Immunodetection of multiple species of retinoic acid receptor alpha: evidence for phosphorylation

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 alpha 1 (hRAR-alpha 1 and mRAR-alpha 1, respectively). Two rabbit polyclonal antibodies directed against either the F region fused to DHFR [RP alpha (F)] or the D2 region [RP alpha (D2)] were selected. Using either immunocytochemistry, Western blotting analysis, or immunoprecipitation, they were found to be specific for human and mouse RAR-alpha 1 proteins produced by COS-1 cells transiently transfected with vectors expressing the RAR-alpha 1 cDNA. Three mouse monoclonal antibodies directed against either the F region [(Ab9 alpha (F) and Ab12 alpha (F)] or the A1 region [Ab10 alpha 1(A1)] recognized transiently expressed human and mouse RAR-alpha 1 proteins, when either immunocytochemistry or immunoprecipitation was used. In addition, Ab9 alpha (F) and Ab12 alpha (F), but not Ab10 alpha 1(A1), revealed the RAR-alpha 1 proteins by Western blotting analysis. Ab9 alpha (F) was also able to "supershift" RAR-alpha 1 protein-RARE oligonucleotide probe complexes in gel retardation assays. All these antibodies recognized also the transiently expressed mRAR-alpha 2 isoform, with the exception of Ab10 alpha 1 (A1), which is specific for the A1 region of RAR-alpha 1. These antibodies have enabled us to detect the presence of mRAR-alpha 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.