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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...

SummaryPhosphoinositide-specific phospholipases C are critical enzymes in the transduction of hormonal and environmental signals into animal cells. Several different isozymes of phospholipese C are known in mammalian systems which differ in their expression and regulation. Elucidation of the regulation of these phospholipases C has greatly advanced our understanding of the control of mammalian cell growth and development. Plant cells, too, contain phospholipeses C specific for phosphoinositides, and there is evidence that they may be involved in plant cell responses to environmental stimuli. This paper reports that there are at least four variants of phosphoinositidespecific phospholipase C in the roots of oat seedlings, two cytosolic and two plasma membrane-associated. The two cytosolic and two plasma membrane variants can be separated on the basis of their affinity for binding to heperin. Both the cytosolic and the plasma membrane heparin-binding forms have apparent molecular weights of about 50-70 kDa by size exclusion chromatography. The two heparin-binding forms have been partially purified. The partially purified enzymes are activated by micromolar calcium and are specific for phosphorylated phosphoinositides; in their substrate specificities they resemble mammalian phospholipases C s.