Characterization of heterologously expressed recombinant retinoic acid receptors with natural or synthetic retinoids

The first step in retinoid action is binding to their nuclear receptors. Therefore, characterization of binding characteristics of retinoids is of major importance. Human retinoic acid receptors α (hRARα), hRARβ, and mouse RARγ (mRARγ) were expressed heterologously in Escherichia coli as a recombinant glutathione S-transferase (GST) fusion protein. The expressed fusion proteins were functional and bound specifically to the all-trans-retinoic acid (RA). The dissociation constants (K_d) for RA were 1.4 nM for GST-hRARα, 1.4 nM for GST-hRARβ, and 3.3 nM for GST-mRARγ, respectively. The fusion proteins were further used for competitive displacement assays to determine the displacement constant (DC50) for other selected retinoids. All-trans-RA and 4-oxo-all-trans-RA have high affinity with all three receptors (DC50 = 0.8 ～ 55 nM). The 13-cis RA binds to hRARα with low affinity, but not to other RARs evaluated here. All-trans-N-ethylretinamide, all-trans-retinylacetate, and an ethyl ester of tetrahydronaphthalene derivative had no affinity to any RARs. The hRARα and mRARγ receptors did not bind a naphthalene carboxylic acid derivative of RA, but hRARβ binds this chemical with high affinity. Results indicated that the three recombinant proteins were functional in binding various RA congeners. The affinity and binding data of these retinoids were compared to their observed teratogenic activity.