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F Hucho

Publications and source records attributed to F Hucho.

At least 55 records · Page 3Linked to original sources
PubMed1997

Signal transduction and protein kinases: the long way from the plasma membrane into the nucleus.

All living cells must be able to receive information from the extracellular space and to react to it by processing and converting it into intracellular effects. If the properties of cells are to change in the long term, some signals must reach the nucleus in order to bring about changes in gene transcription. Three of the pathways, beginning with an extracellular signal and ending with the nucleus, serve to illustrate some principles of signal transduction such as signal conversion, signal cascade, cross-talk, and on/off switch. One element common to most of the pathways is the activation of protein kinases. One example of these kinases, the protein kinase C, is discussed as a vehicle of signal transport toward the nucleus and as a means of cross-talk between different signaling pathways.

Animals↗
PubMed1997

Mapping of exposed surfaces of the nicotinic acetylcholine receptor by identification of iodinated tyrosine residues.

Here we report on the use of iodination of the membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo californica electric tissue in order to define surface-exposed portions of the receptor molecule. Membrane-bound nAChR was 125I-iodinated using the oxidation agent Iodo-Gen. The iodinated subunits were separated by preparative gel electrophoresis, desalted, and cleaved with trypsin. The resulting peptides were separated by reverse-phase HPLC and the radioactive peptides were identified by mass spectrometry and protein sequencing. For the delta-subunit, we identified five iodinated peptides containing the tyrosine residues deltaTyr17, deltaTyr74, deltaTyr365, deltaTyr372, and deltaTyr428. The surface exposition of these amino acids is in agreement with the four-transmembrane-segment model (4TM model) of the nAChR, but the assignment to the intra- or extracellular surface is doubtful. According to this model, the N-terminal portion of the receptor subunits including the iodinated residues deltaTyr17 and deltaTyr74 is extracellular and deltaTyr372 as a site of tyrosine phosphorylation is located on the cytoplasmic side. But since this latter residue is among the first to be iodinated using an immobilized iodination agent, its true position with respect to the membrane bilayer is not clear.

Amino Acid Sequence↗
PubMed1996

The emerging three-dimensional structure of a receptor. The nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor is the neurotransmitter receptor with the most-characterized protein structure. The amino acid sequences of its five subunits have been elucidated by cDNA cloning and sequencing. Its shape and dimensions (approximately 12.5 nm x 8 nm) were deduced from electron-microscopy studies. Its subunits are arranged around a five-fold axis of pseudosymmetry in the order (clockwise) alpha H gamma alpha L delta beta. Its two agonist/competitive-antagonist-binding sites have been localized by photolabelling studies to a deep gorge between the subunits near the membrane surface. Its ion channel is formed by five membrane-spanning (M2) helices that are contributed by the five subunits. This finding has been generalized as the Helix M2 model for the superfamily of ligand-gated ion channels. The binding site for regulatory non-competitive antagonists has been localized by photolabelling and site-directed-mutagenesis studies within this ion channel. Therefore a three-dimensional image of the nicotinic acetylcholine receptor is emerging, the most prominent feature of which is an active site that combines the agonist/ competitive-antagonist-binding sites, the regulatory site and the ion channel within a relatively narrow space close to and within the bilayer membrane.

Acetylcholine↗
PubMed1996

Transport of protein kinase C alpha into the nucleus requires intact cytoskeleton while the transport of a protein containing a canonical nuclear localization signal does not.

Protein kinase C undergoes a redistribution from the cytosol into the nucleus upon various stimuli. Since protein kinase C does not contain any known nuclear localization signal, the exact pathway and mechanism of the translocation into the nucleus is not known. We used immunofluorescence microscopy to investigate the role of the cytoskeleton in this process, and to detect the subcellular distribution of protein kinase C alpha in NIH 3T3 fibroblasts. In these cells protein kinase C alpha is translocated into the nucleus after stimulation with phorbol ester. We observed that cells treated with the cytoskeleton disrupting agents cytochalasin B or colchicine do not show the nuclear translocation of protein kinase C alpha after stimulation. In contrast, the nuclear accumulation of a nuclear localization signal containing reporter protein in an in vitro nuclear transport assay is not affected by these drugs. This observation has been confirmed for intact cells by microinjection experiments: cells which have been incubated with cytochalasin B or colchicine prior to microinjection of the reporter protein show the same accumulation in the nucleus as untreated cells. Our data show that intact cytoskeleton plays an important role in the translocation of protein kinase C alpha into the nucleus but not in the nuclear import of a karyophilic reporter protein.

