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.
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.
Radominska-Pandya
A
A
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA. radominskaanna@exchange.uams.edu
Chen
G
G
Samokyszyn
V M
VM
Little
J M
JM
Gall
W E
WE
Zawada
G
G
Terrier
N
N
Magdalou
J
J
Czernik
P
P
D000077556:Alitretinoin; D000818:Animals; D005965:Glucuronates / Q000737:chemistry; D014453:Glucuronosyltransferase / Q000737:chemistry; D006801:Humans; D008095:Lithocholic Acid / Q000737:chemistry; D008862:Microsomes, Liver / Q000737:chemistry; D019920:Photoaffinity Labels; D051381:Rats; D018168:Receptors, Retinoic Acid / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D014212:Tretinoin / Q000737:chemistry*; D014316:Tritium
D005965:Glucuronates; D019920:Photoaffinity Labels; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; C082927:retinoic acid binding protein I, cellular; C082928:retinoic acid binding protein II, cellular; D014316:Tritium; D000077556:Alitretinoin; D014212:Tretinoin; D008095:Lithocholic Acid; C041558:lithocholate 3-O-glucuronide; C491474:UGT2B7 protein, human; D014453:Glucuronosyltransferase