Application of photoaffinity labeling with [3H] 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 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 β‐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‐β‐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.