Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor

The intracellular basic region/helix-loop-helix (bHLH) dioxin receptor mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) and functions as a ligand-activated DNA binding protein directly interacting with target genes by binding to dioxin response elements. Here we show that the partially purified, ligand-bound receptor alone could not bind target DNA. In contrast, DNA binding by the receptor could be induced by addition of a cytosolic auxiliary activity which functionally and biochemically corresponded to the bHLH factor Arnt. While Arnt exhibited no detectable affinity for the dioxin response element in the absence of the dioxin receptor, it strongly promoted the DNA binding function of the ligand-activated but not the ligand-free receptor forms. Arnt also functionally reconstituted in vitro the DNA binding activity of a mutant, nuclear translocation-deficient dioxin receptor phenotype in cytosolic extracts from a dioxin-resistant hepatoma cell line. Importantly, coimmunoprecipitation experiments showed that Arnt physically interacted in solution with the ligand-activated dioxin receptor but failed to heterodimerize with the ligand-free, hsp90-associated receptor form. Mutational analysis suggested that the functional interaction between these two factors occurred via the bHLH motif of Arnt. These data suggest that dioxin receptor activity is governed by a complex pattern of combinatorial regulation involving repression by hsp90 and then by ligand-dependent recruitment of the positive coregulator Arnt. The dioxin receptor system also provides the first example of signal-controlled dimerization of bHLH factors.     

Distinct nuclear localization and activity of tissue transglutaminase

Tissue transglutaminase is a calcium-dependent transamidating enzyme that has been postulated to play a role in the pathology of expanded CAG repeat disorders with polyglutamine expansions expressed within the affected proteins. Because intranuclear inclusions have recently been shown to be a common feature of many of these codon reiteration diseases, the nuclear localization and activity of tissue transglutaminase was examined. Subcellular fractionation of human neuroblastoma SH-SY5Y cells demonstrated that 93% of tissue transglutaminase is localized to the cytosol. Of the 7% found in the nucleus, 6% copurified with the chromatin-associated proteins, and the remaining 1% was in the nuclear matrix fraction. In situ transglutaminase activity was measured in the cytosolic and nuclear compartments of control cells, as well as cells treated with the calcium-mobilizing agent maitotoxin to increase endogenous tissue transglutaminase activity. These studies revealed that tissue transglutaminase was activated in the nucleus, a finding that was further supported by cytochemical analysis. Immunofluorescence studies revealed that nuclear proteins modified by transglutaminase exhibited a discrete punctate, as well as a diffuse staining pattern. Furthermore, different proteins were modified by transglutaminase in the nucleus compared with the cytosol. The results of these experiments clearly demonstrate localization of tissue transglutaminase in the nucleus that can be activated. These findings may have important implications in the formation of the insoluble nuclear inclusions, which are characteristic of codon reiteration diseases such as Huntington's disease and the spinocerebellar ataxias.     

The nuclear receptor SF-1 mediates sexually dimorphic expression of Mullerian Inhibiting Substance, in vivo

Mullerian Inhibiting Substance (MIS) functions to promote regression of the Mullerian duct during male development. Maintaining the sexually dimorphic pattern of MIS expression is essential for proper mammalian reproductive tract development. Here, we show that the intricate spatial and temporal pattern of MIS expression is directed by a remarkably small proximal promoter of only 180 base pairs in length. Expression of the MIS-human growth hormone transgene (MIS/GH) is restricted to Sertoli cells in embryonic testis and to granulosa cells of postnatal ovary, consistent with the known MIS expression pattern. The proximal MIS promoter is therefore sufficient to direct the initiation and the maintenance of MIS gene expression in both sexes. Moreover, in vivo MIS promoter activity requires an intact binding site for the orphan nuclear receptor SF-1. Taken together, these data strongly suggest that SF-1 directly activates MIS in embryonic and postnatal gonads. Consistent with the proposed role of SF-1 in mammalian sex-determination, our study provides physiological evidence that a SF-1 binding site is essential for gene activation of an embryonic testis-specific marker.     

