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N P Møller

Publications and source records attributed to N P Møller.

At least 19 recordsLinked to original sources
PubMed2001

Comparative study of protein tyrosine phosphatase-epsilon isoforms: membrane localization confers specificity in cellular signalling.

To study the influence of subcellular localization as a determinant of signal transduction specificity, we assessed the effects of wild-type transmembrane and cytoplasmic protein tyrosine phosphatase (PTP) epsilon on tyrosine kinase signalling in baby hamster kidney (BHK) cells overexpressing the insulin receptor (BHK-IR). The efficiency by which differently localized PTPepsilon and PTPalpha variants attenuated insulin-induced cell rounding and detachment was determined in a functional clonal-selection assay and in stable cell lines. Compared with the corresponding receptor-type PTPs, the cytoplasmic PTPs (cytPTPs) were considerably less efficient in generating insulin-resistant clones, and exceptionally high compensatory expression levels were required to counteract phosphotyrosine-based signal transduction. Targeting of cytPTPepsilon to the plasma membrane via the Lck-tyrosine kinase dual acylation motif restored high rescue efficiency and abolished the need for high cytPTPepsilon levels. Consistent with these results, expression levels and subcellular localization of PTPepsilon were also found to determine the phosphorylation level of cellular proteins including focal adhesion kinase (FAK). Furthermore, PTPepsilon stabilized binding of phosphorylated FAK to Src, suggesting this complex as a possible mediator of the PTPepsilon inhibitory response to insulin-induced cell rounding and detachment in BHK-IR cells. Taken together, the present localization-function study indicates that transcriptional control of the subcellular localization of PTPepsilon may provide a molecular mechanism that determines PTPepsilon substrate selectivity and isoform-specific function.

Animals↗
PubMed2000

Structure-based design of a low molecular weight, nonphosphorus, nonpeptide, and highly selective inhibitor of protein-tyrosine phosphatase 1B.

Several protein-tyrosine phosphatases (PTPs) have been proposed to act as negative regulators of insulin signaling. Recent studies have shown increased insulin sensitivity and resistance to obesity in PTP1B knockout mice, thus pointing to this enzyme as a potential drug target in diabetes. Structure-based design, guided by PTP mutants and x-ray protein crystallography, was used to optimize a relatively weak, nonphosphorus, nonpeptide general PTP inhibitor (2-(oxalyl-amino)-benzoic acid) into a highly selective PTP1B inhibitor. This was achieved by addressing residue 48 as a selectivity determining residue. By introducing a basic nitrogen in the core structure of the inhibitor, a salt bridge was formed to Asp-48 in PTP1B. In contrast, the basic nitrogen causes repulsion in other PTPs containing an asparagine in the equivalent position resulting in a remarkable selectivity for PTP1B. Importantly, this was accomplished while retaining the molecular weight of the inhibitor below 300 g/mol.

Animals↗
PubMed2000

2-(oxalylamino)-benzoic acid is a general, competitive inhibitor of protein-tyrosine phosphatases.

Protein-tyrosine phosphatases (PTPs) are critically involved in regulation of signal transduction processes. Members of this class of enzymes are considered attractive therapeutic targets in several disease states, e.g. diabetes, cancer, and inflammation. However, most reported PTP inhibitors have been phosphorus-containing compounds, tight binding inhibitors, and/or inhibitors that covalently modify the enzymes. We therefore embarked on identifying a general, reversible, competitive PTP inhibitor that could be used as a common scaffold for lead optimization for specific PTPs. We here report the identification of 2-(oxalylamino)-benzoic acid (OBA) as a classical competitive inhibitor of several PTPs. X-ray crystallography of PTP1B complexed with OBA and related non-phosphate low molecular weight derivatives reveals that the binding mode of these molecules to a large extent mimics that of the natural substrate including hydrogen bonding to the PTP signature motif. In addition, binding of OBA to the active site of PTP1B creates a unique arrangement involving Asp(181), Lys(120), and Tyr(46). PTP inhibitors are essential tools in elucidating the biological function of specific PTPs and they may eventually be developed into selective drug candidates. The unique enzyme kinetic features and the low molecular weight of OBA makes it an ideal starting point for further optimization.

Binding Sites↗
PubMed1998

Mutant forms of the protein tyrosine phosphatase alpha show differential activities towards intracellular substrates.

