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W Möller

Publications and source records attributed to W Möller.

At least 55 records · Page 3Linked to original sources
PubMed1992

Relation between the insulin receptor number in cells, autophosphorylation and insulin-stimulated Ras.GTP formation.

We showed previously that upon insulin stimulation of an insulin receptor overexpressing cell line, most of the p21ras was rapidly converted into the GTP bound state (Burgering, B. M. T., Medema, R. H., Maassen, J. A., Van de Wetering, M. L., Van der Eb, A. J., McCormick, F., and Bos, J. L. (1991) EMBO J. 10, 1103-1109). To determine whether this process also occurs in cells expressing physiologically relevant numbers of insulin receptors, insulin stimulated Ras.GTP formation was quantitated in Chinese hamster ovary (CHO)-derived cell lines expressing varying numbers of insulin receptors. In the parental CHO9 cells, expressing only 5.10(3) insulin receptors, insulin stimulation for 3 min increased Ras.GTP levels with 10%. Upon increasing the number of insulin receptors in these cells, Ras.GTP levels increased almost proportionally until a plateau value of 60% is reached at high receptor numbers. These data show that receptor overexpression is not a prerequisite for insulin-stimulated Ras.GTP formation. The yield of Ras.GTP generated is 0.2-1.0 mol/mol autophosphorylated insulin receptor in CHO9- and NIH3T3-derived cell lines, respectively. These values argue against signal-amplifying processes between the insulin receptor and p21ras. To determine whether receptor autophosphorylation is required for Ras.GTP formation, NIH3T3 cells overexpressing insulin receptors were stimulated with a monoclonal antibody which activates the receptor and subsequent glucose transport without inducing detectable autophosphorylation. Also, CHO cells expressing the mutant Ser1200 receptor, which has markedly impaired tyrosyl autophosphorylation but is capable of mediating insulin-stimulated metabolic effects in CHO cells, were used. In both cases, no Ras.GTP formation was observed. Furthermore, Rat-1-derived cell lines expressing mutant p21ras, which is permanently in the active GTP-bound form, still responded to insulin by increasing the glucose uptake. These results support our hypothesis that Ras.GTP formation is activated by the tyrosyl-phosphorylated insulin receptor and suggest that an active Ras.GTP complex does not mediate metabolic signaling.

3T3 Cells↗
PubMed1992

An anti-peptide antibody that recognizes the dopamine D2 receptor from bovine striatum.

The bovine dopamine D2 receptor was purified by wheat-germ-agglutinin-Sepharose chromatography and affinity chromatography, using the D2-receptor-specific agonist N-0434. Purification yields a preparation with a major protein band of 95 kDa. In order to ascertain the identity of this protein, polyclonal antibodies against the dopamine D2 receptor have been raised using synthetic peptides based on the predicted amino acid sequence of the cloned D2 receptor. For the initial screening of these antibodies, three fusion proteins consisting of beta-galactosidase and receptor fragments were constructed. One antiserum reacted strongly with the corresponding D2 receptor fusion protein, both on Western blots and in immunoprecipitation experiments. In each case, recognition was inhibited by competition with free peptide. On Western blots of partially purified receptor preparations from bovine striatum, the antiserum specifically recognized a 95-kDa glycoprotein. From similar preparations, the antiserum precipitated a substantial proportion of active D2 receptor, as determined by a decrease in [3H]spiperone binding in the supernatant. Active receptor could be released from the immunoprecipitate by addition of free peptide. Immunocytochemical analysis of cells transiently transfected with DNA coding for the D2 receptor showed specific staining of transfected cells. The antibody raised against a sequence in the third intracellular loop is able to shift the affinity of the receptor for dopamine from high to low, indicating that the antiserum may be interfering with receptor-GTP-binding-protein interactions.

Animals↗
PubMed1992

An Arg for Gly substitution at position 31 in the insulin receptor, linked to insulin resistance, inhibits receptor processing and transport.

