Binding of type II nuclear receptors and estrogen receptor to full and half-site estrogen response elements in vitro.

The mechanism by which retinoids, thyroid hormone (T3) and estrogens modulate the growth of breast cancer cells is unclear. Since nuclear type II nuclear receptors, including retinoic acid receptor (RAR), retinoid X receptor (RXR) and thyroid hormone receptor (TR), bind direct repeats (DR) of the estrogen response elements (ERE) half-site (5'-AGGTCA-3'), we examined the ability of estrogen receptor (ER) versus type II nuclear receptors, i.e. RARalpha, beta and gamma, RXRbeta, TRalpha and TRbeta, to bind various EREs in vitro . ER bound a consensus ERE, containing a perfectly palindromic 17 bp inverted repeat (IR), as a homodimer. In contrast, ER did not bind to a single ERE half-site. Likewise, ER did not bind two tandem (38 bp apart) half-sites, but low ER binding was detected to three tandem copies of the same half-site. RARalpha,beta or gamma bound both ERE and half-site constructs as a homodimer. RXRbeta did not bind full or half-site EREs, nor did RXRbeta enhance RARalpha binding to a full ERE. However, RARalpha and RXRbeta bound a half-site ERE cooperatively forming a dimeric complex. The RARalpha-RXRbeta heterodimer bound the Xenopus vitellogenin B1 estrogen responsive unit, with two non-consensus EREs, with higher affinity than one or two copies of the full or half-site ERE. Both TRalpha and TRbeta bound the full and the half-site ERE as monomers and homodimers and cooperatively as heterodimers with RXRbeta. We suggest that the cellular concentrations of nuclear receptors and their ligands, and the nature of the ERE or half-site sequence and those of its flanking sequences determine the occupation of EREs in estrogen-regulated genes in vivo .     

The Asymmetric Binding of PGC-1α to the ERRα and ERRγ Nuclear Receptor Homodimers Involves a Similar Recognition Mechanism

Background

PGC-1α is a crucial regulator of cellular metabolism and energy homeostasis that functionally acts together with the estrogen-related receptors (ERRα and ERRγ) in the regulation of mitochondrial and metabolic gene networks. Dimerization of the ERRs is a pre-requisite for interactions with PGC-1α and other coactivators, eventually leading to transactivation. It was suggested recently (Devarakonda et al) that PGC-1α binds in a strikingly different manner to ERRγ ligand-binding domains (LBDs) compared to its mode of binding to ERRα and other nuclear receptors (NRs), where it interacts directly with the two ERRγ homodimer subunits.

Methods/Principal Findings

Here, we show that PGC-1α receptor interacting domain (RID) binds in an almost identical manner to ERRα and ERRγ homodimers. Microscale thermophoresis demonstrated that the interactions between PGC-1α RID and ERR LBDs involve a single receptor subunit through high-affinity, ERR-specific L3 and low-affinity L2 interactions. NMR studies further defined the limits of PGC-1α RID that interacts with ERRs. Consistent with these findings, the solution structures of PGC-1α/ERRα LBDs and PGC-1α/ERRγ LBDs complexes share an identical architecture with an asymmetric binding of PGC-1α to homodimeric ERR.

Conclusions/Significance

These studies provide the molecular determinants for the specificity of interactions between PGC-1α and the ERRs, whereby negative cooperativity prevails in the binding of the coactivators to these receptors. Our work indicates that allosteric regulation may be a general mechanism controlling the binding of the coactivators to homodimers.     

Characterization of the interaction between androgen receptor and a new transcriptional inhibitor, SHP.

SHP (short heterodimer partner) is an orphan nuclear receptor, first described for its interaction with nuclear receptors. This study explores a new way of inhibiting the androgen-signaling pathway. We demonstrated that SHP inhibited up to 97% of AR-induced activity. Characterization of AR/SHP interaction provided evidence of a clear ligand dependency. We also showed that the LXXI/LL motifs previously found on SHP mediated the interaction with the AR ligand-binding domain (AR-LBD), the motif responsible for the interaction being slightly different from that found with ER. The AR N-terminal domain (AR-NTD), in contrast to that of other nuclear receptors, accounts for most of the entire receptor transactivation potential. SHP also interacted with AR-NTD, thus stabilizing the interaction with AR. We demonstrated that SHP inhibited both AR-LBD and NTD-dependent transactivation, which evidenced for the first time a protein capable of inhibiting a steroid receptor amino-terminal-dependent transactivation. We further characterized the SHP mechanism of action by showing that SHP reversed AR coactivator-mediated activation. Conversely, FHL2 and TIF2 counteracted SHP-mediated inhibition of AR. SHP evidences a new way of inhibiting AR activity by competing with AR coactivators. This new type of inhibitor could dictate the activity of nuclear receptors, depending on the equilibrium between activators and inhibitors.