BHK cells overexpressing five million IR (BHK-IR) respond to insulin with reduced growth and detachment from the dish surface. We have recently identified protein tyrosine phosphatase (PTP) alpha as a negative regulator of the insulin receptor (IR) tyrosine kinase that is able to rescue BHK-IR cells from the insulin effect. In this report we describe the effect of several point mutations in PTP alpha on the phosphatase activity and regulation of insulin signaling in BHK-IR cells. Analysis of total cellular phosphotyrosine protein revealed several molecules that were dephosphorylated when PTP alpha or a phosphatase active mutant was overexpressed. By contrast, some proteins were tyrosine phosphorylated as strong or to an even higher extent as in the parental line when PTP alpha Y798F was present. We conclude that mutation of the carboxyterminal tyrosine in PTP alpha uncovers a dual function of this phosphatase in BHK cells: reduction of the IR signal and activation of an endogenous kinase.

Animals↗
PubMed1997

Expression of protein-tyrosine phosphatases in the major insulin target tissues.

Protein-tyrosine phosphatases (PTPs) are key regulators of the insulin receptor signal transduction pathway. We have performed a detailed analysis of PTP expression in the major human insulin target tissues or cells (liver, adipose tissue, skeletal muscle and endothelial cells). To obtain a representative picture, all tissues were analyzed by PCR using three different primer sets corresponding to conserved regions of known PTPs. A total of 24 different PTPs were identified. A multiprobe RNase protection assay was developed to obtain a semiquantitative measure of the expression levels of selected PTPs. Surprisingly, PTP-LAR, previously suggested to be a major regulator of the insulin receptor tyrosine kinase, was expressed in extremely low levels in skeletal muscle, whereas the related receptor-type PTP-sigma and PTP-alpha were expressed in relatively high levels in all four tissues. The low levels of LAR PTP mRNA in skeletal muscle were further confirmed by Northern blot analysis.

Adipose Tissue↗
PubMed1996

ERK6, a mitogen-activated protein kinase involved in C2C12 myoblast differentiation.

ERK6, a mitogen-activated protein (MAP) kinase-related serine/threonine kinase, is highly expressed in human skeletal muscle and appears to function as a signal transducer during differentiation of myoblasts to myotubes. In transfected 293 cells, activation of the 45-kDa enzyme results in tyrosine-phosphorylated 46- and 56-kDa forms, which phosphorylate myelin basic protein. Overexpression of wild-type ERK6 or the inactive mutant Y185F has no effect on fibroblast and myoblast proliferation, but it enhances or inhibits C2C12 cell differentiation to myotubes, respectively. Our findings suggest ERK6 to be a tissue-specific, differentiation signal-transducing factor that is connected to phosphotyrosine-mediated signaling pathways distinct from those activating other members of the MAP kinase family such as LRK1 and ERK2.

3T3 Cells↗
PubMed1995

Selective down-regulation of the insulin receptor signal by protein-tyrosine phosphatases alpha and epsilon.

Binding of insulin to its receptor (IR) causes rapid autophosphorylation with concomitant activation of its tyrosine kinase which transmits the signal by phosphorylating cellular substrates. The IR activity is controlled by protein-tyrosine phosphatases, but those directly involved in regulating the insulin receptor and its signaling pathways have not yet been identified. Using baby hamster kidney cells overexpressing the IR and a novel insulin-based selection principle, we established stable cell lines with functionally coupled expression of the IR and protein-tyrosine phosphatases. The two closely related protein-tyrosine phosphatases alpha and epsilon were identified as negative regulators of IR tyrosine kinase.

Animals↗
PubMed1994

Src kinase associates with a member of a distinct subfamily of protein-tyrosine phosphatases containing an ezrin-like domain.

A 6.2-kb full-length clone encoding a distinct protein-tyrosine phosphatase (PTP; EC 3.1.3.48), PTPD1, was isolated from a human skeletal muscle cDNA library. The cDNA encodes a protein of 1174 amino acids with N-terminal sequence homology to the ezrin-band 4.1-merlin-radixin protein family, which also includes the two PTPs H1 and MEG1. The PTP domain is positioned in the extreme C-terminal part of PTPD1, and there is an intervening sequence of about 580 residues without any apparent homology to known proteins separating the ezrin-like and the PTP domains. Thus, PTPD1 and the closely related, partially characterized, PTPD2 belong to the same family as PTPH1 and PTPMEG1, but because of distinct features constitute a different PTP subfamily. Northern blot analyses indicate that PTPD1 and PTPD2 are expressed in a variety of tissues. In transient coexpression experiments PTPD1 was found to be efficiently phosphorylated by and associated with the src kinase pp60src.

Amino Acid Sequence↗
PubMed1994

Expression, purification and crystallization of human phosphotyrosine phosphatase 1B.

