Vitamin A (retinol) and its natural derivatives are required for many physiological processes. The activity of retinoids is thought to be mediated by interactions with two subfamilies of nuclear retinoic acid receptors, RAR and RXR. The RARs bind all-trans retinoic acid (t-RA) with high affinity and alter gene expression as a consequence of this direct ligand interaction. RXR alpha is activated by t-RA, yet has little binding affinity for this ligand. t-RA may be converted to a more proximate ligand that directly binds and activates RXR alpha, and we have developed a method of nuclear receptor-dependent ligand trapping to test this hypothesis. Here we report the identification of a stereoisomer of retinoic acid, 9-cis retinoic acid, which directly binds and activates RXR alpha. These results suggest a new role for isomerization in the physiology of natural retinoids.

The binding of endogenous retinoids and stereoisomers of retinoic add (RA) to the retinoid nuclear receptors, RA receptor (RARs) and retinoid X receptors (RXRs), was characterized using nucleosol preparations from transiently transfected COS-1 cells. Among several stereoisomers of RA tested, including 7-cis-, 9-cis-, 11-cis-, 13-cis-, and all-trns-RA, only 9-cis-RA effectively competes with 9-cis-[3HJRA binding to the RXRs. Additionally, the endogenous retinoid trans-didehydro-RA (t-ddRA) does not interact with RXRs, whereas the 9-cis form of ddRA competes effectively. RXRs (a, (, and 'y) bind 9-cts-RA with dissociation constants (Kd) of 15.7, 18.3, and 14.1 nM, respectively. In contrast to the selectivity of RXRs for 9-cis-RA, RARs bind both t-RA and 9-cis-RA with high affinity, exhibitng Kd values in the 0.2-0.7 nM range for both ligands. Unlike RARs, the cellular RA bindig proteins CRARPI or CRABPH bind t-RA but do not bind 9-cis-RA. Consistent with the binding data, 9-cis-RA and 9-cis-ddRA tnscriptionally activate both GAL4-RXR and GAL4-RAR chimeric receptors with ECso values of 3-20 nM for 9-cis-RA and 9-cis-ddRA, whereas t-RA and t-ddRA efficiently activate only GAIA-RAR chimeric receptors. Thus, 9-cis forms of en s retinoids can contribute to the pleiotropic effects of retinoids by interacting with both the RARs and RXRs.Retinoids have a broad spectrum of biological activities in growth and differentiation of epithelia (1), embryonic development (2), and spermatogenesis (3). These effects are thought to result from interactions of retinoids with nuclear receptors (4, 5) that are members of the steroid-thyroid hormone superfamily ofreceptors and as such are considered to be ligand-dependent transcription factors (6, 7). One explanation for the diversity of retinoid action resides in the multiplicity of nuclear receptors (for a review, see ref. 8).

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We recently demonstrated in animal models that a new conformationally defined RA isomer (Vaezi et al. J. Med. Chem. 1994, 37, 4499-4507) was as effective as RA in the prevention of skin papillomas but was less toxic. In order to provide more details concerning this improved action, we report here the preparation of a homologous conformationally defined 6-s-trans-retinoid (1) and investigate its ability to interact with proteins and to activate gene expression. Four configurational isomers of 1 were evaluated in binding assays for cellular retinoic acid binding protein, CRABP (isolated from chick skin); CRABP-I and CRABP-II (cloned from mouse); nuclear retinoic acid receptors (RARs); and nuclear retinoid X receptors (RXRs). In each assay the all-E-isomer of this retinoid had an activity that was comparable to that of (all-E)-RA. However, the 9Z-isomer was at least 200-fold less active than (all-E)-RA in binding to different RARs, while it was only 6-20 times less active than (9Z)-RA in binding to different RXRs. In an in vivo transient transfection assay, the all-E-isomer activated a reporter gene containing a retinoic acid response element (RARE) with efficiency similar to (all-E)-RA when expression vectors for either RAR alpha, RAR beta, RAR gamma alone or RAR alpha together with RXR alpha were cotransfected. In contrast, the 9Z-isomer was much less active than (9Z)-RA in the same assay systems. However, (9Z)-1 efficiently enhanced the DNA binding and transactivational activity of RXR alpha homodimers. Taken together, these studies demonstrate that the all-E- and 9Z-isomers of this retinoid are selective and potent agonists of RAR and RXR binding and activation.

Retinoids exert their physiological action by interacting with two families of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), which regulate gene expression by forming transcriptionally active heterodimeric RAR/RXR or homodimeric RXR/RXR complexes on DNA. Retinoid receptor activity resides in several regions, including the DNA and ligand binding domains, a dimerization interface, and both a ligand-independent (AF-1) and a ligand-dependent (AF-2) transactivation function. While 9-cis retinoic acid (RA) alone is the cognate ligand for the RXRs, both 9-cis RA and all-trans RA (t-RA) compete for binding with high affinity to the RARs. This latter observation suggested to us that the two isomers may interact with a common binding site. Here we report that RARa has two distinct but overlapping binding sites for 9-cis RA and t-RA. Truncation of a human RARa to 419 amino acids yields a receptor that binds both t-RA and 9-cis RA with high affinity, but truncation to amino acid 404 yields a mutant receptor that binds only t-RA with high affinity. Remarkably, this region also defines a C-terminal boundary for AF-2, as addition of amino acids 405 to 419 restores receptor-mediated gene activity to a truncated human RARRa lacking this region. It is interesting to speculate that binding of retinoid stereoisomers to unique sites within an RAR may function with AF-2 to cause differential activation of retinoid-responsive gene pathways.Retinoic acids (RAs) are derivatives of vitamin A (retinol) which affect a wide spectrum of biological activities, including cellular differentiation and vertebrate development (for a review, see reference 48). These compounds exert their biological action by interacting with two families of nuclear receptors, the RA receptors (RARs) (8,18,27,42) (10,34,53), and the peroxisome proliferator-activated receptor (24,26).On the basis of the amino acid homologies, six regions of functional identity have been described for the members of this superfamily of nuclear receptors (16,19). Regions A and B contain a ligand-independent transactivation function, AF-1, that may play a role in specifying activity on particular target genes (38,39 (3,23,32), while the RARs bind both 9-cis RA and all-trans-RA (t-RA) with high affinity (3). We have shown that 9-cis RA and t-RA compete with each other for binding to the RARs (4), indicating that they share a common binding site. Considering the differences in configuration between 9-cis RA and t-RA, we reasoned that there may be subtle differences in the RAR binding pocket to accommodate both of these ligands. Here we report that in fact, 9-cis RA and t-RA interact with distinct binding determinants on RARc. Using C-terminal truncation mutants of human RARo, we defined a 15-amino-acid region that is absolutely required for 9-cis RA binding but not for high-affinity t-RA binding. Furthermore, we show that these same amino acids are required for the ligand-inducible, receptor-mediated transactivation of 3RARE and identify the AF...