In a patient with Leprechaunism, we have characterized a new mutation in the insulin receptor substituting Arg for Gly at position 31. The proband, the mother, and the maternal grandfather were heterozygous for the mutation. Fibroblasts of the proband show a strongly reduced number of high affinity insulin receptors on the cell surface, whereas fibroblasts of the healthy mother and grandfather show moderately reduced insulin receptor numbers. In the other family members neither the binding defect nor the Arg31 mutation was found. The Arg31-mutant receptor was overexpressed in Chinese hamster ovary cells. In these cells the mutant alpha beta-proreceptor was not proteolytically cleaved and no transport to the cell surface took place. The proreceptor was unable to bind insulin and to undergo autophosphorylation. In addition, the proreceptor was not recognized by monoclonal antibodies directed against conformation-dependent epitopes. These findings suggest that the Gly31 to Arg31 mutant is involved in the insulin receptor dysfunction seen in the Leprechaun patient. The mutation seems to alter the conformation of the receptor in such way that the transport of the proreceptor to the Golgi compartment, where proteolytical processing occurs, is inhibited.

Alleles↗
PubMed1992

Statistical evidence for remnants of the primordial code in the acceptor stem of prokaryotic transfer RNA.

The specificity of interaction of amino acids with triplets in the acceptor helix stem of tRNA was investigated by means of a statistical analysis of 1400 tRNA sequences. The imprint of a prototypic genetic code at position 3-5 of the acceptor helix was detected, but only for those major amino acids, glycine, alanine, aspartic acid, and valine, that are formed by spark discharges of simple gases in the laboratory. Although remnants of the code at position 3-5 are typical for tRNAs of archaebacteria, eubacteria, and chloroplasts, eukaryotes do not seem to contain this code, and mitochondria take up an intermediary position. A duplication mechanism for the transposition of the original 3-5 code toward its present position in the anticodon stem of tRNA is proposed. From this viewpoint, the mode of evolution of mRNA and functional ribosomes becomes more understandable.

Amino Acyl-tRNA Synthetases↗
PubMed1992

The role of ras proteins in insulin signal transduction.

Ras-proteins are guanine nucleotide binding proteins, which, in the GTP bound state emit a strong mitogenic signal. In the GDP bound state, the protein appears inactive. We have found that stimulation by insulin of cells expressing elevated levels of insulin receptors results in a rapid conversion of Ras-GDP into Ras-GTP. This process is part of the signalling pathway leading to immediate-early gene expression and a mitogenic response. There seems to be no involvement of Ras-GTP formation in the process of insulin stimulated glucose transport. Though the precise mechanism by which Ras is converted to the GTP bound state remains to be established, a tight correlation exists between receptor autophosphorylation and Ras-GTP formation.

Animals↗
PubMed1992

Investigation of the defense system of the human lungs with ferrimagnetic particles.

Magnetic microparticles were used to investigate the defense system of the human lungs against foreign material. About 0.5 mg of spherical monodisperse magnetite particles were deposited in the alveolar region of the human lung by voluntary inhalation. After primary magnetization a remanent magnetic field (rmf) of the lung can be measured that allows estimation of the amount of dust retained in the lung. The decay of this rmf, called relaxation, results from a misalignment of the dipole particles due to the activity of pulmonary macrophages. This macrophage activity was characterized by a cell energy E z. With a secondary magnetization the lung can be remagnetized by rotation of the dipole particles. This allows estimation of the intracellular viscosity and motility of the alveolar macrophages in vivo. Secondary magnetization and relaxation curves of spherical monodisperse magnetite particles are presented. Intracelluar viscosity was estimated to be n approximately equal to 100 Paxs by a shear-rate near 0.01 s -1, macrophage activity was E z approximately equal to 5x10 -18 J. Aerosol exposure resulted in a faster relaxation, which was interpreted to be due to activation of the macrophages. The magnetite particles were cleared with a half-time of approximately equal to 110 days.

Humans↗
PubMed1992

Interactions between the dopamine D2 receptor and GTP-binding proteins.

