Binding affinities of CRBPI and CRBPII for 9-cis-retinoids

BackgroundCellular retinol binding-protein I (CRBPI) and cellular retinol binding-protein II (CRBPII) serve as intracellular retinoid chaperones that bind retinol and retinal with high affinity and facilitate substrate delivery to select enzymes that catalyze retinoic acid (RA) and retinyl ester biosynthesis. Recently, 9-cis-RA has been identified in vivo in the pancreas, where it contributes to regulating glucose-stimulated insulin secretion. In vitro, 9-cis-RA activates RXR (retinoid × receptors), which serve as therapeutic targets for treating cancer and metabolic diseases. Binding affinities and structure–function relationships have been well characterized for CRBPI and CRBPII with all-trans-retinoids, but not for 9-cis-retinoids. This study extended current knowledge by establishing binding affinities for CRBPI and CRBPII with 9-cis-retinoids.MethodsWe have determined apparent dissociation constants, K′d, through monitoring binding of 9-cis-retinol, 9-cis-retinal, and 9-cis-RA with CRBPI and CRBPII by fluorescence spectroscopy, and analyzing the data with non-linear regression. We compared these data to the data we obtained for all-trans- and 13-cis-retinoids under identical conditions.ResultsCRBPI and CRBPII, respectively, bind 9-cis-retinol (K′d, 11 nM and 68 nM) and 9-cis-retinal (K′d, 8 nM and 5 nM) with high affinity. No significant 9-cis-RA binding was observed with CRBPI or CRBPII.ConclusionsCRBPI and CRBPII bind 9-cis-retinol and 9-cis-retinal with high affinities, albeit with affinities somewhat lower than for all-trans-retinol and all-trans-retinal.General significanceThese data provide further insight into structure–binding relationships of cellular retinol binding-proteins and are consistent with a model of 9-cis-RA biosynthesis that involves chaperoned delivery of 9-cis-retinoids to enzymes that recognize retinoid binding-proteins.

Research highlights
► Kd values for 9-cis-retinoids and CRBPI and CRBPII.
► Accuracy ensured by non-linear regression analysis for all ligands.
► Concentrations of retinoid binding proteins were an order of magnitude lower than used previously, to enhance accuracy.