Protein phosphotyrosine phosphatases are believed to be involved in the regulation of the activity of cellular proteins, such as receptor tyrosine kinases, by controlling their phosphorylation status. One of the best described and characterized protein of this class of enzymes is the phosphotyrosine phosphatase 1B. To obtain sufficient quantities for structural investigations, truncated forms of PTP1B encompassing the catalytic domain were over-expressed in Escherichia coli and purified to apparent homogeneity by conventional chromatography. The activity of these purified enzymes has been compared with the wild-type enzyme expressed in mammalian cells. By measuring the activities against p-nitrophenyl phosphate, the pH dependence of this activity, and responses to different modulators, it could be demonstrated that the truncated forms of PTP1B retained the same characteristics as the full-length mammalian enzyme, but are not subject to inhibition of enzymic activity mediated by the C-terminus. Due to their improved solubility, it can be assumed that the catalytic domains are advantageous for crystallization studies in comparison to the natural enzyme. In a screening for crystallization conditions, we obtained protein crystals indicating that the quality of the purified protein is sufficient for crystallographic studies.

Base Sequence↗
PubMed1994

Amino acid sequence of human pregnancy-associated plasma protein-A derived from cloned cDNA.

The amino acid sequence of human pregnancy-associated plasma protein-A (PAPP-A), a component of the circulating complex with the proform of eosinophil major basic protein (proMBP), has been determined from partial protein sequencing and from sequencing of cloned cDNA. The PAPP-A monomer contains 1547 amino acid residues, but is derived from a larger precursor of placental origin. PAPP-A contains 82 Cys residues, which are all bridged, 14 putative sites for N-glycosylation, and 7 putative sites for attachment of glycosaminoglycan groups. The C-terminal part of PAPP-A contains 5 approximately 60-residue motifs related to the short consensus repeats of complement proteins and selectins. The SCRs presently known can be grouped into three classes: complement-type, class I; selectin-type, class II; PAPP-A-type, class III. PAPP-A further contains three approximately 26-residue motifs, related to the lin-notch motifs of proteins regulating early tissue differentiation, and, in addition, a putative Zn2+ binding site similar to that found in many metalloproteinases has been identified. Apart from these features, the PAPP-A sequence is not related to other known protein sequences.

Amino Acid Sequence↗
PubMed1993

High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis.

Examination of flk-1 receptor tyrosine kinase mRNA expression by in situ hybridization analysis revealed specific association with endothelial cells at all stages of mouse development, including the blood islands in the yolk sac of day 8.5-10.5 embryos, in which the early progenitors of this lineage originate. flk-1 transcripts were abundant in proliferating endothelial cells of vascular sprouts and branching vessels of embryonic and early postnatal brain, but were drastically reduced in adult brain, where proliferation has ceased. Identification of the angiogenic mitogen, vascular endothelial growth factor (VEGF), as the high affinity ligand of Flk-1 and correlation of the temporal and spatial expression pattern of Flk-1 and VEGF suggest a major role of this ligand-receptor signaling system in vasculogenesis and angiogenesis.

Amino Acid Sequence↗
PubMed1992

Identification of determinants that confer ligand specificity on the insulin receptor.

We have previously shown, using truncated soluble recombinant receptors, that substituting the 62 N-terminal amino acids of the alpha subunit from the insulin-like growth factor I receptor (IGFIR) with the corresponding 68 amino acids from the insulin receptor (IR) results in a chimeric receptor with an approximately 200-fold increase in affinity for insulin and only a 5-fold decrease in insulin-like growth factor I (IGFI) affinity (Kjeldsen, T., Andersen, A. S., Wiberg, F. C., Rasmussen, J. S., Schäffer, L., Balschmidt, P., Møller, K. B., and Møller, N. P. H. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4404-4408). We demonstrate that these 68 N-terminal amino acids of the IR also confer insulin affinity on the intact IGFI holoreceptor both in the membrane-bound state and when solubilized by Triton X-100. Furthermore, this domain can be subdivided into two regions (amino acids 1-27 and 28-68 of the IR alpha subunit) that, when replacing the corresponding IGFIR sequences, increases the insulin affinity of truncated soluble receptor chimeras 8- and 20-fold, respectively, with only minor effects on the IGFI affinity. Within the latter of these two regions, we found that amino acids 38-68 of the IR, representing 13 amino acid differences from IGFIR, confer the same 20-fold increase in insulin affinity on the IGFIR. Finally, the amino acids from position 42 to 50 are not responsible for this increase in insulin affinity. We thus propose that at least two determinants within the 68 N-terminal amino acids of the insulin receptor are involved in defining the ligand specificity of the insulin receptor, and that one or a combination of the remaining seven amino acid differences between position 38 and 68 are involved in conferring insulin affinity on the insulin receptor.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA.