3T3 Cells↗
PubMed1996

[Effective binding of alpha-bungarotoxin with the solubilized alpha-subunit of Torpedo californica acetylcholine receptor].

alpha-Subunit of the Torpedo californica nicotinic acetylcholine receptor was isolated by preparative SDS-PAGE followed by reversed-phase HPLC on a C4 column in an acetonitrile-isopropanol gradient in water. After removal of the organic solvents and solubilization in beta-octylglucoside, the purified alpha-subunit binds alpha-bungarotoxin with high affinity (Kd 28 nM).

Animals↗
PubMed1995

The handedness of the subunit arrangement of the nicotinic acetylcholine receptor from Torpedo californica.

Cross-linking an alpha-neurotoxin with a known three-dimensional structure and with photoactivatable groups in known positions to native membrane-bound acetylcholine receptor reveals its quaternary structure, including the handedness of its circular subunit arrangement. Photolabelling with alpha-neurotoxin carrying the photoactivatable group at position Lys46 is inhibited by the competitive antagonist (+)-tubocurarine in a biphasic manner, indicating that it reacts with both alpha-subunits that were shown to have different affinities for this antagonist [Neubig, R. R. & Cohen, J. B. (1979) Biochemistry 18, 5464-5475]. Lys46 is located on loop III of the neurotoxin. The other information necessary for the elucidation of the handedness was provided by the recent finding that the central loop of the toxin (loop II) is oriented towards the central pore of the receptor, securing the overall orientation of the bound toxin [Machold, J., Utkin, Y. N., Kirsch, D., Kaufmann, R., Tsetlin, V. & Hucho, F. (1995b) Proc. Natl Acad. Sci. USA 92, 7282-7286]. Looking at the receptor from the synaptic side of the postsynaptic membrane, it was concluded that the clockwise subunit arrangement is alpha H-gamma-alpha L-delta-beta (alpha H and alpha L are the alpha-subunits binding (+)-tubocurarine with high and low affinity, respectively). Its mirror image alpha alpha L-gamma-alpha H-beta-delta could thus be excluded.

Animals↗
PubMed1995

Photolabeling reveals the proximity of the alpha-neurotoxin binding site to the M2 helix of the ion channel in the nicotinic acetylcholine receptor.

A photoactivatable derivative of neurotoxin II from Naja naja oxiana containing a 125I-labeled p-azidosalicylamidoethyl-1,3'-dithiopropyl label at Lys-25 forms a photo-induced cross-link with the delta subunit of the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR). The cross-linked radioactive receptor peptide was isolated by reverse-phase HPLC after tryptic digestion of the labeled delta subunit. The sequence of this peptide, delta-(260-277), and the position of the label at Ala-268 were established by matrix-assisted laser-desorption-ionization mass spectrometry based on the molecular mass and on post-source decay fragment analysis. With the known dimensions of the AChR molecule, of the photolabel, and of alpha-neurotoxin, finding the cross-link at delta Ala-268 (located in the upper part of the channel-forming transmembrane helix M2) means that the center of the alpha-neurotoxin binding site is situated at least approximately 40 A from the extracellular surface of the AChR, proximal to the channel axis.

Affinity Labels↗
PubMed1995

A new class of photoactivatable and cleavable derivatives of neurotoxin II from Naja naja oxiana. Synthesis, characterisation, and application for affinity labelling of the nicotinic acetylcholine receptor from Torpedo californica.

A new series of photoactivatable and cleavable derivatives of neurotoxin II from the cobra Naja naja oxiana is investigated which can be used for mapping the surface topology of the nicotinic acetylcholine receptor from Torpedo electric tissue. The preparation and characterisation of five toxin derivatives, each with a radioactive 125I-azidosalicylamidoethyl-1,3'-dithiopropyl group in a defined position within the primary structure, are described. The photoinduced cross-linking reaction of the toxin derivatives with membrane-bound receptor is investigated. The photoactivatable group located at position K25 reacts almost exclusively with the delta subunit of the receptor, whereas the K15 derivative reacts with the alpha and beta subunits. The other derivatives did not react with the receptor to any significant extent. It is shown that, with respect to the receptor subunits, the cross-linking pattern depends on the length and chemical nature of the cross-linking group.

Affinity Labels↗
PubMed1995

Chamber-specific expression of human myocardial proteins detected by two-dimensional gel electrophoresis.

High resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by computer-assisted image analysis (PDQUEST) was used to screen atrial and ventricular protein patterns for quantitative and qualitative differences in protein expression. Myocardial proteins from left ventricular (LV) and right atrial (RA) samples from end-stage, failing explanted hearts and from a healthy donor heart (control) were separated by 2-D large gel electrophoresis. Ten RA versus ten LV gels from explanted dilated cardiomyopathic (DCM) hearts were analyzed for quantitative differences in their spot patterns. Of the 197 spots matched to every gel, 40 spots differed significantly in intensity between RA and LV for DCM patients. A larger number of atrial and ventricular gels (20 RA, 20 LV) from DCM patients and from a healthy donor heart (4 RA, 4 LV gels) were analyzed for qualitative differences in protein expression. Three protein spots (SSP 1120: M(r)/pI:20.5 kDa/4.6; SSP 1119: M(r)/pI:20.6 kDa/4.5; SSP 0117:M(r)/pI:20.7/ < 4.5) that are present in all RA gels for DCM patients are absent in all LV gels. Two protein spots (SSP 0112: M(r)/pI:17.2 kDa,/ < 4.4; SSP 0114:M(r)/pI:17.6 kDa/ < 4.4) occur only in all LV gels but not in the RA gels. These five qualitatively differing spots are identical in DCM patients and in the healthy donor heart. Some of the differing spots were internally sequenced and identified as myosin light chain isoforms (myosin light chain 2, atrial; myosin light chain 2, ventricular; myosin light chain 1, atrial) with the Protein Identification Resource (PIR) accession numbers A44451, S03708, A30881, respectively. Additionally, phosphoglycerate mutase (PIR: JQ0750) and ATP synthase alpha chain (PIR: S17193) were identified. Thus, quantitative and qualitative differences between atrial and ventricular protein patterns were identified by 2-D PAGE. A characteristic distribution of myosin light chains between atrial and ventricular human myocardium was found using our approach.

Amino Acid Sequence↗
PubMed1995

Synthesis of nitrodiazirinyl derivatives of neurotoxin II from Naja naja oxiana and their interaction with the Torpedo californica nicotinic acetylcholine receptor.

Five singly modified nitrodiazirine derivatives of neurotoxin II (NT-II) from Naja naja oxiana were obtained after NT-II reaction with N-hydroxysuccinimide ester of (2-nitro-4-[3-(trifluoromethyl)-3H-diazirin-3yl]phenoxy)acet ic acid followed by chromatographic separation of the products. To localize the label positions, each derivative was first UV-irradiated and then subjected to reduction, carboxymethylation, and trypsinolysis. Tryptic digests were separated by reversed phase-HPLC, the labeled peptides being identified by mass spectrometry. The derivatives containing the photolabel at the position Lys 25, Lys 26, Lys 44, or Lys 46 were [125I]iodinated by the chloramine T procedure. Each iodinated derivative was found to form photoinduced cross-links with the membrane-bound nicotinic acetylcholine receptor (AChR) from Torpedo californica. The pattern of labeling the receptor's alpha, beta, gamma, or delta subunits was dependent on the photolabel position in the NT-II molecule and differed from that obtained earlier with an analogous series of p-azidobenzoyl derivatives of NT-II. The results obtained indicate that (i) different sides of the neurotoxin molecule are involved in the AChR binding, and (ii) fragments of the different AChR subunits are located close together at the neurotoxin-binding sites.

Affinity Labels↗
PubMed1995

A novel nuclear pore protein Nup82p which specifically binds to a fraction of Nsp1p.

Nsp1p interacts with nuclear pore proteins Nup49p, Nup57p and Nic96p in a stable complex which participates in nucleocytoplasmic transport. An additional p80 component is associated with Nsp1p, but does not co-purify with tagged Nup57p, Nup49p and Nic96p. The p80 gene was cloned and encodes a novel essential nuclear pore protein named Nup82p. Immunoprecipitation of tagged Nup82p reveals that it is physically associated with a fraction of Nsp1p which is distinct from Nsp1p found in a complex with Nup57p, Nic96p and Nup49p. The Nup82 protein can be divided into at least two different domains both required for the essential function, but it is only the carboxy-terminal domain, exhibiting heptad repeats, which binds to Nsp1p. Yeast cells depleted of Nup82p stop cell growth and concomitantly show a defect in poly(A)+RNA export, but no major alterations of nuclear envelope structure and nuclear pore density are seen by EM. This shows that Nsp1p participates in multiple interactions at the NPC and thus has the capability to physically interact with different NPC structures.

Amino Acid Sequence↗
PubMed1995

The neurochemistry of Parkinson's disease--comments on present and future developments.

