Molecular determinants of the interactions between LXR/RXR heterodimers and TRAP220

Dimerization-induced activation of LXR is a unique allosteric mechanism described only for LXR/RXR heterodimers. Previously, we demonstrated that RXR functions as an allosteric activator of LXR binding to ASC-2 coactivator rather than as a direct interaction partner. Here, we investigated the molecular basis of the interaction between LXR/RXR and TRAP220 fragment (TN1/2) harboring two NR boxes. We found that either LXR binding to NR box-2 or RXR binding to NR box-1 was sufficient for optimal LXR/RXR binding to TN1/2, indicating that both receptors contribute equally in this interaction. Notably, the AF2 deletion of either receptor completely abolished LXR/RXR-TN1/2 interaction, suggesting dual roles for both AF2 domains in direct interaction with target NR boxes as well as in allosteric activation of partner receptors. We also found specific residues within NR box-2 required for LXR binding using one- plus two-hybrid system and identified Pro⁶⁴³ residue as a major determinant for NR specificity.     

The thyroid hormone receptor-associated protein TRAP220 is required at distinct embryonic stages in placental, cardiac, and hepatic development

Recent work indicates that thyroid hormone receptor-associated protein 220 (TRAP220), a subunit of the multiprotein TRAP coactivator complex, is essential for embryonic survival. We have generated TRAP220 conditional null mice that are hypomorphic and express the gene at reduced levels. In contrast to TRAP220 null mice, which die at embryonic d 11.5 (E11.5), hypomorphic mice survive until E13.5. The reduced expression in hypomorphs results in hepatic necrosis, defects in hematopoiesis, and hypoplasia of the ventricular myocardium, similar to that observed in TRAP220 null embryos at an earlier stage. The embryonic lethality of null embryos at E11.5 is due to placental insufficiency. Tetraploid aggregation assays partially rescues embryonic development until E13.5, when embryonic loss occurs due to hepatic necrosis coupled with poor myocardial development as observed in hypomorphs. These findings demonstrate that, for normal placental function, there is an absolute requirement for TRAP220 in extraembryonic tissues at E11.5, with an additional requirement in embryonic tissues for hepatic and cardiovascular development thereafter.     

An extended LXXLL motif sequence determines the nuclear receptor binding specificity of TRAP220

The interaction of coactivators with the ligand-binding domain of nuclear receptors (NRs) is mediated by amphipathic alpha-helices containing the signature motif LXXLL. TRAP220 contains two LXXLL motifs (LXM1 and LXM2) that are required for its interaction with NRs. Here we show that the nuclear receptor interaction domain (NID) of TRAP220 interacts weakly with Class I NRs. In contrast, SRC1 NID binds strongly to both Class I and Class II NRs. Interaction assays using nine amino acid LXXLL core motifs derived from SRC1 and TRAP220 revealed no discriminatory NR binding preferences. However, an extended LXM1 sequence containing amino acids -4 to +9, (where the first conserved leucine is +1) showed selective binding to thyroid hormone receptor and reduced binding to estrogen receptor. Replacement of either TRAP220 LXXLL motif with the corresponding 13 amino acids of SRC1 LXM2 strongly enhanced the interaction of the TRAP220 NID with the estrogen receptor. Mutational analysis revealed combinatorial effects of the LXM1 core and flanking sequences in the determination of the NR binding specificity of the TRAP220 NID. In contrast, a mutation that increased the spacing between TRAP220 LXM1 and LXM2 had little effect on the binding properties of this domain. Thus, a 13-amino acid sequence comprising an extended LXXLL motif acts as the key determinant of the NR binding specificity of TRAP220. Finally, we show that the NR binding specificity of full-length TRAP220 can be altered by swapping extended LXM sequences.