Location of two photoaffinity-labeled sites on the ligand-binding domain of retinoic acid receptor alpha.

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.