The neurochemistry of Parkinson's disease is currently undergoing considerable changes. From the analysis of diseased tissue and of lesion models, attention has turned to basic questions of neuronal development and neuronal death including the role played by trophic factors in the protection and regeneration of nervous tissue. Here some aspects are highlighted which emphasize molecular targets of possible causes and of future therapies, e.g., transmitter transporters and novel trophic effectors.

Acetylcholinesterase↗
PubMed1994

Differential nuclear localization of protein kinase C isoforms in neuroblastoma x glioma hybrid cells.

The protein kinase C (PKC) alpha, beta and epsilon isoforms have distinct nuclear localizations in neuroblastoma x glioma hybrid cells NG 108-15. We found by immunoblotting that PKC alpha, beta II, delta and epsilon are the predominant isoforms in these cells. In contrast to other neuronal cell lines, none of these isoforms is down-regulated during differentiation. Confocal immunofluorescence microscopy revealed that in undifferentiated cells PKC alpha is located in the cytoplasm and in the nucleus excluding nucleoli. In differentiated cells PKC alpha was almost exclusively located in the cytoplasm. Stimulation of the cells with phorbol ester resulted in translocation to the plasma membrane. PKC beta II was not detectable in the nuclei. PKC delta was found in the nucleoli and in the cytoplasm, in differentiated cells particularly in the neurites. Phorbol ester failed to induce a translocation to other compartments. PKC epsilon was localized with the nuclear-pore complexes at the nuclear envelope. In differentiated cells after stimulation with phorbol ester, partial translocation to the plasma membrane was observed.

Animals↗
PubMed1994

All potential glycosylation sites of the nicotinic acetylcholine receptor delta subunit from Torpedo californica are utilized.

All possible N-glycosylation sites of the delta subunit of the nicotinic acetylcholine receptor from Torpedo californica electric tissue are utilized. By a combination of microsequencing and mass spectrometry, it was shown that a high-mannose-type oligosaccharide is bound at Asn143 of the delta subunit. The oligosaccharides at positions Asn70 and Asn208 of the delta subunit are probably of the complex type. The utilized glycosylation sites pose restrictions on possible transmembrane folding models of the subunit.

Amino Acid Sequence↗
PubMed1994

The transmembrane domains of the nicotinic acetylcholine receptor contain alpha-helical and beta structures.

The transmembrane domain of the nicotinic acetylcholine receptor (nAChR) from Torpedo californica electric tissue contains both alpha-helical and beta structures. The secondary structure was investigated by Fourier transform infrared (FTIR) spectroscopy after the extramembrane moieties of the protein from the extracellular and intracellular sides of the membrane were removed by proteolysis using proteinase K. The secondary structure composition of this membrane structure was: alpha-helical 50%, beta structure and turns 40%, random 10%. The alpha-helices are shown to be oriented with respect to the membrane plane in a way allowing them to span the membrane, while no unidirectional structure for the beta structures was observed. These findings contradict previous secondary structure models based on hydropathy plots alone.

Animals↗
PubMed1994

Relationship between the binding sites for an alpha-conotoxin and snake venom neurotoxins in the nicotinic acetylcholine receptor from Torpedo californica.

Photoinduced cross-links between the iodinated Lys26-p-azidobenzoyl derivative of neurotoxin II from Naja naja oxiana cobra venom and nicotinic acetylcholine receptor from Torpedo californica (AChR) have been studied in the presence of alpha-conotoxin GI from the marine snail C. geographus. Preincubation of the AChR-enriched membranes with increasing concentrations of alpha-conotoxin GI protects first the gamma subunit from photolabelling and then the delta subunit, the IC50 values being 0.76 and 5.01 microM, respectively. The results obtained, in view of the relevant data in literature, demonstrate that the (alpha + gamma) site, which is the high affinity site for d-tubocurarine, has also a higher affinity for an alpha-conotoxin than the (alpha + delta) containing site. The latter has a somewhat higher affinity than the (alpha + gamma) site towards some naturally occurring snake venom alpha-neurotoxins or their derivatives.

Animals↗
PubMed1994

Beta-structure in the membrane-spanning part of the nicotinic acetylcholine receptor (or how helical are transmembrane helices?).

The 'four-transmembrane-helix receptors' transmit their signals from the extracellular space to the cytoplasm via an intramembrane domain. In the case of the nicotinic acetylcholine receptor this domain comprises an ion channel formed by homologous secondary structure elements in the receptor subunits. It was believed to be exclusively alpha-helical, but recent experimental evidence questions the widely accepted model: beta-strands seem to be part of the membrane-spanning domain.

Amino Acid Sequence↗