Retinoic Acid Receptor -y : Specific Immunodetection and Phosphorylation

Synthetic peptides corresponding to cDNAdeduced amino acid sequences unique to the human and mouse retinoic acid receptor yl (hRAR-yl and mRAR-yl, respectively) were used to generate antiRAR-yl antibodies . Four mAbs were selected, which were directed against peptides found in region Al (Ably(Al)), region F (Ab2y(mF) and Ab4y(hF)) and region D2 (Ab5y(D2)) . These antibodies specifically immunoprecipitated and recognized by Western blotting RAR-yl proteins in COS-1 cells transfected with expression vectors containing the RAR-yl cDNAs. They all reacted with both human and mouse RARyl proteins, except Ab4y(hF) that was specific for hRAR yl . Rabbit polyclonal antibodies, directed against a peptide from the mRAR-yl F region were also obtained (RPy(mF)) and found to be specific for mouse RTINoic acid (RA),' a vitamin A derivative, which is thought to be a natural morphogen (Maden, 1982 ; Thaller and Eichele, 1987), can act as a signaling molecule in a number ofdevelopmental systems . The pleiotropic effects of RA are likely to be mediated by specific nuclear RA receptors (RARs) which have been discovered in mouse and human (designated RARa, -ß, and -y) (Petkovich et al ., 1987; Giguère et al ., 1987 ; Brand et al ., 1988 ; Benbrook et al ., 1988 ; Krust et al ., 1989 ; Zelent et al ., 1989) . RARs belong to the steroid/thyroid hormone receptor superfamily, whose members act as ligand-inducible transcriptional enhancer factors (for reviews see Evans, 1988 ; Green and Chambon, 1988; Beato, 1989 and references herein) and can be divided into six distinct regions designated A through F (see Petkovich et al ., 1987 ; Green and Chambon, 1988 ; Brand et al ., 1988) . The complexity of RARs has been further illustrated by the finding of multiple cDNA isoforms for each RAR (Krust et al ., 1989 ; Gigu6re et al ., 1990; Kastner et al ., 1990 ; Leroy et al ., 1991 ; Zelent et al ., 1991) . For each RAR gene (either a, ,ß, or y) 1 . Abbreviations used in this paper: NE, nuclear extracts ; p .c., post-coitum ; RA, retinoic acid ; RAR, retinoic acid receptor ; RARE, retinoic acid response element ; NC, nitrocellulose ; WCE, whole cell extracts . ® The Rockefeller University Press, 0021-9525/91/10/535/11 $2 .00 TheJoumal of Cell Biology, Volume 115, Number 2, October 1991535-545 RARyl 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 RARy2 isoform which differs from RARyI only in the A region . On the other hand, antibodies directed against the Al region of RAR-yl (Ably(Al)) only reacted with the RARyl protein . The antibodies characterized here allowed us to detect the presence of mRARyl and 72 isoforms in mouse embryos and F9 embryonal carcinoma cells nuclear extracts . They were also used to demonstrate that the mRARy1 protein can be phosphorylated and that the phosphorylation occurs mainly in the NH2terminal A/B region . the corresponding isoform messenger RNAs are generated from two promoters and differential splicing ofexons encoding the A region . Specific spatial and temporal patterns ofdistribution ofthe RAR-a, -ß, and -y transcripts have been demonstrated in adult mouse tissues (Krust et al ., 1989 ; Zelent et al ., 1989 ; Kastner et al ., 1990) and during mouse embryogenesis (Dollé et al ., 1989,1990 ; Ruberte et al ., 1990, 1991) . In particular, the localization of RARy transcripts during embryogenesis as determined by in situ hybridization, suggests that RAR-y plays an important role during early morphogenesis and differentiation of cartilage and cornified squamous epithelia (Dollé et al ., 1989, 1990; Ruberte et al ., 1990, 1991) . Furthermore, the two predominant RAR-y isoforms, RAR-yl and RAR-y2, appear to be differentially expressed in adult tissues and during the course of embryogenesis, as determined by Northern blot analysis (Kastner et al ., 1990) . In this paper, we describe the preparation and characterization ofrabbit polyclonal and mouse mAbs directed against synthetic peptides specific to mouse and/or human RAR-y isoforms . These antibodies specifically immunoprecipitate and recognize by Western blotting mouse or human RARy in cells transfected with expression vectors containing the corresponding cDNAs. They also specifically retard the 535 on A uust 6, 2017 jcb.rress.org D ow nladed fom migration of RAR-,y/RARE (RA response element) complexes in gel shift assays . Additionally, these antibodies allowed us to detect the presence ofRAR--y isoforms in F9 embryonal carcinoma cells and mouse embryos, despite the low amount of these proteins in such cells and tissues . Finally, using our antibodies, we have been able to demonstrate that the RAR-y protein is posttranslationally modified by phosphorylation . Materials and Methods