BINDING OF 9-CIS-RETINOIC ACID AND ALL-TRANS-RETINOIC ACID TO RETINOIC ACID RECEPTOR-ALPHA, RECEPTOR-BETA, AND RECEPTOR-GAMMA - RETINOIC ACID RECEPTOR-GAMMA BINDS ALL-TRANS-RETINOIC ACID PREFERENTIALLY OVER 9-CIS-RETINOIC ACID

Both 9-cis-retinoic acid (RA) and all-trans-RA (t-RA) compete for [H-3 ]9-cis-RA binding to RA receptors (RAR alpha, beta, and gamma) in nucl eosol fractions from transiently transfected COS-1 cells with IC50 val ues of approximately 12 and 5 nM, respectively. Curiously, 9-cis-RA co mpetes for [H-3]t-RA binding to mouse RAR alpha, beta, and gamma with IC50 values of 31, 8, and 60 nM, respectively, while t-RA itself does not exhibit such differential competition (IC50 values for RARs, 5 nM) . A similar pattern is observed with human retinoic acid receptors (RA Rs). Differential binding of 9-cis-RA to the RAR beta and gamma recept ors is also found following in vitro transcription and translation of these receptors. Displacement assays demonstrate that t-RA exhibits si milar off-rates for RAR alpha, beta, and gamma. However, 9-cis-RA is 6 -fold more rapidly displaced from RAR gamma than from RAR beta. When R AR-transfected COS-1 cells are incubated with [H-3]t-RA, [H-3]-9-cis-R A or various mixtures of these two radioligands, high performance liqu id chromatography analysis demonstrates that the ligands bound in nucl eosol fractions from RAR beta-transfected cells reflect the isomer con tent of the media. However, in identical whole cell assays, nucleosol fractions from RAR gamma-transfected cells preferentially bind t-RA ov er 9-cis-RA, consistent with the in vitro data. These binding kinetics in vitro and in whole cells suggest that there could be differences i n the interactions of the receptor subtypes with the endogenous retino ic acids under physiologic conditions.