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S Olsnes

Publications and source records attributed to S Olsnes.

At least 37 records · Page 2Linked to original sources
PubMed1998

Modulation by interleukin-2 of cellular response to fibroblast growth factor-1 in F69-3 fibrosarcoma cells.

FGF-1 stimulated DNA synthesis and induced expression of IL-2 receptors in the murine fibrosarcoma cell line, F69-3. Concomitant treatment with IL-2 abolished the stimulation of DNA synthesis, but not binding of FGF-1 to the FGF-receptors or subsequent endocytosis of the bound growth factor. Also, it did not inhibit activation of the FGF-receptor tyrosine kinase or stimulation of the downstream effector, MAP kinase. Treatment with IL-2 prevented transport of FGF-1 to the nuclear fraction in a time- and dose-dependent manner that parallelled the inhibition of FGF-1 stimulated DNA synthesis. The data support our earlier finding that transport of FGF-1 to the nucleus is an important event in the mechanism of stimulation of DNA synthesis induced by the growth factor, and they demonstrate that treatment with a cytokine can modulate the cellular response to FGF-1.

Animals↗
PubMed1998

Variation in the proregion structure of heparin-binding EGF-like growth factor precursors.

In a previous study, we have isolated and characterized cDNA encoding a novel 'short form' of heparin-binding EGF-like growth factor (SF HB-EGF) (Loukianov et al., 1997). In the present work, we have found that cDNA for SF HB-EGF and for full-length HB-EGF are each represented by two variants, which we refer to as L and P forms. The L form is the previously known form of HB-EGF cDNA and encodes a leucine in position 33. The P form described in this report, encodes a proline in codon 33. The L33P substitution is predicted to cause a significant alteration in the proregion structure of SF HB-EGF and HB-EGF.

Amino Acid Sequence↗
PubMed1998

Inability of the acidic fibroblast growth factor mutant K132E to stimulate DNA synthesis after translocation into cells.

Acidic fibroblast growth factor (aFGF) is a potent mitogen. It acts through activation of specific cell surface receptors leading to intracellular tyrosine phosphorylation cascades, but several reports also indicate that aFGF enters cells and that it has an intracellular function as well. The aFGF(K132E) mutant binds to and activates fibroblast growth factor receptors equally strongly as the wild-type, but it is a poor mitogen. We demonstrate that aFGF(K132E) enters NIH 3T3 cells and is transported to the nuclear fraction like wild-type aFGF. A fusion protein of aFGF(K132E) and diphtheria toxin A-fragment (aFGF(K132E)-DT-A) and a similar fusion protein containing wild-type aFGF (aFGF-DT-A) were reconstituted with diphtheria toxin B-fragment. Both fusion proteins were translocated to the cytosol by the diphtheria toxin pathway and subsequently recovered from the nuclear fraction. Whereas translocation of aFGF-DT-A stimulated DNA synthesis in U2OSDR1 cells lacking functional fibroblast growth factor receptors, aFGF(K132E)-DT-A did not. The mutation disrupts a protein kinase C phosphorylation site in the growth factor making it unable to be phosphorylated. The data indicate that a defect in the intracellular action of aFGF(K132E) is the reason for its strongly reduced mitogenicity, possibly due to inability to be phosphorylated.

3T3 Cells↗
PubMed1998

Modulation of the intracellular stability and toxicity of diphtheria toxin through degradation by the N-end rule pathway.

The enzymatically active A-fragment of diphtheria toxin enters the cytosol of sensitive cells where it inhibits protein synthesis by inactivating elongation factor 2 (EF-2). We have constructed a number of diphtheria toxin mutants that are degraded by the N-end rule pathway in Vero cells, and that display a wide range of intracellular stabilities. The degradation could be inhibited by the proteasome inhibitor lactacystin, indicating that the proteasome is responsible for N-end rule-mediated degradation in mammalian cells. Previously, the N-end rule has been investigated by studying the co-translational degradation of intracellularly expressed beta-galactosidase. Our work shows that a mature protein entering the cytosol from the exterior can also be degraded by the N-end rule pathway with a similar, but not identical specificity to that previously found. We found a correlation between the intracellular stability of the mutants and their toxic effect on cells, thus demonstrating a novel manner of modulating the toxicity of a protein toxin. The data also indicate that the inactivation of EF-2 is the rate-limiting step in the intoxication process.

