RETINOIC ACID RECEPTOR-BETA - IMMUNODETECTION AND PHOSPHORYLATION ON TYROSINE RESIDUES

Polyclonal (RP) and monoclonal (Ab) antibodies were raised against synthetic peptides (or fusion proteins) corresponding to amino acid sequences unique to human and mouse retinoic acid receptor-beta (RARbeta) isoforms. Antibodies directed against the A2 region [Ab6beta2(A2), Ab7beta2(A2), and RPbeta2(A2)], the D2 region [RPbeta(D2)], or the F region [Ab8beta(F)2, RPbeta(F)1, and RPbeta(F)2] were selected. The monoclonal and polyclonal antibodies directed against the D2 and F regions specifically immunoprecipitated and recognized by Western blotting all human and mouse RARbeta isoforms (mRARbeta1, -beta2, -beta3, and -beta4), produced in COS-1 cells transfected with expression vectors containing the corresponding RARbeta cDNA. Furthermore, in gel retardation assays, the monoclonal antibodies supershifted RARbeta protein-RA response element oligonucleotide complexes. Antibodies directed against the A2 region were specific for the RARbeta2 isoform. The above antibodies allowed us to detect the presence of mRARbeta2 proteins in mouse embryos and to show that their presence in embryonal carcinoma cells (F9 and P19 cell lines) is dependent upon RA treatment. The antibodies were also used to demonstrate that RARbeta proteins produced by transfection in COS-1 cells are phosphorylated. RARbeta2 phosphorylation was not affected by RA treatment, whereas the phosphorylation of RARbeta1 and RARbeta3 isoforms was greatly enhanced by RA. We also show that, in contrast to RARalpha1 and RARgamma1, RARbeta2 proteins contain phosphotyrosine residues and are only weakly phosphorylated in vitro by cAMP-dependent protein kinase. These results support our previous proposal that the various receptors have distinct functions in the RA-signaling pathway.