Abstract
The mouse orphan nuclear receptor TR2-11 functions as a repressor for reporter genes containing a direct repeat-5 or direct repeat-4 hormone response element. The functional domains responsible for its suppressive activity are defined, including the DNA-binding domain and the ligand-binding domain. The C-terminal 30 amino acid residues can be deleted without compromising its suppressive activity, whereas a deletion for 40 amino acids completely abolishes the suppressive activity and receptor dimerization, and reduces the DNA-binding affinity. Point mutation at three conserved leucine residues located on the predicted dimer interface abolishes the suppressive activity, receptor dimerization and its DNA binding property. However, mutation at two consecutive glutamate residues located within the hinge between the last two helices of the ligand-binding domain (helix 10 and helix 11 according to the human retinoid receptor X alpha structure) drastically reduces its DNA-binding affinity and abrogates the suppressive activity without compromising its ability to dimerize, indicating that receptor dimerization property can be functionally uncoupled from its suppressive activity. A transferable, active silencing activity is encoded within the DEF segment of the receptor molecule, as evidenced by the suppression of a GAL4 reporter by a chimeric protein containing the DNA-binding domain of GAL4 and the DEF segment of TR2-11. Moreover, the C-terminal 49 amino acid sequence is required for this trans-suppressive activity. It is suggested that TR2-11 functions as a repressor, mediated by mechanisms requiring high affinity DNA binding, receptor dimerization, and active silencing.
Highlights
Binding domain (DBD)1 in the middle of the receptor molecule, and a C-terminal ligand-binding domain (LBD) [1, 2]
Dissection of Functional Domains for the Suppressive Activity of TR2-11—In our previous studies, we have demonstrated that TR2-11 exerted a strongly suppressive activity on the reporters containing either a direct repeat-5 (DR5) type RARE [18] or a DR4 type hormone response element [22]
To first define the functional domains of TR2-11 that were required for its suppressive activity, we began by systematic deletions from the N
Summary
Binding domain (DBD) in the middle of the receptor molecule, and a C-terminal ligand-binding domain (LBD) [1, 2]. The LBD determines the ligand specificity by formation of a highly specified binding pocket and mediates receptor dimerization by interacting at the dimerization interface located on the LBD surface. In their apo-forms, nuclear receptors function as repressors by interacting with corepressors, such as the nuclear receptor corepressor (N-CoR) [7] and silencing mediator for retinoid and thyroid hormone receptors (SMRT) [8]. Our previous studies have shown that the full-length receptor strongly suppresses reporters containing either a direct repeat-5 (DR5) [18, 19] derived from the human RAR gene promoter [21], or a DR4 type response element [22] derived from the mouse cellular retinoic acid-binding protein-I gene promoter [23]. We explored the possibility of its interaction with the common corepressor NCoR as well as heterodimerization with RAR/RXR family
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