinoid-dependent transactivation activity. This later effect was also observed with mutation of Tyr(277) into phenylalanine. It is unlikely that major conformational changes are responsible for the lower affinity of RA binding and RA-dependent transactivation since these mutants displayed wild-type dimerization and DNA-binding activities. Limited proteolysis revealed that upon ligand binding, the Y277A mutant induced a conformational change slightly different from that obtained with the wild-type protein. These data could suggest that Tyr(277) play a critical role in the ligand-induced conformational changes required for the activation of RARalpha.

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“…An assay of [ 3 H]RA binding to M6P͞IGF-II receptor was performed as described (27), with some modifications. Briefly, partially purified M6P͞IGF-II receptors from neonatal rat serum [by a fast protein liquid chromatography system using a Sepharose-12 column (Pharmacia)] were diluted in binding buffer [20 Assay of ␤-Glucuronidase Binding and Endocytosis.…”

Section: Methodsmentioning

confidence: 99%

“…To determine the affinity of the binding of [ to partially purified rat M6P͞IGF-II receptor proteins using the dextran-charcoal absorption technique (27). Scatchard analysis of the binding data revealed a single class of highaffinity binding sites for RA, with a K D of 2.5 Ϯ 0.3 nM (n ϭ 3; Fig.…”

Section: Ra Binds To the M6p͞igf-ii Receptor With High Affinitymentioning

confidence: 99%

Mannose-6-phosphate/insulin-like growth factor-II receptor is a receptor for retinoic acid

Kang

Li²,

Leaf³

1997

Proc. Natl. Acad. Sci. U.S.A.

116

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Retinoic acid (RA) exerts diverse biological effects in the control of cell growth in embryogenesis and oncogenesis. These effects of RA are thought to be mediated by the nuclear retinoid receptors. Mannose-6-phosphate (M6P)/insulin-like growth factor-II (IGF-II) receptor is a multifunctional membrane glycoprotein that is known to bind both M6P and IGF-II and function primarily in the binding and trafficking of lysosomal enzymes, the activation of transforming growth factor-β, and the degradation of IGF-II. M6P/IGF-II receptor has recently been implicated in fetal development and carcinogenesis. Despite the functional similarities between RA and the M6P/IGF-II receptor, no direct biochemical link has been established. Here, we show that the M6P/IGF-II receptor also binds RA with high affinity at a site that is distinct from those for M6P and IGF-II, as identified by a photoaffinity labeling technique. We also show that the binding of RA to the M6P/IGF-II receptor enhances the primary functions of this receptor. The biological consequence of the interaction appears to be the suppression of cell proliferation and/or induction of apoptosis. These findings suggest that the M6P/IGF-II receptor mediates a RA response pathway that is important in cell growth regulation. This discovery of the interaction of RA with the M6P/IGF-II receptor may have important implications for our understanding of the roles of RA and the M6P/IGF-II receptor in development, carcinogenesis, and lysosomal enzyme-related diseases.

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Physicochemical Parameters Affecting the Charcoal Adsorption Assay for Quantitative Retinoid-Binding Measurement

Cited by 9 publications

References 0 publications

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

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

Critical Role of Tyrosine 277 in the Ligand-Binding and Transactivating Properties of Retinoic Acid Receptor α

Mannose-6-phosphate/insulin-like growth factor-II receptor is a receptor for retinoic acid

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