Importin 7 and importin alpha/importin beta are nuclear import receptors for the glucocorticoid receptor

The vertebrate glucocorticoid receptor (GR) is cytoplasmic without hormone and localizes to the nucleus after hormone binding. GR has two nuclear localization signals (NLS): NL1 is similar in sequence to the SV40 NLS; NL2 is poorly defined, residing in the ligand-binding domain. We found that GR displayed similar hormone-regulated compartmentalization in Saccharomyces cerevisiae and required the Sxm1 nuclear import receptor for NL2-mediated import. Two metazoan homologues of Sxm1, importin 7 and importin 8, bound both NL1 and NL2, whereas importin alpha selectively bound NL1. In an in vitro nuclear import assay, both importin 7 and the importin alpha-importin beta heterodimer could import a GR NL1 fragment. Under these conditions, full-length GR localized to nuclei in the presence but not absence of an unidentified component in cell extracts. Interestingly, importin 7, importin 8, and importin alpha bound GR even in the absence of hormone; thus, hormonal control of localization is exerted at a step downstream of import receptor binding.     

Evidence for NL1-independent nuclear translocation of the mineralocorticoid receptor

In the absence of hormone, corticosteroid receptors are primarily located in the cytoplasm, and they rapidly accumulate in the nucleus (t0.5 = 5 min) upon ligand binding. It is generally believed that the dissociation of hsp90 from the receptor is an absolute requirement for allowing its nuclear translocation. However, recent evidence suggests that hsp90 may remain associated with the glucocorticoid receptor during this process, and thus, the receptor nuclear localization signal (NLS) is not obscured by its presence. To determine the requirements for mineralocorticoid receptor (MR) nuclear transport, it was first shown that in rat kidney collecting duct cells, nuclear localization of MR in the presence of aldosterone was complete in 10 min. Although the hsp90 inhibitor radicicol delayed nuclear translocation, it did not prevent complete nuclear accumulation of MR at longer incubation times (t0.5 = 30-40 min). MR carbamylation generates a non-steroid-transformed receptor that, in contrast to native MR, is very stable in cell-free systems. In contrast to the full nuclear translocation of aldosterone-transformed MR, only a fraction of the carbamylated MR became nuclear in digitonin-permeabilized cells even though its NLS is exposed. Furthermore, while preincubation of permeabilized cells with NL1 peptide or anti-NL1 antibody fully inhibited the nuclear translocation of NL1-tagged albumin, neither treatment fully inhibited MR nuclear translocation. We postulate that there are at least two possible mechanisms for MR nuclear translocation. One of them is hsp90- and NL1-dependent, and the other functions in a manner that is independent of the classical pathway.     

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

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 G₀ are mediated through NL1 and correlate with the binding of GR to pendulin/importin α. 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 α. 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.     

Identification of nuclear import and export signals within Fli-1: roles of the nuclear import signals in Fli-1-dependent activation of megakaryocyte-specific promoters

The Ets factor Friend leukemia integration 1 (Fli-1) is an important regulator of megakaryocytic (Mk) differentiation. Here, we demonstrate two novel nuclear localization signals (NLSs) within Fli-1: one (NLS1) is located at the N terminus, and another (NLS2) is within the Ets domain. Nuclear accumulation of Fli-1 reflected the combined functional effects of the two discrete NLSs. Each NLS can independently direct nuclear transport of a carrier protein, with mutations within the NLSs affecting nuclear accumulation. NLS1 has a bipartite motif, whereas the NLS2 region contains a nonclassical NLS. Both NLSs bind importin alpha (IMPalpha) and IMPbeta, with NLS1 and NLS2 being predominantly recognized by IMPalpha and IMPbeta, respectively. Fli-1 also contains one nuclear export signal. Leptomycin B abolished its cytoplasmic accumulation, showing CRM1 dependency. We demonstrate that Ets domain binding to specific target DNA effectively blocks IMP binding, indicating that the targeted DNA binding plays a role in localizing Fli-1 to its destination and releasing IMPs for recycling back to the cytoplasm. Finally, by analyzing full-length Fli-1 carrying NLS1, NLS2, and combined NLS1-NLS2 mutations, we conclude that two functional NLSs exist in Fli-1 and that each NLS is sufficient to target Fli-1 to the nucleus for activation of Mk-specific genes.     