The dopamine D2 receptor in bovine brain striatum exists in a high- and a low-affinity state for dopamine. The high-affinity state is believed to arise from coupling with a guanine nucleotide-binding protein, to be disrupted by the addition of GTP. We found that antibodies specific to the C-terminal region of the alpha-subunit of Go and Gi cause a shift from high- towards low-affinity, demonstrating that the C-terminus of G alpha is involved in the receptor G protein contact. Purification of the D2 receptor by affinity chromatography via immobilized agonists or antagonists results in the copurification of both Go and Gi proteins. However, this copurification is of a nonspecific nature and prevents the detection of putative specific receptor.G protein complexes.

Animals↗
PubMed1991

A leucine to proline mutation at position 233 in the insulin receptor inhibits cleavage of the proreceptor and transport to the cell surface.

We have previously shown that a homozygous mutation encoding a substitution of proline for leucine at position 233 in the insulin receptor is linked with the syndrome of leprechaunism, being a lethal form of insulin resistance in newborn children. Specific binding of insulin and insulin-stimulated autophosphorylation of the insulin receptor are nearly absent in fibroblasts from the leprechaun patient. To examine the molecular basis of the observed insulin receptor abnormalities, CHO cell lines overexpressing mutant insulin receptors were made by transfection. The results show that the mutation inhibits cleavage and transport of the proreceptor from intracellular sites to the cell surface. As the mutant receptor is poorly precipitated by two different monoclonal antibodies recognizing epitopes on undenatured wild-type alpha-subunits, the mutation probably affects overall folding of the alpha-subunit. The mutant proreceptor is unable to bind insulin and exhibits no insulin-stimulated autophosphorylation. These data explain the abnormalities seen in the patient's fibroblasts. Pulse-chase labeling experiments on transfected cells show that the mutant precursor has an extended half-life (approximately 5 h) compared to the precursor of wild-type insulin receptors (approximately 2 h). This mutation is the first example of a naturally occurring mutation in the insulin receptor which completely blocks cleavage of the proreceptor and transport to the cell surface.

Animals↗
PubMed1991

A major substrate of maturation promoting factor identified as elongation factor 1 beta gamma delta in Xenopus laevis.

Protein synthesis is believed to be under control of the cell cycle during meiosis and mitosis. Any relationship between substrates for cdc2 kinase and components of the protein synthetic apparatus would therefore be of prime importance. During meiosis of Xenopus laevis oocytes one of the substrates for this kinase is a p47 protein, which is complexed to two other proteins, P36 and P30. Judged from partial amino acid sequence data on P47 and P30, the P30 and P47 proteins were reported to resemble the protein synthetic elongation factors (EF) 1 beta and 1 gamma from Artemia salina (Bellé, R., Derancourt, J., Poulhe, R., Capony, J.P., Ozon, R., and Mulner-Lorillon, O. (1989) FEBS Lett. 255, 101-104). This paper shows that the complex composed of P30, P47, and P36 from Xenopus is identical to the complex of EF-1 beta, EF-1 gamma, and EF-1 delta from Artemia according to two criteria. 1) Both stimulate elongation factor 1 alpha-mediated transfer RNA binding to ribosomes and exchange of guanine nucleotides on elongation factor 1 alpha to a comparable degree. 2) Each of the three subunits of the protein complex P30.P47.P36 from Xenopus shows a structural homology with one of the corresponding subunits of EF-1 beta gamma delta from Artemia. Presumably the phosphorylation of EF-1 gamma, which associates with tubulin at least in vitro, is important in processes following the onset of meiosis which is accompanied by a rise of protein synthesis.

Amino Acid Sequence↗
PubMed1991

Mapping the functional domains of the eukaryotic elongation factor 1 beta gamma.

