Nuclear hormone receptor-specific interactions of the coactivator TRAP220

The TRAP220 subunit of the metazoan mediator complex contains two LXXLL motifs (LXM1 and LXM2) that are required for its ligand-dependent interaction with NRs. Transient transfection experiments revealed that whilst over-expression of TRAP220 in mammalian cells resulted in enhanced transcriptional activity of the class II NR, thyroid hormone receptor ss (TRss), TRAP220 had little effect on the transcriptional activity of the class I NR, estrogen receptor alpha (ERalpha). TRAP220 interacts weakly with class I NRs, whereas interaction with class II NRs is strong. By contrast SRC1 bound strongly to both class I and class II NRs. Hence TRAP220 displays NR-class specific binding properties. Interaction assays using LXXLL core motifs (9 amino acids) derived from SRC1 and TRAP220 showed no discriminatory NR-binding preferences. However an extended TRAP220 LXM1 sequence containing amino acids -4 to +9 (where the first conserved leucine of the LXXLL motif is +1) showed selective binding to TRss and reduced binding to ERalpha. Taken together this suggested that the amino acids immediately adjacent to the core LXM1 sequence were contributing to the NR-binding selectivity of TRAP220. Mutational analyses revealed that exchange of either TRAP220 extended LXXLL sequences (13 amino acids) with the SCR1 extended LXM2 sequence, strongly enhanced interaction with ERalpha, and that amino acids within and flanking the LXM1 core sequence cooperated to achieve this change in NR-binding specificity. In contrast, a mutation that increased the spacing between TRAP220 LXM1 and LXM2 had little effect on the binding properties of the nuclear receptor interaction domain (NID).