Application of photoaffinity labeling with [(3)H] all trans- and 9-cis-retinoic acids for characterization of cellular retinoic acid--binding proteins I and II |
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| Authors: | Radominska-Pandya A Chen G Samokyszyn V M Little J M Gall W E Zawada G Terrier N Magdalou J Czernik P |
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| Affiliation: | Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA. radominskaanna@exchange.uams.edu |
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| Abstract: | Cellular retinoic acid-binding proteins (CRABPs) are carrier proteins thought to play a crucial role in the transport and metabolism of all-trans-retinoic acid (atRA) and its derivatives within the cell. This report describes a novel photoaffinity-based binding assay involving competition between potential ligands of CRABP and [(3)H]atRA or [(3)H]-9-cis-RA for binding to the atRA-binding sites of CRABP I and II. Photoaffinity labeling of purified CRABPs with [(3)H]atRA was light- and concentration-dependent, saturable, and protected by several retinoids in a concentration-dependent manner, indicating that binding occurred in the CRABP atRA-binding site. Structure-function relationship studies demonstrated that oxidative changes to the atRA beta-ionone ring did not affect ligand potency. However, derivatives lacking a terminal carboxyl group and some cis isomers did not bind to CRABPs. These studies also identified two novel ligands for CRABPs: 5,6-epoxy-RA and retinoyl-beta-D-glucuronide (RAG). The labeling of both CRABPs with 9-cis-RA occurred with much lower affinity. Experimental evidence excluded nonspecific binding of RAG to CRABPs and UDP-glucuronosyltransferases, the enzymes responsible for RAG synthesis. These results established that RAG is an effective ligand of CRABPs. Therefore, photoaffinity labeling with [(3)H]atRA can be used to identify new ligands for CRABP and retinoid nuclear receptors and also provide information concerning the identity of amino acid(s) localized in the atRA-binding site of these proteins. |
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| Keywords: | Cellular retinoic acid-binding protein photoaffinity labeling all-trans-retinoic acid 9-cis-retinoic acid retinoic acid glucuronide 5 6-epoxy-retinoic acid |
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