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

Cellular retinoic acid-binding proteins (CRABPs) are carrier proteins thoug ht to play a crucial role in the transport and metabolism of all-trans-reti noic acid (atRA) and its derivatives within the cell. This report describes a novel photoaffinity-based binding assay involving competition between po tential ligands of CRABP and [H-3]atRA or [H-3]-9-cis-RA for binding to the atRA-binding sites of CRABP I and II. Photoaffinity labeling of purified C RABPs with [H-3]atRA was light- and concentration-dependent, saturable, and protected by several retinoids in a concentration-dependent manner, indica ting that binding occurred in the CRABP atRA-binding site. Structure-functi on relationship studies demonstrated that oxidative changes to the atRA bet a -ionone ring did not affect ligand potency. However, derivatives lacking a terminal carboxyl group and some cis isomers did not bind to CRABPs, Thes e studies also identified two novel ligands for CRABPs: 5,6-epoxy-RA and re tinoyl-beta -D-glucuronide (RAG), The labeling of both CRABPs with 9-cis-RA occurred with much lower affinity. Experimental evidence excluded nonspeci fic binding of RAG to CRABPs and UDP-glucuronosyltransferases, the enzymes responsible for RAG synthesis. These results established that RAG is an eff ective ligand of CRABPs. Therefore, photoaffinity labeling with [H-3]atRA c an be used to identify new ligands for CRABP and retinoid nuclear receptors and also provide information concerning the identity of amino acid(s) loca lized in the atRA-binding site of these proteins.