Phosphorylation of Srp1p, the yeast nuclear localization signal receptor, in vitro and in vivo

Srp1p, the protein encoded by SRP1 of the yeast Saccharomyces cerevisiae, is a yeast nuclear localization signal (NLS) receptor protein. We have previously reported isolation of a protein kinase from yeast extracts that phosphorylates Srp1p complexed with NLS peptides/proteins. From partial amino acid sequences of the four subunits of the purified kinase, we have now identified this protein kinase to be identical to yeast casein kinase II (CKII). It was previously thought that autophosphorylation of the 36 kDa subunit of the yeast enzyme was stimulated by the substrate, GST-Srp1p. However, with the use of a more refined system, no stimulation of autophosphorylation of the 36 kDa subunit of yeast CKII was observed. Biochemical and mutational analyses localized the in vitro phosphorylation site of Srp1p by CKII to serine 67. It was shown that, in the absence of NLS peptides/proteins, phosphorylation of the intact Srp1p protein is very weak, but deletion of the C-terminal end causes great stimulation of phosphorylation without NLS peptides/proteins. Thus, the CKII phosphorylation site is apparently masked in the intact protein structure by the presence of a C-terminal region, probably between amino acids 403 and 516. Binding of NLS peptides/proteins most likely causes a change in protein conformation, exposing the CKII phosphorylation site. Mutational alterations of serine 67, the CKII phosphorylation site, to valine (S67V) and aspartic acid (S67D) were not found to cause any significant deleterious effects on cell growth. Analysis of in vivo phosphorylation showed that at least 30% of the wild type Srp1p molecules are phosphorylated in growing cells, and that the phosphorylation is mostly at the serine 67 CKII site. The ability of Srp1p purified from E coli and treated with calf intestinal phosphatase to bind a SV40 T-antigen NLS peptide was compared with that of Srp1p which was almost fully phosphorylated by CKII. No significant difference was observed. It appears that NLS binding does not require any phosphorylation of Srp1p, either by CKII or by some other protein kinase.     

Nucleocytoplasmic recycling of the nuclear localization signal receptor alpha subunit in vivo is dependent on a nuclear export signal, energy, and RCC1

Nuclear protein import requires a nuclear localization signal (NLS) receptor and at least three other cytoplasmic factors. The alpha subunit of the NLS receptor, Rag cohort 1 (Rch1), enters the nucleus, probably in a complex with the beta subunit of the receptor, as well as other import factors and the import substrate. To learn more about which factors and/or events end the import reaction and how the import factors return to the cytoplasm, we have studied nucleocytoplasmic shuttling of Rch1 in vivo. Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm. Rch1 injected into the nucleus was rapidly exported in a temperature-dependent manner. In contrast, a mutant of Rch1 lacking the first 243 residues accumulated in the nuclei of Vero cells after cytoplasmic injection. After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207-217 or a heterologous nuclear export signal, but not a mutant form of residues 207-217, restored nuclear export. Loss of the nuclear transport factor RCC1 (regulator of chromosome condensation) at the nonpermissive temperature in the thermosensitive mutant cell line tsBN2 caused nuclear accumulation of wild-type Rch1 injected into the cytoplasm. However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported. These results suggested that RCC1 acts at an earlier step in Rch1 recycling, possibly the disassembly of an import complex that contains Rch1 and the import substrate. Consistent with this possibility, incubation of purified RanGTP and RCC1 with NLS receptor and import substrate prevented assembly of receptor/substrate complexes or stimulated their disassembly.     

