Abstract
The aryl hydrocarbon receptor nuclear translocator (ARNT) serves as the obligate heterodimeric partner for bHLH-PAS proteins involved in sensing and coordinating transcriptional responses to xenobiotics, hypoxia, and developmental pathways. Although its C-terminal transactivation domain is dispensable for transcriptional activation in vivo, ARNT has recently been shown to use its N-terminal bHLH and/or PAS domains to interact with several transcriptional coactivators that are required for transcriptional initiation after xenobiotic or hypoxic cues. Here we show that ARNT uses a single PAS domain to interact with two coiled coil coactivators, TRIP230 and CoCoA. Both coactivators interact with the same interface on the ARNT PAS-B domain, located on the opposite side of the domain used to associate with the analogous PAS domain on its heterodimeric bHLH-PAS partner HIF-2alpha. Using NMR and biochemical studies, we identified the ARNT-interacting motif of one coactivator, TRIP230 as an LXXLL-like nuclear receptor box. Mutation of this motif and proximal sequences disrupts the interaction with ARNT PAS-B. Identification of this ARNT-coactivator interface illustrates how ARNT PAS-B is used to form critical interactions with both bHLH-PAS partners and coactivators that are required for transcriptional responses.
Highlights
aryl hydrocarbon receptor nuclear translocator (ARNT) PAS-A is considerably larger than PAS-B because of the presence of several long loops compared with the canonical PAS domain, and care was taken during subcloning to maintain the required secondary structure elements of the PAS fold
Simultaneous recruitment of multiple coactivators by different domains within transcription factor complexes is important for transcriptional activation, whereby interactions of varying stability are important for determining specificity of gene induction and cross-talk with other pathways [44, 45]
Within the basic helix-loop-helix (bHLH)-PAS family, the use of C-terminal activation domains to recruit coactivators is well documented [13,14,15], but PAS domains have been shown to contribute to the specificity of gene induction [22], suggesting that they too interact with coactivators
Summary
We demonstrate that the coiled coil coactivators TRIP230 and CoCoA bind a similar interface on ARNT that maps to the ␣-helical face of PAS-B. The ARNT interaction site was not defined on the 631-residue CoCoA protein [20]; a central coiled coil region critical for interaction with the bHLH-PAS domains of the GRIP1 coactivator is predicted to contain two distinct coiled coil regions [36].
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