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.
Sasaki
T
T
Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
Shimazawa
R
R
Sawada
T
T
Iljima
T
T
Fukasawa
H
H
Shudo
K
K
Hashimoto
Y
Y
Iwasaki
S
S
D000345:Affinity Labels / Q000138:chemical synthesis / Q000737:chemistry*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003619:Dansyl Compounds / Q000138:chemical synthesis / Q000737:chemistry; D005453:Fluorescence; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D018168:Receptors, Retinoic Acid / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012176:Retinoids / Q000138:chemical synthesis / Q000737:chemistry*; D012697:Serine Endopeptidases; D013764:Tetrahydronaphthalenes / Q000138:chemical synthesis / Q000737:chemistry
C091976:ADAM 3; D000345:Affinity Labels; D003619:Dansyl Compounds; D008024:Ligands; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; D012176:Retinoids; D013764:Tetrahydronaphthalenes; C071337:2-(3-(5-dimethylaminonaphthalene-1-sulfonyl)aminopropyl-1-oxyl)-4-((5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido)benzoic acid; C044499:submandibular proteinase A; D012697:Serine Endopeptidases