Distinct dendritic cell subsets differentially regulate the class of immune response in vivo

Dendritic cells (DCs) are unique in their ability to stimulate T cells and initiate adaptive immunity. Injection of mice with the cytokine Flt3-ligand (FL) dramatically expands mature lymphoid and myeloid-related DC subsets. In contrast, injection of a polyethylene glycol-modified form of granulocyte/macrophage colony-stimulating factor (GM-CSF) into mice only expands the myeloid-related DC subset. These DC subsets differ in the cytokine profiles they induce in T cells in vivo. The lymphoid-related subset induces high levels of the Th1 cytokines interferon gamma and interleukin (IL)-2 but little or no Th2 cytokines. In contrast, the myeloid-related subset induces large amounts of the Th2 cytokines IL-4 and IL-10, in addition to interferon gamma and IL-2. FL- or GM-CSF-treated mice injected with soluble ovalbumin display dramatic increases in antigen-specific antibody titers, but the isotype profiles seem critically dependent on the cytokine used. Although FL treatment induces up to a 10, 000-fold increase in ovalbumin-specific IgG2a and a more modest increase in IgG1 titers, GM-CSF treatment favors a predominantly IgG1 response with little increase in IgG2a levels. These data suggest that distinct DC subsets have strikingly different influences on the type of immune response generated in vivo and may thus be targets for pharmacological intervention.     

Adipocyte differentiation and nuclear receptor]

The roles of nuclear receptors in differentiation and function of adipocytes were reviewed and discussed. Expression of peroxisome proliferator-activated receptor (PPAR) gamma have been reported to be strongly induced during adipocyte differentiation and maintained in matured adipocytes. Forced expression of PPAR gamma converted NIH3T3 fibroblasts to adipocytes, indicating PPAR gamma regulates essential genes to obtain the adipocyte phenotype. Newly developed antidiabetic thiazolidinediones known as high affinity ligands for PPAR gamma improved insulin resistance. This finding suggests that PPAR gamma contributes regulation of insulin action. Several genes regulated by troglitazone, one of the most potent thiazolidinediones, in matured 3T3-L1 adipocytes-were obtained by differential display PCR method. Orphan receptors ROR alpha/gamma and Rev-ErbA which bind to the same response element are also induced during adipocyte differentiation but their function is still to be investigated.     

In vivo coupling of insulin-like growth factor II/mannose 6-phosphate receptor to heteromeric G proteins. Distinct roles of cytoplasmic domains and signal sequestration by the receptor

We examined the signaling function of the IGF-II/mannose 6-phosphate receptor (IGF-IIR) by transfecting IGF-IIR cDNAs into COS cells, where adenylyl cyclase (AC) was inhibited by transfection of constitutively activated G alpha i cDNA (G alpha i2Q205L). In cells transfected with IGF-IIR cDNA, IGF-II decreased cAMP accumulation promoted by cholera toxin or forskolin. This effect of IGF-II was not observed in untransfected cells or in cells transfected with IGF-IIRs lacking Arg2410-Lys2423. Thus, IGF-IIR, through its cytoplasmic domain, mediates the Gi-linked action of IGF-II in living cells. We also found that IGF-IIR truncated with C-terminal 28 residues after Ser2424 caused G beta gamma-dominant response of AC in response to IGF-II by activating Gi. Comparison with the G alpha i-dominant response of AC by intact IGF-IIR suggests that the C-terminal 28-residue region inactivates G beta gamma. This study not only provides further evidence that IGF-IIR has IGF-II-dependent signaling function to interact with heteromeric G proteins with distinct roles by different cytoplasmic domains, it also suggests that IGF-IIR can separate and sequestrate the G alpha and G beta gamma signals following Gi activation.     

Fluorescent labeling of NK2 receptor at specific sites in vivo and fluorescence energy transfer analysis of NK2 ligand-receptor complexes

A fluorescent unnatural amino acid was introduced biosynthetically at known sites into the G protein-coupled neurokinin (tachykinin) NK2 receptor by suppression of UAG nonsense codons with the aid of a chemically misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. A systematic UAG-scanning mutagenesis in NK2 extra- or intracellular loops and proximal transmembrane domains established that readthrough at some UAG sites may represent a limitation to the range of applicability of the nonsense suppression methodology. Fluorescence-labeled NK2 mutants containing an unique fluorescent nitrobenzoxadiazoyl-diaminopropionic acid residue at known sites were shown to be functionnally active. Intermolecular distances were determined by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist in a native membrane environment. These distances confirmed the seven transmembrane topology for G protein-coupled receptors and determined a structural model for NK2 ligand-receptor interactions. The peptide is inserted between the fifth and sixth transmembrane domains, thus suggesting that antagonism may be caused by preventing correct packing of the helices required for receptor function.     

