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

Abstract

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.

Importin 7 and Importin α/Importin β Are Nuclear Import Receptors for the Glucocorticoid Receptor

“…Although NL2 has not been precisely mapped, the NL2 region lacks sequences homologous to NL1 or to any of the identified importin 7 and importin 8 substrates. NL1 is closely related to the well-characterized NLS sequences of SV40 T-antigen and nucleoplasmin (Figure 1; Picard and Yamamoto, 1987;Tang et al, 1997). NL1 binds importin ␣ and Figure 3), which together with importin ␤ confer nuclear localization in digitonin permeablized cells ( Figure 5B).…”

“…It is not known how glucocorticoids regulate GR intracellular localization. GR has two nuclear localization signals and one nuclear export signal (Picard and Yamamoto, 1987;Tang et al, 1997;Black et al, 2001). The first nuclear localization signal, NL1 (amino acids 497-524 of rat GR), is located within the DNA-binding domain (DBD) and is similar in amino acid sequence to the SV40 NLS ( Figure 1A).…”

Nucleocytoplasmic Trafficking of Steroid-free Glucocorticoid Receptor

“…First, active retention mechanisms may limit the accessibility of 8 S GR to the nuclear-cytoplasmic transport machinery. For example, it has recently been shown that following withdrawal of hormonal ligand GRs become concentrated in a discrete nuclear subcompartment prior to their relocalization to the cytoplasm (12). Targeting 8 S GR to this subcompartment could promote the maintenance of the GR population in the nucleus by decreasing its access to the nuclear export machinery in a manner similar to that discussed above for DNA binding.…”

Thematic review series: Lipid Posttranslational Modifications. Farnesyl transferase inhibitors

“…This facilitates the folding of newly synthesized proteins and the folding of proteins as they translocate into organelles (111,112). These cochaperones also facilitate Hsp70-mediated nuclear trafficking and steroid receptor signaling (113,114).…”