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Sensitive and specific detection of retinoid receptor subtype proteins in cultured cell and tumor extracts
M W Titcomb1, M M Gottardis, J W Pike
1Ligand Pharmaceuticals, Inc., Department of Biochemistry, La Jolla, California 92037.
Abstract:
Subtype-specific antipeptide antibodies have been developed against each of the retinoic acid receptors (RARs alpha, beta, and gamma) and each of the retinoid X receptors (RXRs alpha, beta, and gamma). Each antibody reacts specifically with its respective recombinantly expressed protein but not with any of the other retinoid receptor subtypes, by both immunoblot and immunoprecipitation technology. We describe a sensitive and specific assay that combines the binding of cultured cell and tumor extracts to [3H]all-trans-retinoic acid or [3H]9-cis-retinoic acid with immunoprecipitation of the hormone-receptor complexes by the subtype-specific antibodies to determine the levels of functional retinoid receptor subtype proteins that are present. We also report the use of a hormone-binding assay that uses RAR- and RXR-selective compounds as competitors of the tritiated retinoids to ascertain the RAR and RXR subfamily profiles of these cells. HeLa cells contain all six retinoid receptor proteins ranging in concentration from 9 fmol/mg total protein for RAR beta and RXR gamma to 50 fmol/mg for RXR alpha. Hep G2 and HL60 cells express RAR alpha and RXR alpha proteins at approximately 20-60 fmol receptor/mg protein, and RAR beta is expressed at lower levels (approximately 5 fmol/mg) in Hep G2 cells. MCF-7 cells in culture express RAR alpha (approximately 32 fmol/mg), RAR gamma (approximately 35 fmol/mg), and RXR alpha (approximately 60 fmol/mg).(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Researchers developed subtype-specific antibodies for retinoic acid receptors (RARs) and retinoid X receptors (RXRs). A new assay quantifies functional RAR and RXR proteins in cells, revealing varying levels across different cell lines.
Area of Science:
- Molecular Biology
- Endocrinology
- Cell Biology
Background:
- Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) are crucial nuclear receptors involved in gene regulation.
- Understanding the specific subtypes and their expression levels is vital for deciphering retinoid signaling pathways.
Purpose of the Study:
- To develop subtype-specific antibodies for all RAR and RXR proteins.
- To establish a sensitive assay for quantifying functional RAR and RXR subtype proteins in cellular extracts.
- To determine the expression profiles of RAR and RXR subtypes in various human cell lines.
Main Methods:
- Development of antipeptide antibodies specific for RAR alpha, beta, gamma and RXR alpha, beta, gamma.
- Utilized immunoblot and immunoprecipitation techniques to confirm antibody specificity.
- Established a combined hormone-binding and immunoprecipitation assay using radiolabeled retinoids and subtype-specific antibodies.
- Employed hormone-binding assays with selective competitors to profile RAR and RXR subfamily expression.
Main Results:
- Successfully generated subtype-specific antibodies for all six retinoid receptor subtypes.
- Demonstrated the ability to quantify functional RAR and RXR proteins in cell extracts.
- HeLa cells express all six receptor subtypes, with RXR alpha at the highest concentration (50 fmol/mg).
- Hep G2 and HL60 cells show significant RAR alpha and RXR alpha expression (20-60 fmol/mg), with lower RAR beta levels.
- MCF-7 cells express RAR alpha, RAR gamma, and RXR alpha.
Conclusions:
- The developed antibodies and assay provide a powerful tool for studying retinoid receptor expression.
- Quantification of specific RAR and RXR subtypes reveals differential expression patterns in various cancer cell lines.
- This methodology facilitates further research into the roles of specific retinoid receptor subtypes in cellular processes and disease.