Identification of opiate/receptor binding in vivo

The displacement of small amounts of tritiumlabbelled antagonists or agonists by increasing amounts of unlabelled antagonists in mouse brain in vivo offered the possibility of characterizing properties of opiate receptors in the intact animal. The displacement effect was stereospecific, saturable and dependent upon the affinity of the substance investigated. At brain concentrations of 0.3 nM, 75% of 3H-diprenorphine, 60% of 3H-naltrexone and 50% of 3H-naloxone were displaced by high amounts of the respective unlabelled drug. Comparison of the in vivo data with receptor binding in vitro revealed similar results in respect to binding sites and receptor affinity. The displacement was different in various brain areas. The time course of the displacement was also different for the various substances used and seems to reflect differences in the speed of association and dissociation to and from the receptors. The displacement of 3H-etorphine by naltrexone could be correlated with the reversal of analgesia.     

Distinct T cell receptor signaling requirements for perforin- or FasL-mediated cytotoxicity

A diverse array of signals are generated in a cytotoxic T lymphocyte (CTL) after the T cell receptor (TCR) engages the class I major histocompatibility complex (MHC) peptide complex. These signals result in a multitude of CTL effector functions, including cellular cytotoxicity, cell surface receptor expression, and cytokine secretion. We have examined signaling through the TCR in a wild type CD8+, MHC-restricted, antigen-specific CTL clone, 14-7, and its interleukin 2-dependent variant clone 14-7FD. We report here that 14-7FD is unable to kill via the perforin mechanism of killing, yet is able to kill via the Fas ligand/Fas mechanism and secrete interferon-gamma in an antigen-specific manner. 14-7FD has cytolytic granules that contain perforin and serine esterases, which are secreted after phorbol ester and Ca2+ ionophore treatment. Lastly, to investigate which TCR signaling requirements were operational in 14-7FD, we examined TCR-triggered intracellular Ca2+ mobilization in the two clones. After TCR engagement, 14-7FD failed to mobilize intracellular Ca2+, which may be the cause for its inability to trigger the perforin/granule exocytosis mechanism of killing. These results indicate that the signal transduction events that trigger perforin killing and the signaling requirements to induce FasL expression are distinct. We hypothesize that these two distinct TCR signal transduction requirements allow for separate activation of these two mechanisms of killing relating to their role in eradication of infected cells or regulation of immune responses.     

Proteasomal regulation of nuclear receptor corepressor-mediated repression

We analyzed the clinical results of 195 Harris Design-2 total hip replacements performed with so-called second-generation cementing techniques in 166 consecutive patients who had osteoarthrosis. The mean age of the patients at the time of the replacement was sixty-seven years and nine months (range, thirty-one to eighty-nine years). Forty-eight patients (fifty-four hips) died before the time of the latest follow-up, but the implants were apparently functioning well at the time of death. Three patients (four hips) were lost to follow-up. Five patients (five hips; 3 percent) had a revision because of aseptic loosening of the acetabular or femoral component, or both, that was related to wear-induced osteolysis. The mean Harris hip score for the 131 hips that were available at the latest follow-up examination at a mean of twelve years (range, ten to fifteen years) after the operation was 89 +/- 10 points. On the basis of the Harris hip score, seventy-six hips had an excellent result, thirty-four had a good result, fifteen had a fair result, and six had a poor result at the latest follow-up examination. Radiographically, twelve (9 percent) of the 131 acetabular components and three (2 percent) of the 131 femoral components were probably or definitely loose. At a mean of twelve years, 186 (97 percent) of 191 Harris Design-2 implants were in situ or had been in situ at the time of the patient's death.     

