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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The synthesis of dilithium pentalenide, the reduction product of pentalene, by the reaction of dihydropentalene with n-butyllithium is described. Spectral properties of the pentalenyl dianion are presented.(1) Preliminary communication: T . J Katz and M . Rosenberger, J . A m . Chem. So< , 84, 865 (1962).( 2 ) For reviews see (a) the chapters hy E . D .