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

Abstract

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.

Retinoic Acid Restores Adult Hippocampal Neurogenesis and Reverses Spatial Memory Deficit in Vitamin A Deprived Rats

“…Species differences in RA sensitivity and/or differences in the dose of RA (150 µg vs 1 mg/kg/day) may explain the discrepancy between these studies. Furthermore, because 13-cis RA has a low affinity for RA receptors [48], the biological effects of these two retinoic acid isomers may also differ.…”

Cellular retinoid binding-proteins, CRBP, CRABP, FABP5: Effects on retinoid metabolism, function and related diseases

“…For example, 13- cis -retinoic acid (13cRA) binds CRABP1 and CRABP2 with affinities an order of magnitude or more lower than atRA, and has little if any affinity for RAR (Fiorella et al, 1993; Kim et al, 1994). These observations indicate that 13cRA, which converts into atRA in vivo, serves as a slow-release reservoir of atRA (i.e.…”

13-cis-Retinoic acid specific down-regulation of angiotensin type 1 receptor in rat liver epithelial and aortic smooth muscle cells

“…The primary isomers of retinoic acid formed in vivo are all-transretinoic acid (atRA) and 9-cis-retinoic acid (9cRA); each binds separate retinoic acid receptor types, thus acting upon a select subset of genes (Chambon 1996). The 13-cis-retinoic acid (13cRA) is a synthetic form that may function similar to the other isoforms, although the exact mechanism of action is unclear; in other words, its activity may be due to spontaneous isomerization to atRA or a novel activity of its own (Kim et al 1994, Levin 1995, Ganceviciene & Zouboulis 2007. 13cRA has been used in the past as a dermatological preparation applied orally or topically to influence skin growth factors in such a way as to lower sebum secretion (Landthaler et al 1980).…”