Nuclear localization signal receptor affinity correlates with in vivo localization in Saccharomyces cerevisiae

Nuclear localization signals (NLSs) target proteins into the nucleus through mediating interactions with nuclear import receptors. Here, we perform a quantitative analysis of the correlation between NLS receptor affinity and the steady-state distribution of NLS-bearing cargo proteins between the cytoplasm and the nucleus of live yeast, which reflects the relative import rates of various NLS sequences. We find that there is a complicated, but monotonic quantitative relationship between the affinity of an NLS for the import receptor, importin alpha, and the steady-state accumulation of the cargo in the nucleus. This analysis takes into consideration the impact of protein size. In addition, the hypothetical upper limit to an NLS affinity for the receptors is explored through genetic approaches. Overall, our results indicate that there is a correlation between the binding affinity of an NLS cargo for the NLS receptor, importin alpha, and the import rate for this cargo. This correlation, however, is not maintained for cargoes that bind to the NLS receptor with very weak or very strong affinity.     

Nuclear localization and the heat shock proteins

The highly conserved heat shock proteins (HSP) belong to a subset of cellular proteins that localize to the nucleus. HSPs are atypical nuclear proteins in that they localize to the nucleus selectively, rather than invariably. Nuclear localization of HSPs is associated with cell stress and cell growth. This aspect of HSPs is highly conserved with nuclear localization occurring in response to a wide variety of cell stresses. Nuclear localization is likely important for at least some of the heat shock proteins’ protective functions; little is known about the function of the heat shock proteins in the nucleus. Nuclear localization is signalled by the presence of a basic nuclear localization sequence (NLS) within a protein. Though most is known about HSP 72’s nuclear localization, the NLS(s) has not been definitively identified for any of the heat shock proteins. Likely more is involved than presence of a NLS; since the heat shock proteins only localize to the nucleus under selective conditions, nuclear localization must be regulated. HSPs also function as chaperons of nuclear transport, facilitating the movement of other macromolecules across the nuclear membrane. The mechanisms involved in chaperoning of proteins by HSPs into the nucleus are still being identified.     

Nuclear Targeting of the Cauliflower Mosaic Virus Coat Protein

The entry of the viral genomic DNA of cauliflower mosaic virus into the nucleus is a critical step of viral infection. We have shown by transient expression in plant protoplasts that the viral coat protein (CP), which is processed from the product of open reading frame IV, contains an N-terminal nuclear localization signal (NLS). The NLS is exposed on the surface of the virion and is thus available for interaction with a putative NLS receptor. Phosphorylation of the matured CP did not influence the nuclear localization of the protein but improved protein stability. Mutation of the NLS completely abolished viral infectivity, thus indicating its importance in the virus life cycle. The NLS seems to be regulated by the N terminus of the precapsid, which inhibits its nuclear targeting. This regulation could be important in allowing virus assembly in the cytoplasm.     

Molecular evidence for the nuclear localization of FADD

The Fas-associated death domain (FADD) adaptor protein FADD/Mort-1 is recruited by several members of the tumor necrosis factor receptor (TNFR) superfamily during cell death activated via death receptors. Since most studies have focused on the interaction of FADD with plasma membrane proteins, FADD's subcellular location is thought to be confined to the cytoplasm. In this report, we show for the first time that FADD is present in both the cytoplasm and the nucleus of cells, and that its nuclear localization relies on strong nuclear localization and nuclear export signals (NLS and NES, respectively) that reside in the death-effector domain (DED) of the protein. Specifically, we found that a conserved basic KRK35 sequence of the human protein is necessary for FADD's nuclear localization, since disruption of this motif leads to the confinement of FADD in the cytoplasm. Furthermore, we show that the leucine-rich motif LTELKFLCL28 in the DED is necessary for FADD's nuclear export. Functionally, mutation of the NES of FADD and its seclusion in the nucleus reduces the cell death-inducing efficacy of FADD reconstituted in FADD-deficient T cells.     

A nuclear localization domain in the hnRNP A1 protein

The heterogeneous nuclear RNP (hnRNP) A1 protein is one of the major pre-mRNA/mRNA binding proteins in eukaryotic cells and one of the most abundant proteins in the nucleus. It is localized to the nucleoplasm and it also shuttles between the nucleus and the cytoplasm. The amino acid sequence of A1 contains two RNP motif RNA-binding domains (RBDs) at the amino terminus and a glycine-rich domain at the carboxyl terminus. This configuration, designated 2x RBD-Gly, is representative of perhaps the largest family of hnRNP proteins. Unlike most nuclear proteins characterized so far, A1 (and most 2x RBD-Gly proteins) does not contain a recognizable nuclear localization signal (NLS). We have found that a segment of ca. 40 amino acids near the carboxyl end of the protein (designated M9) is necessary and sufficient for nuclear localization; attaching this segment to the bacterial protein beta- galactosidase or to pyruvate kinase completely localized these otherwise cytoplasmic proteins to the nucleus. The RBDs and another RNA binding motif found in the glycine-rich domain, the RGG box, are not required for A1 nuclear localization. M9 is a novel type of nuclear localization domain as it does not contain sequences similar to classical basic-type NLS. Interestingly, sequences similar to M9 are found in other nuclear RNA-binding proteins including hnRNP A2.     