Human beta 2-glycoprotein (beta 2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature beta 2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 kb, respectively, hybridizing specifically with the beta 2gpI cDNA. Upon isoelectric focusing, recombinant beta 2gpI obtained from expression of beta 2gpI cDNA in baby hamster kidney cells showed the same pattern of bands as beta 2gpI isolated from plasma, and at least 5 polypeptides were visible.

Amino Acid Sequence↗
PubMed1991

The ligand specificities of the insulin receptor and the insulin-like growth factor I receptor reside in different regions of a common binding site.

To identify the region(s) of the insulin receptor and the insulin-like growth factor I (IGF-I) receptor responsible for ligand specificity (high-affinity binding), expression vectors encoding soluble chimeric insulin/IGF-I receptors were prepared. The chimeric receptors were expressed in mammalian cells and partially purified. Binding studies revealed that a construct comprising an IGF-I receptor in which the 68 N-terminal amino acids of the insulin receptor alpha-subunit had replaced the equivalent IGF-I receptor segment displayed a markedly increased affinity for insulin. In contrast, the corresponding IGF-I receptor sequence is not critical for high-affinity IGF-I binding. It is shown that part of the cysteine-rich domain determines IGF-I specificity. We have previously shown that exchanging exons 1, 2, and 3 of the insulin receptor with the corresponding IGF-I receptor sequence results in loss of high affinity for insulin and gain of high affinity for IGF-I. Consequently, it is suggested that the ligand specificities of the two receptors (i.e., the sequences that discriminate between insulin and IGF-I) reside in different regions of a binding site with common features present in both receptors.

Animals↗
PubMed1990

Changing the insulin receptor to possess insulin-like growth factor I ligand specificity.

To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, we prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the alpha-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor.

Amino Acid Sequence↗
PubMed1989

Human lung cancer--a comparative study of the levels of circulating immune complexes in pulmonary blood draining the tumor area and peripheral venous blood.

Our objective was to investigate whether low levels of circulating immune complexes (cICs) in peripheral venous blood of cancer patients could be due to removal of cIC released at the tumor site during passage to the peripheral veins. In 54 patients with primary lung cancer, we therefore compared the cIC levels as detected by 3 different assays in paired samples from the pulmonary vein draining the tumor area and from a peripheral vein. Only 6 of the 54 patients had significantly increased pulmonary vein cIC levels as compared to the corresponding peripheral vein levels. The peripheral vein levels of these 6 patients were all within the normal range, and in none of these patients was the difference between the 2 sites of analysis--although significant--of such a magnitude that the pulmonary vein cIC level appeared higher than the normal range, i.e., "positive" for cIC. Positive cIC levels were only found in 11% of lung cancer patients (irrespective of the site of measurement). Thus, our present data, together with our previous findings indicating no significant difference between peripheral venous blood cIC levels in cancer patients and normal controls, contradict the theory of tumor cells expressing new antigens resulting in the formation of tumor-associated cICs.

Antigen-Antibody Complex↗
PubMed1989

Analysis of the decreased NK (natural killer) activity in lung cancer patients, using whole blood versus separated mononuclear cells.

The aim of this study was to analyze whether a whole blood assay would give a more correct measure of NK activity than assays using separated mononuclear cells (SMNC). We found that the NK activity of whole blood was higher than the NK activity of SMNC in the 28 lung cancer patients investigated (p = 0.01), whereas this difference between the assays could not be demonstrated in the 29 healthy controls. Since no differences were found between the NK activity of washed blood, SMNC, and monocyte-depleted lymphoid cells, there was no indication that the lower NK activity of SMNC in comparison with whole blood was due to cell loss or to a systematic disturbing effect due to monocytes. The possible effect of plasma factors on the whole blood NK activity was analyzed by comparing whole blood and washed blood. The NK activity of whole blood was increased in comparison with washed blood in the lung cancer patients (p less than 0.0001) indicating a stimulatory effect of plasma. Further, the finding that the reactive capability of lymphocytes from cancer patients was higher than in controls could indicate preactivation of the lymphocytes from the cancer patients due to the presence of stimulatory plasma factors. The NK activity of lung cancer patients was lower than the NK activity of healthy controls. The difference was found to be smaller with whole blood than with SMNC as effector cells, although both differences were significant. The decreased NK activity of cancer patients could be due to blocking immune complexes (IC), but we found no evidence for circulating or cell-bound IC in the lung cancer patients.

Adult↗