The functional domains of the eukaryotic elongation factor (EF) 1 beta gamma have been delineated with the use of limited proteolysis, protein microsequencing, gel electrophoresis under non-denaturing conditions and antibodies against EF-1 beta and EF-1 gamma. By means of limited proteolysis, it was possible to obtain large fragments of EF-1 beta. In contrast to amino-terminal fragments, those derived from the carboxy-terminal part of EF-1 beta were still active in enhancing the guanine nucleotide exchange of GDP bound to EF-1 alpha. With the same technique of limited proteolysis, it was possible to isolate a trypsin-resistant core from EF-1 beta gamma containing polypeptide chain fragments derived from both subunits. A polyvalent antiserum against EF-1 beta and two monoclonal antibodies against EF-1 gamma were used to identify the protein fragments in this core. The monoclonal antibodies were shown to recognize different epitopes, one localized on the amino-terminal and another on the carboxy-terminal half of EF-1 gamma. The antiserum against EF-1 beta and one of the monoclonal antibodies (mAb 36E5), which recognized the amino-terminal half of EF-1 gamma, reacted with this trypsin-resistant core. We conclude that the amino-terminal halves of both EF-1 beta and EF-1 gamma are firmly attached to each other, and that the carboxy-terminal part of EF-1 beta interacts with EF-1 alpha.

Antibodies, Monoclonal↗
PubMed1990

The primary structure of artemin from Artemia cysts.

The primary structure of artemin, a major protein isolated from Artemia cysts, has been determined by direct Edman degradation of the purified protein. The amino-terminal acetylated protein has 229 amino acid residues and a high content of histidine and cysteine/cystine. A search in the GenBank Data Base at Los Alamos, using the FASTA program [Pearson, W. R. & Lipman, D. J. (1988) Proc. Natl. Acad. Sci. USA 85, 2444-2448] revealed a limited but unmistakable similarity to ferritin from vertebrates.

Amino Acid Sequence↗
PubMed1990

Elongation factor 1 beta of artemia: localization of functional sites and homology to elongation factor 1 delta.

Elongation factor (EF)-1 beta, a 26 kDa protein, is the eukaryotic equivalent of bacterial EF-Ts, the nucleotide exchange factor in protein synthesis. EF-1 beta catalyzes the exchange of guanine nucleotides bound to EF-1 alpha; the latter protein is the eukaryotic equivalent of bacterial EF-Tu. Limited proteolytic cleavage studies on EF-1 beta lead to the following picture: the protein is composed of two domains, an aminoterminal and a carboxyterminal domain, connected to each other by a stretch of hydrophilic, charged amino acids situated in the middle of the molecule. The carboxyterminal domain supplies the catalytic site for the nucleotide exchange reaction, whereas the aminoterminal domain interacts with EF-1 gamma, the third component of elongation factor 1. The regulatory, serine phosphate residue, Ser-89, localized in the hydrophilic stretch of EF-1 beta, does not appear to be necessary for the basic exchange reaction. The fourth component of the high molecular weight elongation factor complex (EF-1H), named EF-1 delta or 28 K protein, is homologous to EF-1 beta and contains regions very similar to the carboxyterminal part. EF-1 delta was found to be active in the nucleotide exchange reaction.

Amino Acid Sequence↗
PubMed1990

Elongation factor EF-1 alpha from Artemia is specifically labeled at Lys-63 by the guanine nucleotide analog pyridoxal-5'-diphospho-5'-guanosine.

We have synthesized the guanine nucleotide analog pyridoxal-5'-diphospho-5'-guanosine. This compound specifically modifies a single lysine residue in elongation factor 1 alpha from Artemia, indicating that this residue is in close contact with the reactive part of the guanine nucleotide analog. This result is discussed in terms of the structure of the nucleotide-binding domain of the factor.

Affinity Labels↗
PubMed1990

Structural and functional differentiation of the embryonic chick pineal organ in vivo and in vitro. A scanning electron-microscopic and radioimmunoassay study.

