The nuclear localization of a putative neurotransmitter receptor

A high affinity strychnine binding site has been identified within a membrane fraction prepared from partially purified rat brain nuclei. This interaction appears similar in its characteristics to that occurring in the non-nuclear membrane fraction which is thought to occur at the synaptic glycine receptor complex. Both the nuclear and non-nuclear membrane binding of tritiated strychnine is greater within the pons-medulla region than in the cerebral cortex. Nuclear membrane binding sites for dopamine, norepinephrine (-adrenergic), acetylcholine (muscarinic), GABA, and diazepam were not detected.     

Mechanisms of nuclear localization of the progesterone receptor

Deletion mutants of the rabbit progesterone receptor were used to identify two major mechanisms of its nuclear localization. A putative signal sequence, homologous to that of the SV40 large T antigen, was localized around amino acids 638–642 and was shown to be constitutively active. When amino acids 638–642 were deleted, the receptor became cytoplasmic but could be shifted into the nucleus by the addition of hormone (or antihormone), it was almost fully active. A second putative nuclear localization signal is located in the DNA binding domain activated either through ligand binding or through production of constitutive receptor. By deleting epitopes recognized by monoclonal antibodies, it was possible to follow different receptor mutants inside the same cells. In the absence of ligand the receptor was transferred into the nucleus as a monomer. After administration of hormone (or antihormone) a “cytoplasmic” monomer was transferred into the nucleus through interaction with a “nuclear” monomer. These interactions occurred through the steroid binding domains of both monomers.     

Nuclear localization of the phosphatidylserine receptor protein via multiple nuclear localization signals

The interaction between phosphatidylserine and its receptor on phagocytic cells plays a critical role in the clearance of apoptotic bodies under normal physiological condition. A specific receptor for phosphatidylserine (PSR) has recently been identified by phage display and shown to mediate phosphatidylserine dependent phagocytosis. Here we show that the protein encoded by the PSR cDNA is localized in the nuclei through multiple nuclear localization signals. First, a fusion between PSR and GFP is localized in the nuclei of transfected cells, suggesting that PSR have intrinsic nuclear localization capability. Indeed, affinity-purified anti-PSR antibodies identified a 47 kDa protein species in cells transfected with untagged PSR and localized this protein in the nuclei by immunofluorescent confocal microscopy. In NIH3T3 cells, which express endogenous PSR mRNA, a similar 47 kDa species was detected and localized in the nuclei. Finally, multiple nuclear localization signals were identified in PSR sequence, each capable of targeting GFP to the nuclei. Together, these results suggest that PSR may serve a dual role both on the cell surface and in the nuclei.     

Dynamic localization of the nuclear import receptor and its interactions with transport factors

Characterization of the interactions between soluble factors required for nuclear transport is key to understanding the process of nuclear trafficking. Using a synthetic lethal screen with the rna1-1 strain, we have identified a genetic interaction between Rna1p, a GTPase activating protein required for nuclear transport, and yeast importin- beta, a component of the nuclear localization signal receptor. By the use of fusion proteins, we demonstrate that Rna1p physically interacts with importin-beta. Mutants in importin-beta exhibit in vivo nuclear protein import defects, and importin-beta localizes to the nuclear envelope along with other proteins associated with the nuclear pore complex. In addition, we present evidence that importin-alpha, but not importin-beta, mislocalizes to the nucleus in cells where the GTPase Ran is likely to be in the GDP-bound state. We suggest a model of nuclear transport in which Ran-mediated hydrolysis of GTP is necessary for the import of importin-alpha and the nuclear localization signal- bearing substrate into the nucleus, while exchange of GDP for GTP on Ran is required for the export of both mRNA and importin-alpha from the nucleus.     