Amino Acid Sequence↗
PubMed1997

Expression of mRNA for a short form of heparin-binding EGF-like growth factor.

In this paper we report the cloning and characterization of cDNA encoding a novel, short form of heparin-binding EGF-like growth factor (SF HB-EGF), and show expression of specific mRNA in various tissues and cell types. Our data suggest that SF HB-EGF mRNA is a product of alternative splicing. Like normal HB-EGF, SF HB-EGF contains the signal peptide, the propeptide, the heparin-binding domain and the first two conservative disulfide loops of the EGF unit. Instead of the third disulfide loop, the spacer, the transmembrane and the cytoplasmic domains, SF HB-EGF has a nine amino acid tail.

Amino Acid Sequence↗
PubMed1997

Effect of mutation of cytoplasmic receptor domain and of genistein on transport of acidic fibroblast growth factor into cells.

Acidic fibroblast growth factor (aFGF) binds to specific transmembrane receptors and is partly transported to a nuclear location. To study this transport we made a kinase-negative mutant of FGF receptor 4 as well as one where the major part of the cytoplasmic receptor domain was deleted, and expressed them in U2OSDr1 cells that lack functional FGF receptors. All receptors mediated endocytic uptake of aFGF. Translocation of the growth factor across cellular membranes was assayed using aFGF with a C-terminal CAAX-motif, which signals addition of a farnesyl group onto the protein once in the cytosol. CAAX-tagged aFGF was farnesylated when incubated with cells containing wild-type or kinase-negative receptors. It was not farnesylated in cells expressing the deleted receptor, or when the incubation was in the presence of genistein. aFGF incubated with cells transfected with wild-type or kinase-negative receptors, but not with the deleted receptor, was partly recovered from the nuclear fraction in the absence, but not in the presence of genistein. The data indicate that the cytoplasmic receptor domain, but not the active kinase, is required for transport of the growth factor into cells, and that genistein inhibits the process.

3T3 Cells↗
PubMed1997

Role of the disulfide bond in Shiga toxin A-chain for toxin entry into cells.

Shiga toxin consists of an enzymatically active A-chain and a pentameric binding subunit. The A-chain has a trypsin-sensitive region, and upon cleavage two disulfide bonded fragments, A1 and A2, are generated. To study the role of the disulfide bond, it was eliminated by mutating cysteine 242 to serine. In T47D cells this mutated toxin was more toxic than wild type toxin after a short incubation, whereas after longer incubation times wild type toxin was most toxic. Cells cleaved not only wild type but also mutated A-chain into A1 and A2 fragments. The mutated A-chain was more sensitive than wild type toxin to Pronase, and it was degraded at a higher rate in T47D cells. Subcellular fractionation demonstrated transport of both wild type and mutated toxin to the Golgi apparatus. Brefeldin A, which disrupts the Golgi apparatus, protected not only against Shiga toxin but also against the mutated toxin, indicating involvement of the Golgi apparatus. After prebinding of Shiga(C242S) toxin to wells coated with the Shiga toxin receptor, Gb3, trypsin treatment induced dissociation of A1 from the toxin-receptor complex demonstrating that in addition to stabilizing the A-chain, the disulfide bond prevents dissociation of the A1 fragment from the toxin-receptor complex.

Amino Acid Sequence↗
PubMed1997

Retrograde transport of mutant ricin to the endoplasmic reticulum with subsequent translocation to cytosol.

Translocation of ricin A chain to the cytosol has been proposed to take place from the endoplasmic reticulum (ER), but attempts to visualize ricin in this organelle have failed. Here we modified ricin A chain to contain a tyrosine sulfation site alone or in combination with N-glycosylation sites. When reconstituted with ricin B chain and incubated with cells in the presence of Na(2)(35)SO(4), the modified A chains were labeled. The labeling was prevented by brefeldin A and ilimaquinone, and it appears to take place in the Golgi apparatus. This method allows selective labeling of ricin molecules that have already been transported retrograde to this organelle. A chain containing C-terminal N-glycosylation sites became core glycosylated, indicating retrograde transport to the ER. In part of the toxin molecules, the A chain was released from the B chain and translocated to the cytosol. The finding that glycosylated A chain was present in the cytosol indicates that translocation takes place after transport of the toxin to the ER.

