Photoaffinity Labeling of Human Retinoid X Receptor β (RXRβ) with 9-cis-Retinoic Acid: Identification of Phytanic Acid, Docosahexaenoic Acid, and Lithocholic Acid as Ligands for RXRβ

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Abstract
We utilized [20-methyl-3H]-9-cis-retinoic acid ([3H]9-cis-RA) as a direct photoaffinity probe for the characterization of human recombinant retinoid X receptor β protein (RXRβ). The photoaffinity labeling was light- and concentration-dependent, saturable, and protected by unlabeled 9-cis-RA in a concentration-dependent manner, indicating that binding occurred in the RXR retinoid binding site. all-trans-Retinoic acid (atRA) did not affect labeling with the 9-cis derivative, confirming that atRA does not compete for the 9-cis-RA binding site. Several retinoid, fatty acid, and bile acid ligands were evaluated for their ability to recognize the 9-cis-RA binding site. Retinol, atRA glucuronide, 13-cis-RA, dolichol, 5,6-epoxy-RA, and vitamin D3 did not compete for the 9-cis-RA binding site. However, the saturated diterpenoid phytanic acid (PA) and docosahexaenoic acid, which have been recently shown to activate the nuclear receptor, RXR, competed with 9-cis-RA labeling, showing high affinity for the 9-cis-RA binding site. Oleic acid, arachidonic acid, and butyric acid did not interact. However, the bile acid lithocholic acid competed efficiently with 9-cis-RA for the binding site. These data validated the photoaffinity assay as an excellent system for the identification and evaluation of ligands for RXR.