Two Receptor Interaction Domains in the Corepressor, N-CoR/RIP13, Are Required for an Efficient Interaction with Rev-erbA  and RVR: Physical Association is Dependent on the E Region of the Orphan Receptors

Abstract

Rev-erbA alpha and RVR/Rev-erb beta/BD73 are orphan steroid receptors that have no known ligands in the 'classical sense'. These 'orphans' do not activate transcription, but function as dominant transcriptional silencers. The thyroid hormone receptor (TR) and the retinoic acid receptor (RAR) act as transcriptional silencers by binding corepressors (e.g. N-CoR/RIP13 and SMRT/TRAC-2) in the absence of ligands. The molecular basis of repression by orphan receptors, however, remains obscure, and it is unclear whether these corepressors mediate transcriptional silencing by Rev-erbA alpha and RVR. Recently, two new variants of N-CoR have been described, RIP13a and RIP13delta1. The characterisation of these splice variants has identified a second receptor interaction domain (ID-II), in addition to the previously characterised interaction domain (ID-I). This investigation utilised the mammalian two hybrid system and transfection analysis to demonstrate that Rev-erbA alpha and RVR will not efficiently interact with either ID-I or ID-II separately from RIP13a or RIP13delta1. However, they interact efficiently with a domain composed of ID-I and ID-II from RIP13a. Interestingly, the interaction of Rev-erbA alpha and RVR is strongest with ID-I and ID-II from RIP13delta1. Detailed deletion analysis of the orphan receptor interaction with RIP13/N-CoR rigorously demonstrated that the physical association was critically dependent on an intact E region of Rev-erbA alpha and RVR. Over-expression of the corepressor interaction domains (i.e. dominant negative forms of N-CoR/RIP13) could alleviate orphan receptor-mediated repression of transactivation by GALVP16. This demonstrated that these regions could function as anti-repressors. In conclusion, these data from two independent approaches demonstrate that repression by Rev-erbA alpha and RVR is mediated by an interaction of ID-I and ID-II of N-CoR, RIP13a and delta1 with the putative ligand binding domain of the orphan receptors.

Functional Domains of the Human Orphan Receptor ARP-1/ COUP-TFII Involved in Active Repression and Transrepression

“…This finding is in line with the fact that the conserved motif Ala223-His224-Thr227 (66), located at the carboxy-terminal end of H1 in TR␤, which is essential for binding of the corepressor, is absent in H1 of ARP-1/COUP-TFII. This observation, however, does not preclude interaction of a corepressor protein with a different domain within the carboxy terminus of ARP-1/COUP-TFII, as has been demonstrated for Rev-erbA␣ or RVR (13). Second, deletion of the AF-2 AD core of TR␤ results in a strong constitutive silencer, which is unable to release a corepressor protein and to activate transcription upon binding of a ligand (3,4,66), while the presence of the AF-2 AD core is critical for repressor activity of ARP-1/COUP-TFII.…”

“…Several potential coactivator proteins, which are able to interact with AF-2 domains of various nuclear receptors in a ligand-dependent fashion, and two putative corepressors contacting the hinge regions of TR and RAR have been identified (8,9,21,28,29,46). Recently Downes and coworkers reported that integrity of the E region of Rev-erbA␣ is essential for its interaction with N-CoR/RIP13 (13).…”

Alternative mRNA Splicing of SMRT Creates Functional Diversity by Generating Corepressor Isoforms with Different Affinities for Different Nuclear Receptors

“…Alternative mRNA Splicing Is a General Means of Diversifying Corepressor Expression-In common with SMRT, there are also alternatively spliced isoforms of N-CoR that differ in their interaction with nuclear receptors (87)(88)(89). Intriguingly, this alternative mRNA splicing of N-CoR operates through a very distinct mode from that described here for SMRT.…”

“…Instead, N-CoR is expressed as two isoforms (denoted N-CoR and RIP13⌬1) that differ by the inclusion or exclusion of a third receptor interaction domain (N3) that is absent from all known SMRT isolates (32). The longer N-CoR isoform interacts strongly with TRs, primarily through N3 and N2 contacts, whereas the alternatively spliced RIP13⌬1 isoform lacks the N3 receptor interaction domain and interacts only weakly with TRs (32,89). Conversely, RIP13⌬1 interacts more strongly than does N-CoR with the orphan nuclear receptors COUP-TF (chicken ovalbumin upstream promoter transcription factor) and Rev-ErbA (90,91).…”

Retinoid Isomers Differ in the Ability to Induce Release of SMRT Corepressor from Retinoic Acid Receptor-α

“…The bimodal transcriptional properties of the nuclear hormone receptors reflect the ability of these receptors to physically recruit auxiliary proteins, denoted corepressors and coactivators, that help mediate the actual transcriptional response (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). In the absence of cognate hormone, thyroid hormone receptors and RARs bind to a corepressor complex composed of SMRT and/or N-CoR, mSin3A or B, histone deacetylase 1 or 2, Rb-associated proteins p46 and p48, and a number of additional proteins of as-yet unknown function (reviewed in Refs.…”