The Specificity of Interactions between Nuclear Hormone Receptors and Corepressors Is Mediated by Distinct Amino Acid Sequences within the Interacting Domains

Abstract

The thyroid hormone receptor (TR) and retinoic acid receptor (RAR) isoforms interact with the nuclear corepressors [NCoR (nuclear corepressor protein) and SMRT (silencing mediator for retinoid and thyroid hormone receptors)] in the absence of ligand to silence transcription. NCoR and SMRT contain C-terminal nuclear hormone receptor (NHR) interacting domains that each contain variations of the consensus sequence I/L-x-x-I/V-I (CoRNR box). We have previously demonstrated that TRbeta1 preferentially interacts with NCoR, whereas RARalpha prefers SMRT. Here, we demonstrate that this is due, in part, to the presence of a novel NCoR interacting domain, termed N3, upstream of the previously described domains. An analogous domain is not present in SMRT. This domain is specific for TR and interacts poorly with RAR. Our data suggest that the presence of two corepressor interacting domains are necessary for full interactions with nuclear receptors in cells. Interestingly, mutation of N3 alone specifically decreases binding of NCoR to TR in cells but does not decrease NCoR-RAR interactions. In addition, while the exact CoRNR box sequence of a SMRT interacting domain is critical for recruitment of SMRT by RAR, the CoRNR box sequences themselves do not explain the strong interaction of the N2 domain with TRbeta1. Additional regions distal to the CoRNR box sequence are needed for optimal binding. Thus, through sequence differences in known interacting domains and the presence of a newly identified interacting domain, NCoR is able to preferentially bind TRbeta1. These preferences are likely to be important in corepressor action in vivo.

Thyroid Hormone Receptor DNA Binding Is Required for Both Positive and Negative Gene Regulation

“…Construction of nuclear receptor corepressor (NCoR), steroid receptor coactivator-1 (SRC-1), and RXR-␣ Gal4BD fusion expression vectors was described previously (37). The VP16AD-GS125 mutant TR-␤2 was created by exchanging appropriate restriction fragments within the VP16AD-TR-␤2 construct, which has been reported previously (38).…”

The nuclear corepressor, NCoR, regulates thyroid hormone action in vivo

“…Extensive analysis has disclosed the presence in both molecules of 3 RIDs that can be expressed alternatively in a tissue-specific manner (18)(19)(20)(21). Despite their similarity the RIDs do not function equivalently.…”

“…Despite their similarity the RIDs do not function equivalently. TR has been shown to preferably bind NCoR both in vitro and in mammalian cell lines (22,23) via the most 5Ј of its RIDs, N3, which is required for strong interactions with DNA-bound TR (18,21,24). Importantly, N3 must cooperate with another RID, preferably N2, to interact with a TR homodimer on DNA as each TR binds to 1 of the RIDs (18,(21)(22)(23)(24)(25).…”

Multiple mutations contribute to repression by the v-Erb A oncoprotein

“…Whereas c-Erb A binds N-CoR more strongly than SMRT (Cohen et al, 1998(Cohen et al, , 2000(Cohen et al, , 2001Webb et al, 2000;Makowski et al, 2003;Goodson et al, 2005;Zamir et al, 1997), we found that v-Erb A displayed a nearequal interaction with SMRT as with N-CoR. Restoring the c-Erb A helix 12 to v-Erb A restored the c-Erb A preference for N-CoR.…”

“…As anticipated, both v-Erb A SMRT corepressor is closely related to N-CoR but differs in its affinity for different nuclear receptors (Privalsky, 2004). Although alternative mRNA splicing can produce corepressors with differing receptor affinities, c-Erb A generally interacts more strongly with NCoR than with SMRT (Zamir et al, 1997;Cohen et al, 1998Cohen et al, , 2000Cohen et al, , 2001Webb et al, 2000;Makowski et al, 2003;Goodson et al, 2005). To examine this question for v-Erb A, we tested SMRT constructs in place of NCoR in our EMSA supershifts.…”