Constitutive and ligand-induced nuclear localization of oxytocin receptor

Oxytocin receptor (OTR) is a membrane protein known to mediate oxytocin (OT) effects, in both normal and neoplastic cells. We report here that human osteosarcoma (U2OS, MG63, OS15 and SaOS2), breast cancer (MCF7), and primary human fibroblastic cells (HFF) all exhibit OTR not only on the cell membrane, but also in the various nuclear compartments including the nucleolus. Both an OTR-GFP fusion protein and the native OTR appear to be localized to the nucleus as detected by transfection and/or confocal immunofluorescence, respectively. Treatment with oxytocin causes internalization of OTR and the resulting vesicles accumulate in the vicinity of the nucleus and some of the perinuclear OTR enters the nucleus. Western blots indicate that OTR in the nucleus and on the plasma membrane are likely to be the same biochemical and immunological entities. It appears that OTR is first visible in the nucleoli and subsequently disperses within the nucleus into 4-20 spots while some of the OTR diffuses throughout the nucleoplasm.The behaviour and kinetics of OTR-GFP and OTR are different, indicating interference by GFP in both OTR entrance into the nucleus and subsequent relocalization of OTR within the nucleus. There are important differences among the tested cells, such as the requirement of a ligand for transfer of OTR in nuclei. A constitutive internalization of OTR was found only in osteosarcoma cells, while the nuclear localization in all other tested cells was dependent on ligand binding. The amount of OTR-positive material within and in the vicinity of the nucleus increased following a treatment with oxytocin in both constitutive and ligand-dependent type of cells. The evidence of OTR compartmentalization at the cell nucleus (either ligand-dependent or constitutive) in different cell types suggests still unknown biological functions of this protein or its ligand and adds this G-protein-coupled receptor to other heptahelical receptors displaying this atypical and unexpected nuclear localization.     

G protein-coupled receptor kinase 5 contains a DNA-binding nuclear localization sequence

G protein-coupled receptor kinases (GRKs) mediate desensitization of agonist-occupied G protein-coupled receptors (GPCRs). Here we report that GRK5 contains a DNA-binding nuclear localization sequence (NLS) and that its nuclear localization is regulated by GPCR activation, results that suggest potential nuclear functions for GRK5. As assessed by fluorescence confocal microscopy, transfected and endogenous GRK5 is present in the nuclei of HEp2 cells. Mutation of basic residues in the catalytic domain of GRK5 (between amino acids 388 and 395) results in the nuclear exclusion of the mutant enzyme (GRK5(Delta)(NLS)), demonstrating that GRK5 contains a functional NLS. The nuclear localization of GRK5 is subject to dynamic regulation. Calcium ionophore treatment or activation of Gq-coupled muscarinic-M3 receptors promotes the nuclear export of the kinase in a Ca(2+)/calmodulin (Ca(2+)/CaM)-dependent fashion. Ca(2+)/CaM binding to the N-terminal CaM binding site of GRK5 mediates this effect. Furthermore, GRK5, but not GRK5(Delta)(NLS) or GRK2, binds specifically and directly to DNA in vitro. Consistent with their presence in the nuclei of transfected cells, all the GRK4, but not GRK2, subfamily members contain putative NLSs. These results suggest that the GRK4 subfamily of GRKs may play a signaling role in the nucleus and that GRK4 and GRK2 subfamily members perform divergent cellular functions.     

Mechanisms of nuclear localization of the progesterone receptor: evidence for interaction between monomers

Deletion mutants of the rabbit progesterone receptor were used to identify two major mechanisms of its nuclear localization. A putative signal sequence, homologous to that of the SV40 large T antigen, was localized around amino acids 638-642 and shown to be constitutively active. When amino acids 638-642 were deleted, the receptor became cytoplasmic but could be shifted into the nucleus by the addition of hormone (or anti-hormone); it was almost fully active. The second mechanism consisted of the activation of the DNA binding domain. By deleting epitopes recognized by monoclonal antibodies, it was possible to follow different receptor mutants inside the same cells. In the absence of ligand, the receptor was transferred into the nucleus as a monomer. After administration of hormone (or anti-hormone) a "cytoplasmic" monomer was transferred into the nucleus through interaction with a "nuclear" monomer. These interactions occurred through the steroid binding domains of both monomers.     

