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Retinoid X receptors (RXRα, β, and γ) are essential members of the nuclear receptor (NR) superfamily of ligand-dependent transcriptional regulators that bind DNA response elements and control the expression of large gene networks. As obligate heterodimerization partners of many NRs, RXRs are involved in a variety of pathophysiological processes. However, despite this central role in NR signaling, there is still no consensus regarding the precise biological functions of RXRs and the putative role of the endogenous ligands (rexinoids) previously proposed for these receptors. Based on available crystal structures, we introduced a series of amino acid substitutions into the ligand-binding pocket of all three RXR subtypes in order to alter their binding properties. Subsequent characterization using a battery of cell-based and in vitro assays led to the identification of a double mutation abolishing the binding of any ligand while keeping the other receptor functions intact and a triple mutation that selectively impairs interaction with natural rexinoids but not with some synthetic ligands. We also report crystal structures that help understand the specific ligand-binding capabilities of both variants. These RXR variants, either fully disabled for ligand binding or retaining the property of being activated by synthetic compounds, represent unique tools that could be used in future studies to probe the presence of active endogenous rexinoids in tissues/organs and to investigate their role in vivo. Last, we provide data suggesting a possible involvement of fatty acids in the weak interaction of RXRs with corepressors.

The nuclear receptor (NR) coactivator TIF2 possesses a single NR interaction domain (NID) and two autonomous activation domains, AD1 and AD2. The TIF2 NID is composed of three NR-interacting modules each containing the NR box motif LxxLL. Mutation of boxes I, II and III abrogates TIF2-NR interaction and stimulation, in transfected cells, of the ligand-induced activation function-2 (AF-2) present in the ligand-binding domains (LBDs) of several NRs. The presence of an intact NR interaction module II in the NID is sufficient for both efficient interaction with NR holo-LBDs and stimulation of AF-2 activity. Modules I and III are poorly efficient on their own, but synergistically can promote interaction with NR holo-LBDs and AF-2 stimulation. TIF2 AD1 activity appears to be mediated through CBP, as AD1 could not be separated mutationally from the CBP interaction domain. In contrast, TIF2 AD2 activity apparently does not involve interaction with CBP. TIF2 exhibited the characteristics expected for a bona fide NR coactivator, in both mammalian and yeast cells. Moreover, in mammalian cells, a peptide encompassing the TIF2 NID inhibited the ligand-induced AF-2 activity of several NRs, indicating that NR AF-2 activity is either mediated by endogenous TIF2 or by coactivators recognizing a similar surface on NR holo-LBDs.

The activity of the ligand-inducible activation function 2 (AF-2) contained in the ligand binding domain (LBD) of nuclear receptors (NRs) is thought to be mediated by transcriptional intermediary factors (TIFs). We have recently reported the isolation and characterization of two novel mouse proteins, designated TIF1 and mSUG1, that interact in a ligand-dependent fashion with the LBD (region E) of several NRs in vivo as well as in vitro. Remarkably, these interactions require the conserved core motif of the AF-2 activating domain (AF-2 AD) and can be blocked by AF-2 antagonists. TIF1 and mSUG1 might therefore represent TIFs/mediators for the ligand-dependent AF-2 of NRs. By comparing the interaction properties of these two putative TIFs with different NRs including the oestrogen (ER), thyroid hormone (TR), vitamin D3 (VDR), retinoic acid (RAR alpha) and retinoid X (RXR) receptors, we demonstrate that: (i) RXR alpha efficiently interacts with TIF1, but not with mSUG1, whereas TR alpha interacts much more efficiently with mSUG1 than with TIF1, and RAR alpha, VDR and ER efficiently interact with both TIF1 and mSUG1; (ii) the amphipathic alpha helix core of AF-2 AD is differentially involved in the interactions of RAR alpha with TIF1 and mSUG1; and (iii) the AF-2 AD cores of RAR alpha and ER are similarly involved in their interaction with TIF1, but not with mSUG1. Thus the interaction interfaces between the various NRs and either TIF1 or mSUG1 may vary depending on the nature of both the receptor and the putative mediator of its AF-2 function. We discuss the possible roles of TIF1 and mSUG1 as mediators of the transcriptional activity of the AF-2 of NRs.

The ability of nuclear receptors (NRs) to activate transcription of target genes requires the binding of cognate ligands to their ligand-binding domains (LBDs). Information provided by the three-dimensional structures of the unliganded RXR alpha and the liganded RAR gamma LBDs has been incorporated into a general alignment of the LBDs of all NRs. A twenty amino-acid region constitutes a NR-specific signature and contains most of the conserved residues that stabilize the core of the canonical fold of NR LBDs. A common ligand-binding pocket, involving predominantly hydrophobic residues, is inferred by homology modelling of the human RXR alpha and glucocorticoid receptor ligand-binding sites according to the RAR gamma holo-LBD structure. Mutant studies support these models, as well as a general mechanism for ligand-induced activation deduced from the comparison of the transcriptionally active RAR gamma holo- and inactive RXR alpha apo-LBD structures.