Functional studies on the ligand-binding domain of Ultraspiracle from Drosophila melanogaster

The functional insect ecdysteroid receptor is comprised of the ecdysone receptor (EcR) and Ultraspiracle (USP). The ligand-binding domain (LBD) of USP was fused to the GAL4 DNA-binding domain (GAL4-DBD) and characterized by analyzing the effect of site-directed mutations in the LBD. Normal and mutant proteins were tested for ligand and DNA binding, dimerization, and their ability to induce gene expression. The presence of helix 12 proved to be essential for DNA binding and was necessary to confer efficient ecdysteroid binding to the heterodimer with the EcR (LBD), but did not influence dimerization. The antagonistic position of helix 12 is indispensible for interaction between the fusion protein and DNA, whereas hormone binding to the EcR (LBD) was only partially reduced if fixation of helix 12 was disturbed. The mutation of amino acids, which presumably bind to a fatty acid evoked a profound negative influence on transactivation ability, although enhanced transactivation potency and ligand binding to the ecdysteroid receptor was impaired to varying degrees by mutation of these residues. Mutations of one fatty acid-binding residue within the ligand-binding pocket, 1323, however, evoked enhanced transactivation. The results confirmed that the LBD of Ultraspiracle modifies ecdysteroid receptor function through intermolecular interactions and demonstrated that the ligand-binding pocket of USP modifies the DNA-binding and transactivation abilities of the fusion protein.     

Evidence for DNA-binding domain--ligand-binding domain communications in the androgen receptor

DNA binding as well as ligand binding by nuclear receptors has been studied extensively. Both binding functions are attributed to isolated domains of which the structure is known. The crystal structure of a complete receptor in complex with its ligand and DNA-response element, however, has been solved only for the peroxisome proliferator-activated receptor γ (PPARγ)-retinoid X receptor α (RXRα) heterodimer. This structure provided the first indication of direct interactions between the DNA-binding domain (DBD) and ligand-binding domain (LBD). In this study, we investigated whether there is a similar interface between the DNA- and ligand-binding domains for the androgen receptor (AR). Despite the structural differences between the AR- and PPARγ-LBD, a combination of in silico modeling and docking pointed out a putative interface between AR-DBD and AR-LBD. The surfaces were subjected to a point mutation analysis, which was inspired by known AR mutations described in androgen insensitivity syndromes and prostate cancer. Surprisingly, AR-LBD mutations D695N, R710A, F754S, and P766A induced a decrease in DNA binding but left ligand binding unaffected, while the DBD-residing mutations K590A, K592A, and E621A lowered the ligand-binding but not the DNA-binding affinity. We therefore propose that these residues are involved in allosteric communications between the AR-DBD and AR-LBD.     

Ligand-independent orphan receptor TR2 activation by phosphorylation at the DNA-binding domain

In a previous report we demonstrated protein kinase C (PKC)-mediated phosphorylation of the ligand-binding domain (LBD) of orphan nuclear receptor TR2. In this report, we provide the evidence of PKC-mediated phosphorylation of the DNA-binding domain (DBD) of TR2. Two PKC target sites were predicted within the DBD, at Ser-170 and Ser-185, but only Ser-185 was confirmed by MS. Phosphorylation of DBD facilitated DNA binding of the TR2 receptor and its recruiting of coactivator p300/CBP-associated factor (P/CAF). Ser-185 was required for DNA binding, whereas both Ser-170 and Ser-185 were necessary for receptor interaction with P/CAF. The P/CAF-interacting domain of TR2 was located in its DBD. A double mutant (Ser-170 and Ser-185) of TR2 significantly lowered the activation of its target gene RARbeta2. This study provides the first evidence for ligand-independent activation of TR2 orphan receptor through PTM at the DBD, which enhanced its DNA-binding ability and interaction with coactivator P/CAF.     

A role for HDJ-2/HSDJ in correcting subnuclear trafficking, transactivation, and transrepression defects of a glucocorticoid receptor zinc finger mutant.

All steroid receptors possess a bipartite nuclear localization signal sequence (NLS) that localizes within the second zinc finger of their DNA-binding domain. Fine-structure mapping of the rat glucocorticoid receptor (rGS) NLS identified a composite signal composed of three distinct proto-NLSs that function effectively when present in unique pairs. At least one of the rGR proto-NLSs appears to influence receptor trafficking within the nucleus, as revealed by a unique nuclear staining pattern of receptors possessing a point mutation (i.e., arginine at position 496; R496), at proto-NLS, pNLS-2. Specifically, carboxyl-terminal-truncated rGRs possessing various point mutations at R496 localized within a limited number of large foci in nuclei of transiently transfected COS-1 cells. R496 mutations did not affect subnuclear targeting when present in full-length rGR, reflecting a protective effect of the receptor's ligand-binding domain that can be exerted in cis and in trans. The effects of rGR R496 mutations on subnuclear targeting were not autonomous because we also observed a coincident localization of hsp70, the 70-kDa heat shock protein, within nuclear foci that include r496 mutant receptors. The elimination of R496 mistargeting by overexpression of an hsp70 partner (i.e., the DnaJ homologue, HDJ-2/HSDJ) suggests that the hsp70/DnaJ chaperone system is mobilized to specific sites within the nucleus in response to inappropriate targeting or folding of specific mutant receptors. HDJ-2/HSDJ overexpression also corrects defective transactivation and transrepression activity of R496 mutant GRs. Thus, molecular chaperones, such as members of the hsp70 and DnaJ families, may survey the nucleus for misfolded proteins and actively participate in their refolding into biologically active conformational states.