Ultraspiracle promotes the nuclear localization of ecdysteroid receptor in mammalian cells

The heterodimer consisting of ecdysteroid receptor (EcR) and ultraspiracle (USP), both of which are members of the nuclear receptor superfamily, is considered to be the functional ecdysteroid receptor. Here we analyzed the subcellular distribution of EcR and USP fused to fluorescent proteins. The experiments were carried out in mammalian COS-7, CHO-K1 and HeLa cells to facilitate investigation of the subcellular trafficking of EcR and USP in the absence of endogenous expression of these two receptors. The distribution of USP tagged with a yellow fluorescent protein (YFP-USP) was almost exclusively nuclear in all cell types analyzed. The nuclear localization remained constant for at least 1 day after the first visible signs of expression. In contrast, the intracellular distribution of EcR tagged with a yellow fluorescent protein (YFP-EcR) varied and was dependent on time and cell type, although YFP-EcR alone was also able to partially translocate into the nuclear compartment. Coexpression of YFP-EcR with USP tagged with a cyan fluorescent protein (CFP-USP) resulted in exclusively nuclear localization of both proteins in all cell types analyzed. The USP-induced nuclear localization of YFP-EcR was stable for at least 20 hours. These experiments suggest that USP has a profound effect on the subcellular distribution of EcR.     

Rad52 multimerization is important for its nuclear localization in Saccharomyces cerevisiae

Rad52 is essential for all homologous recombination and DNA double strand break repair events in Saccharomyces cerevisiae. This protein is multifunctional and contains several domains that allow it to interact with DNA as well as with different repair proteins. However, it has been unclear how Rad52 enters the nucleus. In the present study, we have used a combination of mutagenesis and sequence analysis to show that Rad52 from S. cerevisiae contains a single functional pat7 type NLS essential for its nuclear localization. The region containing the NLS seems only to be involved in nuclear transport as it plays no role in repair of MMS-induced DNA damage. The NLS in Rad52 is weak, as monomeric protein species that harbor this NLS are mainly located in the cytosol. In contrast, multimeric protein complexes wherein each subunit contains a single NLS(Rad52) sort efficiently to the nucleus. Based on the results we propose a model where the additive effect of multiple NLS(Rad52) sequences in a Rad52 ring-structure ensures efficient nuclear localization of Rad52.     

PPAR-gamma: a nuclear receptor with affinity for cannabinoids

An increasing number of cannabinoid actions are being reported that do not appear to be mediated by either CB1 or CB2, the known cannabinoid receptors. One such example is the synthetic analog ajulemic acid (AJA), which shows potent analgesic and anti-inflammatory effects in rodents and humans. AJA binds weakly to CB1 only at concentrations many fold higher than its therapeutic range, and is, therefore, completely free of psychotropic effects in both normal subjects and pain patients suggesting the involvement of a target site other than CB1. AJA as well as several other cannabinoids appear to have profound effects on cellular lipid metabolism as evidenced by their ability to transform fibroblasts into adipocytes where the accumulation of lipid droplets can be readily observed. Such transformations can be mediated by the activation of the nuclear receptor PPAR-gamma. A variety of small molecule ligands including AJA have been shown to induce the activation of PPAR-gamma and, in some cases this has led to the introduction of clinically useful agents. It is suggested that PPAR-gamma may serve a receptor function for certain actions of some cannabinoids.     

Ligands specify estrogen receptor alpha nuclear localization and degradation

Background  

The estrogen receptor alpha (ERα) is found predominately in the nucleus, both in hormone stimulated and untreated cells. Intracellular distribution of the ERα changes in the presence of agonists but the impact of different antiestrogens on the fate of ERα is a matter of debate.     

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.