Protein-protein interactions involvingerbA superfamily members.

Abstract

The erbA superfamily constitutes a group of ligand-inducible transcriptional activators that includes the receptors for steroid and thyroid hormones, retinoic acid and vitamin D. Previous work has demonstrated that thyroid hormone receptors (TRs) require the presence of an additional nuclear factor to achieve high affinity DNA binding in vitro. This factor has been designated TRAP, which stands for T3 Receptor Auxiliary Protein. Using a variety of techniques, an association with TRAP from JEG-3 cells has been demonstrated for retinoic acid receptors (RARs) and vitamin D receptor (VDR), but not the estrogen receptor, v-erbA, or erbA-$\alpha$2. These receptors possess a conserved twenty amino acid region within the ligand binding domain which is required for TRAP activity in the TR. Mutational analysis indicates that RAR$\alpha$ and VDR also require the twenty amino acid domain in order to associate with TRAP. Deletion of this domain abrogates protein-protein interactions between these erbA receptors and TRAP, while point mutations attenuate the association to varying degrees. In a transient transfection system, mutant receptors are impaired in ligand dependent trans-activation without displaying a defect in ligand binding per se. The recent realization that the retinoid X receptor (RXR) possesses TRAP activity, coupled with the identification of 9-cis retinoic acid (RA) as the endogenous ligand for RXR, allowed us to ask whether 9-cis RA would affect T3-dependent trans-activation in transiently transfected cells. Transcriptional synergy was noted when T3 and 9-cis RA were added to cells transfected with TR$\beta$ and RXR$\beta$ only when the DNA response element contained an inverted repeat halfsite motif. For a response element with only directly repeated halfsites, RXR alone enhances T3 dependent trans-activation, but 9-cis RA is without additional effect. These results allow us to define a subset of the erbA superfamily that requires a nuclear protein from JEG-3 cells, possibly RXR, to exhibit high affinity DNA binding in vitro. Additionally, a highly conserved twenty amino acid domain present in all of these receptors is critical in mediating this protein-protein interaction. Finally, the ligand for RXR, 9-cis RA, has been shown to synergize with T3 in mediating trans-activation under certain conditions. These last results allow us to propose a mechanism for specificity of T3-dependent trans-activation within a cell depending upon the type of response element present in a given gene, and between cell types depending upon the availability of RXR and 9-cis RA.