Structure-affinity relationships of retinoids with embryonic cellular retinoic acid-binding protein.

Separation and quantitation of cellular retinoic acid-binding protein (CRABP) in embryonic and fetal hamster tissues was accomplished with high-performance size-exclusion chromatography. Binding affinity of 26 retinoids was established by in vitro displacement of high specific activity all-trans-[3H2]retinoic acid from fetal CRABP. The CRABP concentration in presomite-to-early somite (Day 8) hamster embryos was 1.9 pmol/mg cytosolic protein and increased to 7.5 pmol/mg protein in Day 13 fetuses; CRABP concentrations subsequently declined as gestation progressed. CRABP was located primarily in fetal brain and skin (5.8 +/- 0.3 and 2.2 +/- 0.1 pmol/mg protein, respectively), whereas only trace concentrations were found in fetal liver, placenta, and maternal uterus. Retinoids that could displace all-trans-retinoic acid from CRABP had a free acid at the polar terminus (or were carboxylate esters that were readily hydrolyzed to the corresponding free acid) and had a hydrophobic ring at the distal position. The ligand specificity of the CRABP studied here suggests that this protein was analogous to the CRABP I isoform. The in vitro binding affinities of teratogenic retinoids that competed for embryonic CRABP failed to correlate directly with relative teratogenic potency. In some instances, the latter observation can be related to extensive in vivo biotransformation of retinoids to multiple teratogenic metabolites and to retinoid persistence in the embryo. Three analogs containing a free carboxy terminus, SRI 5898-21, SRI 7323-78, and SRI 6153-40, were identified with high teratogenic potency but failed to bind fetal hamster CRABP. The structure-activity and binding data of the analogs studied here indicate that many, if not most, teratogenic retinoids (or their acidic metabolites) bind with embryonic/fetal CRABP, but the present data question the role for CRABP in their teratogenic mechanism of action.