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 dihydroxylated form of vitamin D3 (1,25-dihydroxy-D3)mediates a biological response by binding to intracellular receptors which belong to the steroid receptor superfamily. These receptors act as ligand-dependent transcription factors that bind to specific DNA sequences (reviewed in refs 6-9). We have identified two classes of vitamin D response elements that are activated either by the vitamin D receptor (VDR) alone or by heterodimers of VDR and the retinoid-X receptor-alpha (RXR-alpha). The motif GGGTGA arranged as a direct repeat with a spacing of six nucleotides or as a palindrome without spacing, or as an inverted palindrome with a 12-nucleotide spacing, confers vitamin D inducibility mediated by VDR alone. A second class of response elements, composed of directly repeated pairs of motifs (GGTCCA, AGGTCA, or GGGTGA) spaced by three nucleotides, is synergistically activated by RXR and VDR, but only in the presence of both ligands. Thus, the RXR ligand and the nature of the response element determine whether a nuclear receptor is co-regulated by RXR.

Lysosomotropic drugs such as NH4Cl have been useful for studying the role of low pH in early events in virus infection. NH4Cl blocks the production of infectious progeny virus in mammalian reovirus-infected cells. The inhibitory effect of NH4C1 is mediated by an inhibition of intracellular digestion of reovirus outer capsid proteins. In vitro digestion of viral outer capsid proteins produces infectious partially uncoated particles, called intermediate subviral particles, which are no longer inhibited by the presence of NH4Cl. These results indicate that proteolytic processing of reovirus outer capsid proteins takes place in a low pH compartment of the cell and is an essential step in the viral infectious cycle.

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