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

Autor: Guangping Chen, Piotr J. Czernik, Gregory Zawada, Victor M. Samokyszyn, Walter E. Gall, Anna Radominska-Pandya, Nadege Terrier, Jacques Magdalou, Joanna M. Little
Rok vydání: 2001
Předmět:
Zdroj: Protein Science. 10:200-211
ISSN: 1469-896X
0961-8368
DOI: 10.1110/ps.26501
Popis: 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.
Databáze: OpenAIRE
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