Sasaki T, Shimazawa R, Sawada T, Iljima T, Fukasawa H, Shudo K, Hashimoto Y, Iwasaki S
Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
Biol Pharm Bull. 1996 May;19(5):659-64. doi: 10.1248/bpb.19.659.
Retinoic acid receptors (RARs) consist of six domain structures. The C-terminal region (D/E/F-domains) is involved in ligand binding, dimerization, and ligand-dependent transactivation. Structural information about RARs is required for understanding its complex function. A photoreactive retinoid denoted as ADAM-3, which was designed as the result of comparison of two fluorescent retinoids (DAM-3 and DAM-15), was synthesized and used for photoaffinity labeling of recombinant protein MBP-RAR alpha/E. The photoaffinity-labeled site was determined by an endoprotease combination method which utilizes four endoproteinases in a two-phase digestion procedure. Two major labeled fragments were detected in each digestion, and the results of two-phase digestion allowed identification of the labeled residues as being located within residues 492-510 and 585-594, which correspond to 288-306 and 381-390 in human RAR alpha, respectively.
维甲酸受体（RARs）由六个结构域组成。C 末端区域（D/E/F 结构域）参与配体结合、二聚化以及配体依赖性反式激活。了解 RARs 的复杂功能需要其结构信息。一种名为 ADAM-3 的光反应性类视黄醇，它是通过比较两种荧光类视黄醇（DAM-3 和 DAM-15）设计而成，经合成后用于重组蛋白 MBP-RARα/E 的光亲和标记。通过一种内切蛋白酶组合方法确定光亲和标记位点，该方法在两阶段消化过程中使用四种内切蛋白酶。每次消化均检测到两个主要的标记片段，两阶段消化的结果使得标记残基被确定位于 492 - 510 位残基和 585 - 594 位残基内，分别对应于人类 RARα中的 288 - 306 位残基和 381 - 390 位残基。