The development of sensory structures in the pineal organ of the chick was examined by means of scanning electron microscopy from embryonic day 10 through day 12 post-hatching. At embryonic day 10, the wall of the tubules within the pineal primordium is composed of cells with unspecialized luminal surface. Differentiation of sensory structures starts at embryonic day 12 when pinealocytes and supporting cells can be distinguished. Pinealocytes are recognized by virtue of an inner segment only rarely endowed with a cilium, whereas supporting cells exhibit numerous short microvilli. Further differentiation of the sensory apparatus is achieved by development of an oval-shaped, biconcave swelling at the tip of the cilium, 1 x 2 microns in size, and a collar of long microvilli at the base of the inner segment. Membrane specializations of sensory cilia, however, were not detected. Since during embryonic life new tubules and follicles are continuously formed, all stages of differentiation of sensory structures are found in the chick pineal organ during the second half of the incubation period and the first two weeks after hatching. In 200-microns-thick Vibratome sections of chick-embryo pineal organs cultured in medium BM 86 Wissler for periods up to 13 days the cytodifferentiation parallels the development in vivo. Using an organ-culture system the 24-h release of melatonin into the culture medium was measured by means of radioimmunoassay after solid-phase extraction. At embryonic day 10, the 24-h secretion of melatonin was at the lower range of detection of the RIA (5 pg). The rapid increase in 24-h secretion in melatonin until hatching (approximately 50 micrograms) is approximated by an exponential curve.

Animals↗
PubMed1990

Transfer RNAs for primordial amino acids contain remnants of a primitive code at position 3 to 5.

Analysis of the nucleotide sequence of 1,400 transfer RNAs has revealed the imprint of a prototypic genetic code in position 3-4-5 of the acceptor stem. It appears only in the transfer RNAs for the primordial amino acids ie those found by chemical condensation of a nitrogen-methane-water-ammonia mixture. The model for primitive protein synthesis as mentioned by Crick assumes a direct interaction between the amino acid and a prototypic adaptor oligonucleotide. This has hitherto appeared irreconcilable with the large spatial separation between the aminoacylation site and the anticodon in present day transfer RNAs. The observations reported here show how this paradox can be resolved by a process of duplication and cleavage of a prototypic adaptor.

Amino Acid Sequence↗
PubMed1989

Affinity chromatography with the immobilized agonist N-0434 yields an active and highly purified preparation of the dopamine D-2 receptor from bovine striatum.

Partial purification of the dopamine D-2 receptor from bovine striatum, solubilized in the presence of 1% digitonin, was obtained by chromatography on wheat germ lectin agarose. The preparation was purified approximately 10-fold. The stability of the receptor preparation was considerably improved and non-specific protein absorption on the affinity gel used later was decreased. Further purification was achieved on a column containing a D-2-selective agonist, N-0434. Approximately 90% of the receptor activity was bound to the gel and 20-40% of the activity could be eluted by pH shock. The total purification factor after one affinity chromatography step was estimated to be at least 1500. An active preparation of at least 20% purity was obtained after a second cycle of affinity chromatography. This corresponds to an enrichment of more than 5000 times compared to the solubilized receptor preparation.

Animals↗
PubMed1989

A leucine-to-proline mutation in the insulin receptor in a family with insulin resistance.

We have determined the primary structure of a mutant insulin receptor of a leprechaun patient born from a consanguineous marriage. A characteristic feature of leprechaunism is an extreme resistance to insulin. In this patient the insulin resistance seems to result from an observed lack of insulin binding to intact cells. Solubilization of cells in non-ionic detergents leads to the appearance of insulin receptors which can bind insulin. However, the insulin-stimulated autophosphorylation of the receptor's beta subunit is markedly reduced. Cloning and sequencing of cDNA derived from insulin receptor mRNA of this patient revealed a leucine-to-proline mutation at position 233 in the alpha subunit. By means of DNA amplification we found that the patient is homozygous for this mutation and that the parents and two grandparents from the consanguineous line are heterozygous. The heterozygous individuals all show decreased insulin binding to cultured fibroblasts. In addition, they are mildly insulin resistant in vivo. These observations show a linkage between the leucine-to-proline mutation and the observed insulin resistance in this family. We therefore conclude that the mutation in the homozygous form is responsible for the extreme insulin resistance in the leprechaun patient. The mutation for the first time characterizes a region in the insulin receptor which seems to be involved in transmitting the insulin binding signal to the tyrosine kinase domain.

Cells, Cultured↗