Animals↗
PubMed1997

Structure-function relationship of the ion channel formed by diphtheria toxin in Vero cell membranes.

Diphtheria toxin (DT) forms cation selective channels at low pH in cell membranes and planar bilayers. The channels formed by wild-type full length toxin (DT-AB), wild-type fragment B (DT-B) and mutants of DT-B were studied in the plasma membrane of Vero cells using the patch-clamp technique. The mutations concerned certain negatively charged amino acids within the channel-forming transmembrane domain (T-domain). These residues might interact electrostatically with cations flowing through the channel, and were therefore exchanged for uncharged amino acids or lysine. The increase in whole-cell conductance induced by toxin, Deltagm, was initially determined. DT-AB induced a approximately 10-fold lower Deltagm than DT-B. The mutations DT-B E327Q, DT-B D352N and DT-B E362K did not affect Deltagm, whereas DT-B D295K, DT-B D352K and DT-B D318K drastically reduced Deltagm. Single channel analysis of DT-B, DT-AB, DT-B D295K, DT-B D318K and DT-B E362K was then performed in outside-out patches. No differences were found for the single-channel conductances, but the mutants varied in their gating characteristics. DT-B D295K exhibited only a very transient channel activity. DT-AB as well as DT-B D318K displayed significantly lower open probability and mean dwell times than DT-B. Hence, the lower channel forming efficiency of DT-AB and DT-B D318K as compared to DT-B is reflected on the molecular level by their tendency to spend more time in the closed position and the fast flickering mode. Altogether, the present work shows that replacements of single amino acids distributed throughout a large part of the transmembrane domain (T-domain) strongly affect the overall channel activity expressed as Deltagm and the gating kinetics of single channels. This indicates clearly that the channel activity observed in DT-exposed Vero cells at low pH is inherent to DT itself and not due to DT-activation of an endogenous channel.

Animals↗
PubMed1997

Membrane translocation of diphtheria toxin fragment A exploits early to late endosome trafficking machinery.

After reaching early endosomes by receptor-mediated endocytosis, diphtheria toxin (DT) molecules have two possible fates. A large pool enters the degradative pathway whereas a few molecules become cytotoxic by translocating their catalytic fragment A (DTA) into the cytosol. Impairment of DT degradation by microtubule depolymerization does not block DT cytotoxicity. Therefore, DTA membrane translocation into the cytosol occurs from an endocytic compartment located upstream of late endosomes. Comparisons between early endosomes and endocytic carrier vesicles in a cell-free translocation assay have demonstrated that early endosomes are the earliest endocytic compartment from which DTA translocates. DTA translocation is ATP-dependent, requires early endosomal acidification, and is increased by the addition of cytosol. Cytosol-dependent DTA translocation is GTP gamma S-insensitive but is blocked by anti-beta COP antibodies.

Animals↗
PubMed1996

GPI-anchored diphtheria toxin receptor allows membrane translocation of the toxin without detectable ion channel activity.

We have investigated the role of the transmembrane and cytoplasmic domains of the diphtheria toxin (DT) receptor [heparin-binding epidermal growth factor (HB-EGF) precursor] in the intoxication pathway. Two mutants were constructed in which these domains were replaced by either a 37 amino acid sequence signalling membrane attachment via a glycosylphosphatidylinositol (GPI) anchor (DTR-GPI) or by the transmembrane and cytoplasmic domains of the human EGF receptor (DTR-EGFR). Similar amounts of DTA fragment were translocated through the plasma membrane of NIH 3T3 cells transfected with the wild-type receptor (DTR), DTR-GPI and DTR-EGFR, but translocation was about six times less efficient in the case of DTR-GPI and DTR-EGFR when taking into account the number of receptors expressed. Interestingly, DT-induced 22Na+ influx was weak in DTR-EGFR cells and not detectable in DTR-GPI cells. Whole cell patch-clamp analysis showed the DT at low pH induced depolarization and decreased input resistance in DTR cells (and to a lesser extent also in DTR-EGFR cells) but not in DTR-GPI cells. These results suggest that the transmembrane and cytoplasmic part of the receptor might be involved in channel activity and that translocation of the A fragment is independent of toxin-induced cation channel activity.

