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Application of photoaffinity labeling with [³H] all trans- and 9-cis-retinoic acids for characterization of cellular retinoic acid–binding proteins I and II

¹ Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA
² Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA
³ Faculté de Medecine, UMR 7561 CNRS-Université Henri Poincaré, Vandoeuvre-lès-Nancy, France

Reprint requests: to Dr. Anna Radominska-Pandya, Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 W. Markham, Slot 516, Little Rock, Arkansas 72205, USA; e-mail: radominskaanna{at}exchange.uams.edufax: 501-603-1146.

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 [³H]atRA or [³H]-9-cis-RA for binding to the atRA-binding sites of CRABP I and II. Photoaffinity labeling of purified CRABPs with [³H]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 ß-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-ß-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 [³H]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.

Keywords: Cellular retinoic acid-binding protein; photoaffinity labeling; all-trans-retinoic acid; 9-cis-retinoic acid; retinoic acid glucuronide; 5,6-epoxy-retinoic acid

Keywords: CRABP, cellular retinoic acid–binding protein; atRA, all-trans-retinoic acid; RAR, nuclear retinoic acid receptor; RXR, nuclear retinoid receptor; 13-cis-RA, 13-cis-retinoic acid; 9-cis-RA, 9-cis-retinoic acid; 4-OH-RA, 4-hydroxy-all-trans-retinoic acid; ROH, all-trans-retinol; ROAc, all-trans-retinyl acetate; 5,6-epoxy-RA, 5,6-epoxy-all-trans-RA; KPFG, ketoprofen glucuronide; LA, lithocholic acid; LAG, lithocholic acid glucuronide; RAG, retinoic acid glucuronide; UGT, UDP-glucuronosyltransferase; UDP-GlcUA, UDP-glucuronic acid

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