Transportin: nuclear transport receptor of a novel nuclear protein import pathway

The main purposes of this study were to investigate the best parameter for describing gallbladder emptying and whether gallbladder bile emptying should be induced with a bolus injection or continuous infusion of cholecystokinin-octapeptide (CCK-8). METHODS: Gallbladder emptying was measured by dynamic cholescintigraphy. Twelve healthy subjects and six patients with gallstones were examined twice with CCK-8 infusion cholescintigraphy, 0.3 ng CCK-8 kg per min for 60 min under identical circumstances. Another six healthy subjects randomly received bolus injection (0.04 microgram/kg) and infusion of CCK-8 (0.3 ng/kg per min for 60 min), respectively, during cholescintigraphy on two separate occasions. The choice of bolus dose was based on recommendations from the CCK-8 manufacturer. The infusion dose was chosen to produce plasma CCK concentrations similar to postprandial plasma CCK levels. RESULTS: A parameter of gallbladder emptying, mean ejection fraction (EF), was defined as 100% minus the area under the time-activity curve normalized to 100% and divided by the time interval from maximum to minimum counts per minute. This parameter proved superior to the well known parameters, EFmax. and EF30, in regard to reproducibility in healthy subjects. The slope of the regression line for the mean EF was 0.998 and the intercept value approximately 0% (p = 0.0001). The mean coefficient of variation was 4%. Apart from a higher mean coefficient of variation, similar reproducibility results were seen in the six patients. The measurements of EF30 in healthy subjects scattered more widely around the mean compared to the mean EF and EFmax, which indicates poorer ability to separate normal from abnormal gallbladder emptying. Intravenous bolus injection of CCK-8 resulted in incomplete gallbladder emptying with a mean EF value of 16% (s.d. 9%; range 7%-32%) compared to 49% (s.d. 7%; range 37%-57%) following CCK-8 infusion (p = 0.004). Abdominal discomfort was observed in all subjects after administration of the bolus injection, whereas no complaints were reported during infusion. CONCLUSION: Mean EF is the best parameter for describing gallbladder emptying. Moreover, slow infusion of a physiological dose of CCK-8 is preferable to induce gallbladder emptying because it results in more complete emptying and has no side effects.     

Hepatitis delta virus antigen forms dimers and multimeric complexes in vivo

Although the hepatitis delta virus genome contains multiple open reading frames, only one of these reading frames is known to be expressed during replication of the virus. This open reading frame encodes two distinct molecular species of hepatitis delta antigen (HDAg), p24 delta and p27 delta, depending on the location of the stop codon which terminates translation. We found antibody specific for p27 delta to be capable of precipitating p24 delta in extracts of infected liver, indicating that p27 delta and p24 delta form heterologous complexes in vivo. After cross-linking with 0.05% glutaraldehyde, specific HDAg dimers were detected in antigen prepared from both the liver and serum of an HDV-infected woodchuck carrier of woodchuck hepatitis virus. Guanidine HCl-denatured HDAg extracted from liver and dialyzed against phosphate-buffered saline sedimented in rate-zonal sucrose density gradients as 15S multimeric complexes. These 15S multimers were stable in the presence of 1.2% Nonidet P-40. After RNase digestion, the 15S complex was reduced to a 12S complex without associated RNA, while boiling for 3 min in 1% sodium dodecyl sulfate-0.5% 2-mercaptoethanol further reduced the 15S complex to 3S HDAg monomers. In the absence of glutaraldehyde cross-linking, HDAg extracted from liver migrated as monomer species in reducing and nonreducing gels, suggesting that the conserved cysteine residue present in p27 delta does not play a role in the formation of either dimers or multimers. On the other hand, an amino-terminal chymotrypsin-digested HDAg fragment, with a predicted length of 81 or less amino acids, retained the ability to form dimers, consistent with the hypothesis that a coiled-coil motif present between residues 27 and 58 may play a role in HDAg protein interactions in vivo.