3T3 Cells↗
PubMed1996

Ability of methotrexate to inhibit translocation to the cytosol of dihydrofolate reductase fused to diphtheria toxin.

A fusion protein consisting of dihydrofolate reductase and diphtheria toxin A-fragment was made by genetically linking cDNA for the two proteins followed by in vitro transcription and translation in a rabbit reticulocyte lysate system. The dihydrofolate reductase in the fusion protein exhibited enzyme activity and, in the presence of methotrexate which imposes a tight structure on dihydrofolate reductase, it was trypsin resistant, indicating that it was correctly folded. When reconstituted with diphtheria toxin B-fragment, it bound specifically to diphtheria toxin receptors and was translocated into cells upon exposure to low pH. Methotrexate prevented the translocation. Protein synthesis was inhibited in cells incubated with the reconstituted fusion protein, but the inhibition was reduced in the presence of methotrexate. We also made a fusion protein containing a mutated dihydrofolate reductase with much lower affinity to methotrexate. Methotrexate did not prevent translocation of this protein. The data indicate that methotrexate prevents translocation of the fusion protein containing wild-type dihydrofolate reductase by imposing a tight structure on to the enzyme.

Animals↗
PubMed1996

Stimulation of proliferation of a human osteosarcoma cell line by exogenous acidic fibroblast growth factor requires both activation of receptor tyrosine kinase and growth factor internalization.

U2OS Dr1 cells, originating from a human osteosarcoma, are resistant to the intracellular action of diphtheria toxin but contain toxin receptors on their surfaces. These cells do not have detectable amounts of fibroblast growth factor receptors. When these cells were transfected with fibroblast growth factor receptor 4, the addition of acidic fibroblast growth factor to the medium induced tyrosine phosphorylation, DNA synthesis, and cell proliferation. A considerable fraction of the cell-associated growth factor was found in the nuclear fraction. When the growth factor was fused to the diphtheria toxin A fragment, it was still bound to the growth factor receptor and induced tyrosine phosphorylation but did not induce DNA synthesis or cell proliferation, nor was any fusion protein recovered in the nuclear fraction. On the other hand, when the fusion protein was associated with the diphtheria toxin B fragment to allow translocation to the cytosol by the toxin pathway, the fusion protein was targeted to the nucleus and stimulated both DNA synthesis and cell proliferation. In untransfected cells containing toxin receptors but not fibroblast growth factor receptors, the fusion protein was translocated to the cytosol and targeted to the nucleus, but in this case, it stimulated only DNA synthesis. These data indicate that the following two signals are required to stimulate cell proliferation in transfected U2OS Dr1 cells: the tyrosine kinase signal from the activated fibroblast growth factor receptor and translocation of the growth factor into the cell.

Cell Division↗
PubMed1995

Translocation of cytosol of exogenous, CAAX-tagged acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF) added externally to cells has been proposed to enter the nucleus and stimulate DNA synthesis, but it has remained controversial whether or not exogenous aFGF has the capability of crossing cellular membranes. To test this, a novel principle to study translocation of proteins to the cytosol was developed by fusing a C-terminal farnesylation signal, a CAAX tag (C = Cys, A = an aliphatic amino acid, and X = any amino acid), onto aFGF. Farnesylation is only known to occur in the cytosol and possibly in the nucleus. When incubated with NIH3T3 cells overnight, about one-third of the cell-associated, CAAX-tagged growth factor was farnesylated, indicating that efficient translocation had taken place. Binding to specific FGF receptors was required for translocation to occur. Part of the farnesylated growth factor was found in the nuclear fraction. The data indicate that CAAX-tagged aFGF added externally to cells is able to cross cellular membranes and enter the cytosol and the nucleus.

3T3 Cells↗
PubMed1995

Farnesylation of CaaX-tagged diphtheria toxin A-fragment as a measure of transfer to the cytosol.