Hormonal regulation of the nuclear localization signals of the human glucocorticosteroid receptor.

Nuclear localization of the rat glucocorticosteroid receptor (rGR) transiently expressed in COS-7 cells appears to be mediated by two nuclear localization signals, NL1 and NL2, in a hormone-dependent mechanism. We investigated the intracellular distribution of the human GR (hGR) expressed in COS-7 cells, by a different immunohistochemical technique involving immunostaining of cell pellet sections, thus avoiding the use of cell permeabilizing agents and allowing rigorous comparison between successive experiments. With a large set of hGR mutants, we could define determinants of the hGR nuclear localization and compare them with those previously reported for rGR. Our study demonstrated two hormone-dependent nuclear localization signals. NL1 activity, overlapping the DNA-binding domain (DBD)-hinge boundary, was repressed by the unliganded ligand-binding domain (LBD), even if the repressed NL1 retained a residual potency to target hGR in the nucleus. Structure/function analysis suggested a bipartite structure of NL1, analogous to that of other nuclear targeting signals (the carboxy-terminal part of DBD between amino acids 478 and 487 and the beginning of the hinge region which includes a basic amino acid stretch between 491 and 498). Upon hormone binding, NL2, located in the LBD, was activated, but was unable by itself to sustain full nuclear localization, which required the derepressed NL1 activity. Only two sequences in the LBD, localized between amino acids 600 and 626 and from amino acid 696 up to the carboxyl-terminal amino acid 777, respectively, were found to inhibit NL1 activity. As previously reported, efficient nuclear retention, mandatory for gene expression, did not required DNA-binding activity. The controversial intracellular localization of the unliganded form of hGR and the role of hsp90 in cytoplasmic localization are further discussed.     

Nucleocytoplasmic shuttling of the progesterone receptor.

The nuclear localization of the progesterone receptor is mediated by two signal sequences: one is constitutive and lies in the hinge region (between the DNA and steroid binding domains), the other is hormone dependent and is localized in the second zinc finger of the DNA binding domain. The use of various inhibitors of energy synthesis in cells expressing permanently or transiently the wild-type receptor or a receptor mutated within the nuclear localization signals, demonstrated that the nuclear residency of the receptor reflects a dynamic situation: the receptor diffusing into the cytoplasm and being constantly and actively transported back into the nucleus. The existence of this nucleo-cytoplasmic shuttle mechanism was confirmed by receptor transfer from one nucleus to the other in heterokaryons. Preliminary evidence was obtained, using oestrogen receptor, that this phenomenon may be of general significance for steroid receptors.     

Subcellular distribution of ADAR1 isoforms is synergistically determined by three nuclear discrimination signals and a regulatory motif

ADAR1 is an RNA-specific adenosine deaminase that edits RNA sequences. We have demonstrated previously that different ADAR1 isoforms are induced during acute inflammation. Here we show that the mouse ADAR1 isoforms are differentially localized in cellular compartments and that their localization is controlled by several independent signals. Nuclear import of the full-length ADAR1 is predominantly regulated by a nuclear localization signal at the C terminus (NLS-c), which consists of a bipartite basic amino acid motif plus the last 39 residues of ADAR1. Deletion of the NLS-c causes the truncated ADAR1 protein to be retained in the cytoplasm. The addition of this sequence to pyruvate kinase causes the cytoplasmic protein to be localized within the nucleus. The localization of nuclear ADAR1 is determined by a dynamic balance between the nucleolar binding activity of the nucleolar localization signal (NoLS) in the middle of the protein and the exporting activity of the nuclear exporter signal (NES) near the N terminus. The NoLS consists of a typical monopartite cluster of basic residues followed by the third double-stranded RNA-binding domain. These signals act independently; however, NES function can be completely silenced by the NLS-c when a regulatory motif within the catalytic domain and the NoLS are deleted. Thus, the intracellular distribution of the various ADAR1 isoforms is determined by NLS-c, NES, NoLS, and a regulatory motif.