Discrimination between NL1- and NL2-Mediated Nuclear Localization of the Glucocorticoid Receptor

Abstract

Glucocorticoid receptor (GR) cycles between a free liganded form that is localized to the nucleus and a heat shock protein (hsp)-immunophilin-complexed, unliganded form that is usually localized to the cytoplasm but that can also be nuclear. In addition, rapid nucleocytoplasmic exchange or shuttling of the receptor underlies its localization. Nuclear import of liganded GR is mediated through a well-characterized sequence, NL1, adjacent to the receptor DNA binding domain and a second, uncharacterized motif, NL2, that overlaps with the ligand binding domain. In this study we report that rapid nuclear import (half-life [t 1/2 ] of 4 to 6 min) of agonist-and antagonist-treated GR and the localization of unliganded, hsp-associated GRs to the nucleus in G 0 are mediated through NL1 and correlate with the binding of GR to pendulin/importin ␣. By contrast, NL2-mediated nuclear transfer of GR occurred more slowly (t 1/2 ‫؍‬ 45 min to 1 h), was agonist specific, and appeared to be independent of binding to importin ␣. Together, these results suggest that NL2 mediates the nuclear import of GR through an alternative nuclear import pathway. Nuclear export of GR was inhibited by leptomycin B, suggesting that the transfer of GR to the cytoplasm is mediated through the CRM1-dependent pathway. Inhibition of GR nuclear export by leptomycin B enhanced the nuclear localization of both unliganded, wild-type GR and hormone-treated NL1 ؊ GR. These results highlight that the subcellular localization of both liganded and unliganded GRs is determined, at least in part, by a flexible equilibrium between the rates of nuclear import and export.The predominant pathway for the nuclear import of transcription factors and other nuclear regulatory proteins originates with the interaction of importin ␣-like proteins (also called karyopherin ␣, Rch1/hSRP␣, hSRP1/NPI-1, and pendulin/OHO31) with specific nuclear localization sequences (NLSs), which contain closely spaced arrangements of five to eight basic amino acids (31,62,64). For DNA sequence-specific transcription factors, NLSs generally colocalize with their DNA binding domains (DBDs), which appears to reflect a coevolutionary selective pressure to ensure that proteins that bind DNA are able to access the nucleus (52). Nuclear export, by contrast, occurs through alternative pathways, which for many proteins involves the binding of CRM1 (exportin 1) to hydrophobic nuclear export sequences (26,90).However, some transcription factors, including the glucocorticoid hormone receptor (GR), contain additional NLSs that occur in other regions of the proteins (69,89,95,99). In at least some instances, the presence of these additional NLSs has been found to reflect a requirement for specialized or tightly regulated nuclear localization of the protein. For example, the nuclear localization potential of one of the two NLSs in the adenovirus E1A protein is active only during early development (92), while two of the three c-abl NLSs promote nuclear localization of c-abl only in certain cell types (97, 99). T...

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

“…Thus, the hormone-regulated step for nuclear localization appears to be downstream of import receptor binding. For example, GR has been proposed to be rapidly exported from the nucleus in the absence of hormone, although evidence for this model remains controversial Savory et al, 1999;Liu and DeFranco, 2000;Holaska et al, 2001). A nuclear export sequence has been identified in GR ), but its role in regulating GR localization in the absence of hormone, remains to be determined.…”

Cross-talk between nuclear receptors and nuclear factor κB

“…However, recent data indicate that, in fact, a continuous dynamic NR shuttling between the nuclear and cytoplasmic compartments takes place, and, as hypothesized for ER, this may allow the receptor to exert both genomic (nuclear) and non-genomic (cytoplasmic) activities (Lo¨sel and Wehling, 2003;Leclercq et al, 2006). For GR too, the subcellular localization of both liganded and unliganded receptors is at any given time the net result of a dynamic equilibrium determined by the rates of nuclear import and export (Savory et al, 1999), although the import/ export mechanisms are not precisely known (Liu and DeFranco, 1999).…”

Nontranscriptional actions of the glucocorticoid receptor

“…Hormone binding to the LBD leads to a conformational change in the receptor followed by dissociation of the multiprotein complex and rapid nuclear entry. Nuclear translocation is mediated by two nuclear localization sequences located adjacent to the DBD, and in the LBD, respectively [31,32]. Interestingly, hormone-bound GR is not permanently localized to the nucleus.…”