Immunodetection of multiple species of retinoic acid receptor α: Evidence for phosphorylation

• Published in: Experimental cell research Vol. 201; no. 2; pp. 335 - 346
• Main Authors: Gaub, M.P., Rochette-Egly, C., Lutz, Y., Ali, S., Matthes, H., Scheuer, I., Chambon, P.
• Format: Journal Article
• Language: English
• Published: Orlando, FL Elsevier Inc 01.08.1992; Elsevier
• Subjects: 2-type; Amino Acid Sequence; Animals; anti bodies; Antibodies; Antibodies, Monoclonal; Biological and medical sciences; Carrier Proteins - analysis; Carrier Proteins - chemistry; Cell Line - chemistry; Cell receptors; Cell structures and functions; Embryo, Mammalian - chemistry; Fundamental and applied biological sciences. Psychology; Genetic Vectors; Hormone receptors. Growth factor receptors. Cytokine receptors. Prostaglandin receptors; Humans; immunodetection; man; Mice; Molecular and cellular biology; Molecular Sequence Data; monoclonal antibodies; mouse; Peptides - chemical synthesis; Phosphorylation; receptors; Receptors, Retinoic Acid; retinoic acid; subtypes; Tretinoin - pharmacology; Human; Molecular form; Peptides; Antibody; Immunocytochemistry; Rodentia; HPLC chromatography; Cell differentiation; Tissue; Vertebrata; Immunoprecipitation reaction; Mammalia; Mouse; Immunoblotting assay; Detection; Embryonal carcinoma; Biological receptor
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1016/0014-4827(92)90282-D

Polyclonal and monoclonal antibodies were raised against synthetic peptides (or fusion protein) corresponding to cDNA-deduced amino acid sequences unique to the human and mouse retinoic acid (RA) receptor α1 (hRAR-α1 and mRAR-α1, respectively). Two rabbit polyclonal antibodies directed against either the F region fused to DHFR [RPα(F)] or the D2 region [RPα(D2)] were selected. Using either immunocytochemistry, Western blotting analysis, or immunoprecipitation, they were found to be specific for human and mouse RAR-α1 proteins produced by COS-1 cells transiently transfected with vectors expressing the RAR-α1 cDNA. Three mouse monoclonal antibodies directed against either the F region [(Ab9α(F) and Ab12α(F)] or the A1 region [Ab10α1(A1)] recognized transiently expressed human and mouse RAR-α1 proteins, when either immunocytochemistry or immunoprecipitation was used. In addition, Ab9α(F) and Ab12α(F), but not Ab10α1(A1), revealed the RAR-α1 proteins by Western blotting analysis. Ab9α(F) was also able to “supershift” RAR-α1 protein-RARE oligonucleotide probe complexes in gel retardation assays. All these antibodies recognized also the transiently expressed mRAR-α2 isoform, with the exception of Ab10α1(A1), which is specific for the A1 region of RAR-α1. These antibodies have enabled us to detect the presence of mRAR-α as multiple species in mouse embryo and adult tissue extracts as well as in embryonal carcinoma (EC) cells. Moreover, we found that one of these species (51 kDa) was phosphorylated in EC cells. This phosphorylation was not affected by RA treatment, but appeared to be dependent on the differentiation state of the EC cells.