Allenby G, Janocha R, Kazmer S, Speck J, Grippo J F, Levin A A
Department of Toxicology and Pathology, Hoffmann-La Roche Inc., Nutley, New Jersey 07110.
J Biol Chem. 1994 Jun 17;269(24):16689-95.
Both 9-cis-retinoic acid (RA) and all-trans-RA (t-RA) compete for [3H]9-cis-RA binding to RA receptors (RAR alpha, beta, and gamma) in nucleosol fractions from transiently transfected COS-1 cells with IC50 values of approximately 12 and 5 nM, respectively. Curiously, 9-cis-RA competes for [3H]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 (RARs). Differential binding of 9-cis-RA to the RAR beta and gamma receptors is also found following in vitro transcription and translation of these receptors. Displacement assays demonstrate that t-RA exhibits similar 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 RAR-transfected COS-1 cells are incubated with [3H]t-RA, [3H]-9-cis-RA or various mixtures of these two radioligands, high performance liquid chromatography analysis demonstrates that the ligands bound in nucleosol fractions from RAR beta-transfected cells reflect the isomer content of the media. However, in identical whole cell assays, nucleosol fractions from RAR gamma-transfected cells preferentially bind t-RA over 9-cis-RA, consistent with the in vitro data. These binding kinetics in vitro and in whole cells suggest that there could be differences in the interactions of the receptor subtypes with the endogenous retinoic acids under physiologic conditions.
9-顺式视黄酸（RA）和全反式视黄酸（t-RA）均可竞争与瞬时转染的COS-1细胞核溶质组分中RA受体（RARα、β和γ）的[3H]9-顺式视黄酸结合，其IC50值分别约为12和5 nM。奇怪的是，9-顺式视黄酸可竞争与小鼠RARα、β和γ的[3H]t-RA结合，其IC50值分别为31、8和60 nM，而t-RA本身并未表现出这种差异竞争（RARs的IC50值为5 nM）。在人类视黄酸受体（RARs）中也观察到类似模式。在这些受体进行体外转录和翻译后，还发现9-顺式视黄酸与RARβ和γ受体存在差异结合。置换实验表明，t-RA对RARα、β和γ表现出相似的解离速率。然而，9-顺式视黄酸从RARγ上的解离速度比从RARβ上快6倍。当用[3H]t-RA、[3H]-9-顺式视黄酸或这两种放射性配体的各种混合物孵育转染了RAR的COS-1细胞时，高效液相色谱分析表明，RARβ转染细胞的核溶质组分中结合的配体反映了培养基中的异构体含量。然而，在相同的全细胞实验中，RARγ转染细胞的核溶质组分优先结合t-RA而非9-顺式视黄酸，这与体外数据一致。这些体外和全细胞中的结合动力学表明，在生理条件下，受体亚型与内源性视黄酸的相互作用可能存在差异。
