Distinct binding determinants for 9-cis retinoic acid are located within AF-2 of retinoic acid receptor alpha.

Abstract

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...

Inhibition of ligand induced promoter occupancy in vivo by a dominant negative RXR

“…This region of TR is missing in v-erbA, thus eliminating the capacity of this receptor to bind to ligand and to activate transcription. However, mutation analysis of the TR as well as RAR indicates that the activation domain is independent of ligand binding (Barretino et al 1994;Tate et al 1994). The C-terminal region of RXRs has a similar function.…”

Retinoid Isomers Differ in the Ability to Induce Release of SMRT Corepressor from Retinoic Acid Receptor-α

“…Notably, the 9-cis-retinoic acid isomer displays a significantly different topography, manifested as an orthogonal bend in the center of the retinoid side chain, from that of the all-trans-isomer. Although the specific conformation of RAR␣ bound to 9-cis-retinoic acid has not been experimentally determined, molecular modeling and genetic analysis suggests that the 9-cis-isomer can be accommodated in the binding pocket of RAR␣ but that the 9-cis-isomer would differ in its contacts with receptor helices 3, 11, and 12 relative to those mediated by the all-trans-isomer (42,61). Given that these helices play important roles in the allosteric changes observed for RAR␣ on binding of all-trans-retinoic acid (41)(42)(43)(44)(45)(46), it is plausible that binding of the 9-cis-isomer may result in a distinct, if subtle, reorientation of one or more of these essential helices, resulting in impaired release of the SMRT corepressor.…”

Characterization of the chimeric retinoic acid receptor RARα/VDR

“…38 All cotransfections contained 0.5 g receptor expression plasmid, 5 g reporter plasmid (eg ␤(RARE) 3 -TK-LUC), 5 g control plasmid-, p␤Ac-lacZ, 4-5 g carrier plasmid-Bluescript IIKS, for a total of 15 g per 100 mm plate. Ligand-mediated transcriptional activity was expressed as either fold induction of normalized luciferase activity above that observed in the absence of ligand, or as a percentage of maximal normalized response.…”

“…40 Gel mobility shift assays COS-1 cells were transfected by the DEAE-dextran procedure with 10 g each of pSG5 (Control) or receptor expression vectors. Whole cell extracts were prepared, 38,43 and protein concentration determined 44 using a commercial Bradford reagent (BioRad, Richmond, VA, USA). Cells transfected with pSG5-RAR␣/ER or pSG5-ER/RAR␣/ER were treated with 10 −8 M ␤-estradiol for 1 h before harvesting.…”

“…45,46 The preincubation and incubation protocols for the whole cell extracts and RXR were as described in detail elsewhere. 38 Protein-DNA complexes were resolved on a native 5% (acrylamide-N,N′-methylenebisacrylamide 29:1) polyacrylamide gel (2 h at 20 mA). Gels were dried (1 h at 80°C) and exposed to film as described above.…”