Keidel S, Lamour F P, Apfel C M
Preclinical Research, Department of Infectious Diseases, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland.
J Biol Chem. 1997 Jul 18;272(29):18267-72. doi: 10.1074/jbc.272.29.18267.
Retinoids exert their pleiotropic effects on cell differentiation and proliferation through specific nuclear receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Two biologically highly active natural retinoids have been identified, all-trans-retinoic acid (t-RA) and 9-cis-retinoic acid (9-cis-RA). The RXRs exclusively bind 9-cis-RA, whereas the RARs bind both isomers of RA with comparable affinity. Recently published results suggest that RARs have the same binding site for t-RA and 9-cis-RA but with different determinants (1-3). Antagonist binding on RARalpha has been suggested to induce distinct conformational changes in comparison with agonist binding. To elucidate the region minimally required for efficient binding of agonist (t-RA and 9-cis-RA) and antagonist Ro 41-5253 to the RARalpha, we generated N- and C-terminally truncated mutants of the receptor. Characterization of these deletion mutant proteins using protease mapping and ligand binding experiments revealed that different parts of the ligand-binding domain are necessary for t-RA, 9-cis-RA, and antagonist binding. Three distinct regions of the ligand-binding domain of the human retinoic acid receptor-alpha are required for binding of t-RA (RARalpha187-402), 9-cis-RA (RARalpha188-409), and the antagonist Ro 41-5253 (RARalpha226-414).
维甲酸通过特定的核受体，即维甲酸受体（RARs）和维甲酸X受体（RXRs），对细胞分化和增殖发挥其多效性作用。已鉴定出两种具有高度生物活性的天然维甲酸，全反式维甲酸（t-RA）和9-顺式维甲酸（9-cis-RA）。RXRs仅结合9-顺式维甲酸，而RARs以相当的亲和力结合维甲酸的两种异构体。最近发表的结果表明，RARs对t-RA和9-顺式维甲酸具有相同的结合位点，但决定因素不同（1-3）。与激动剂结合相比，RARα上的拮抗剂结合被认为会诱导不同的构象变化。为了阐明激动剂（t-RA和9-顺式维甲酸）和拮抗剂Ro 41-5253与RARα有效结合所需的最小区域，我们构建了该受体的N端和C端截短突变体。使用蛋白酶图谱和配体结合实验对这些缺失突变蛋白进行表征，结果表明配体结合结构域的不同部分对于t-RA、9-顺式维甲酸和拮抗剂结合是必需的。人维甲酸受体α的配体结合结构域的三个不同区域分别是t-RA结合（RARα187-402）、9-顺式维甲酸结合（RARα188-409）和拮抗剂Ro 41-5253结合（RARα226-414）所必需的。
