Retinoic acid receptors (RARs) are ligand-activated nuclear transcription factors that belong to the steroid-thyroid hormone receptor superfamily. We have used the transient transfection of a retinoic acid-responsive reporter plasmid (RARECAT) to investigate the potential interactions between the retinoic acid (RA) and cAMP signaling pathways. Cotransfections of expression plasmids for the catalytic (C) subunits of cAMP-dependent protein kinase with RARECAT showed ligand-independent activation in both CV-1 and HeLa cells and a further 2-fold increase in RARECAT activity in the presence of RA. In CV-1 cells, cotransfections of the expression plasmids for RAR and the C-subunits produced increases in RARECAT activity (12- and 8-fold in the absence of ligand and 21- and 15-fold in the presence of RA for the C alpha- and C beta-isoforms, respectively). Cotransfections of both the C beta-subunit and RAR expression plasmids in HeLa cells produced 22- and 114-fold increases in RARECAT activity in the absence and presence of RA, respectively. These results provide evidence to suggest that the RA and cAMP signaling pathways are coupled, and signaling cross-talk may occur through the direct phosphorylation of RARs by the C-subunit as indicated by in vitro phosphorylation of the receptor.

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 alpha (hRAR alpha), hRAR beta, and mouse RAR gamma (mRAR gamma) 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 (Kd) for RA were 1.4 nM for GST-hRAR alpha, 1.4 nM for GST-hRAR beta, and 3.3 nM for GST-mRAR gamma, 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 hRAR alpha 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 hRAR alpha and mRAR gamma receptors did not bind a naphthalene carboxylic acid derivative of RA, but hRAR beta 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.

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