RETINOIC ACID RECEPTOR-GAMMA - SPECIFIC IMMUNODETECTION AND PHOSPHORYLATION

Synthetic peptides corresponding to cDNA-deduced amino acid sequences unique to the human and mouse retinoic acid receptor gamma-1 (hRAR-gamma-1 and mRAR-gamma-1, respectively) were used to generate anti-RAR-gamma-1 antibodies. Four mAbs were selected, which were directed against peptides found in region A1 (Ab1-gamma(A1)), region F (Ab2-gamma(mF) and Ab4-gamma(hF)) and region D2 (Ab5-gamma(D2)). These antibodies specifically immunoprecipitated and recognized by Western blotting RAR-gamma-1 proteins in COS-1 cells transfected with expression vectors containing the RAR-gamma-1 cDNAs. They all reacted with both human and mouse RAR-gamma-1 proteins, except Ab4-gamma(hF) that was specific for hRAR-gamma-1. Rabbit polyclonal antibodies, directed against a peptide from the mRAR-gamma-1 F region were also obtained (RP-gamma(mF)) and found to be specific for mouse RAR-gamma-1 protein. Furthermore, in gel retardation/shift assays the antibodies specifically retarded the migration of complexes obtained with a RA response element (RARE). Antibodies raised against regions D2 and F also recognized the RAR-gamma-2 isoform which differs from RAR-gamma-1 only in the A region. On the other hand, antibodies directed against the A1 region of RAR-gamma-1 (Ab1-gamma(A1)) only reacted with the RAR-gamma-1 protein. The antibodies characterized here allowed us to detect the presence of mRAR-gamma-1 and gamma-2 isoforms in mouse embryos and F9 embryonal carcinoma cells nuclear extracts. They were also used to demonstrate that the mRAR-gamma-1 protein can be phosphorylated and that the phosphorylation occurs mainly in the NH2-terminal A/B region.