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.
Rochette-Egly
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Laboratoire de Génétique Moléculaire des Eucaryotes du Centre National pour la Recherche Scientifique, l'Institut National de la Santé et de la Recherche Medicale, Faculté de Médecine, Strasbourg, France.
Lutz
Y
Y
Saunders
M
M
Scheuer
I
I
Gaub
M P
MP
Chambon
P
P
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D004622:Embryo, Mammalian / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011817:Rabbits; D018168:Receptors, Retinoic Acid; D014162:Transfection / Q000235:genetics; D014212:Tretinoin / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D010446:Peptide Fragments; D018168:Receptors, Retinoic Acid; D014212:Tretinoin; D000469:Alkaline Phosphatase