Diphtheria toxin binds to receptor-positive cells through its B-fragment, the toxin is then endocytosed, and the low pH in endosomes triggers the translocation of the enzymatically active A-fragment to the cytosol. A synchronous release of A-fragments into the cytosol can be induced by exposing cells with surface-bound toxin to low pH. We have used this protein translocation system to develop a novel method to study whether or not a protein is exposed to the cytosol. Protein farnesylation is a cytosolic modification signaled by a C-terminal CaaX motif, and to visualize the translocation process, we added a farnesylation signal to the toxin A-fragment. The A-fragment with an added CaaX motif was farnesylated within 1 h after exposure of cells with surface-bound toxin to low pH, and also A-fragment translocated from endosomes was quantitatively farnesylated. The results indicate that all cell-mediated reduction of the toxin implicates translocation of the A-fragment to the cytosol. The farnesylation was inhibited by lovastatin, the alkylating agent NEM, and the peptidomimetic farnesylation inhibitor B581. Farnesylated A-fragment partitioned preferentially into the detergent phase upon extraction with Triton X-114. Our data suggest that farnesylation of a CaaX tag is generally applicable as a cytosolic marker, and this strategy for monitoring protein transfer to the cytosol may have considerable potential for studying the transport to the cytosol of proteins added externally to cells.

Amino Acid Sequence↗
PubMed1995

Cell-mediated reduction and incomplete membrane translocation of diphtheria toxin mutants with internal disulfides in the A fragment.

Active diphtheria toxin consists of two fragments, A and B, joined by a disulfide bond. The B fragment binds to cell surface receptors and aids in the translocation of the enzymatically active A fragment to the cytosol. Normally, the toxin A fragment enters the cytosol from acidic endosomes, but translocation can also be induced at the level of the plasma membrane by exposing cells with surface-bound toxin to low pH. Recently, we showed that disulfide bonds introduced into the A fragment by mutation are inhibitory for translocation. In the present work, we found that although the complete translocation of the A fragment is blocked, three mutant toxins underwent reduction of the interfragment disulfide bond upon low pH exposure, whereas the internal disulfide in the A fragment remained intact. In the case of two of these mutants, the A fragment was released into the extracellular medium upon exposure of cell-bound toxin to low pH. The pH profile for the release of the mutant A fragments was the same as for translocation of wild-type A fragment to the cytosol, and the release was inhibited by conditions that interfere with A fragment translocation. In the case of the third mutant, which remained cell-associated upon reduction of the interfragment disulfide bond, a translocation intermediate was detected. The results show that the reduction of the interfragment disulfide bond can occur in the absence of complete translocation of the A fragment to the cytosol, and they indicate that the reduction takes place at an early stage in the translocation process. Our findings suggest that the translocation of the A fragment across the membrane is initiated at the C terminus.

Animals↗
PubMed1995

Diphtheria toxin endocytosis and membrane translocation are dependent on the intact membrane-anchored receptor (HB-EGF precursor): studies on the cell-associated receptor cleaved by a metalloprotease in phorbol-ester-treated cells.

Preincubation of Vero cells with 1 microM phorbol 12-myristate 13-acetate (PMA) decreased the specific binding of diphtheria toxin by about 50%, whereas the toxic effect, endocytic uptake and membrane translocation were completely blocked. Toxin bound to PMA-treated cells was released upon incubation with heparinase. The effect of PMA was abrogated in the presence of EDTA or N-(DL-[2-(hydroxyaminocarbonyl)methyl]-4-methyl-pentanoyl)-L-3-(2' - naphthyl)-alanyl-L-alanine 2-aminoethyl-amide (TAPI), a specific inhibitor of matrix metalloproteases. The results indicate that PMA induces proteolytic cleavage of the diphtheria-toxin receptor [heparin-binding EGF-like growth factor (HB-EGF)-precursor] outside the membrane anchor, and that about 50% of the growth-factor ecto-domain remains associated with the cells, due to binding to surface proteoglycans containing heparan sulphates. Although the cleaved cell-associated HB-EGF binds diphtheria toxin, it does not serve as a functional receptor, since neither toxin internalization nor translocation occurs. Thus the intact HB-EGF precursor is of crucial importance for its function as the diphtheria-toxin receptor.

Animals↗