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L Ellis

Publications and source records attributed to L Ellis.

At least 19 recordsLinked to original sources
PubMed1992

Insulin receptor tyrosine kinase domain auto-dephosphorylation.

We have observed dephosphorylation of the soluble, 48 kDa insulin receptor tyrosine kinase domain following its tyrosine autophosphorylation. Dephosphorylation was associated with generation of inorganic phosphate, thereby making catalysis by reversal of the kinase reaction unlikely. The kinase domain preparations could not be shown to contain detectable, contaminating protein tyrosine phosphatase activity. In addition, dephosphorylation was insensitive to protein phosphatase inhibitors. However, it was blocked by the kinase inhibitor staurosporine. These results are consistent with insulin receptor kinase domain auto-dephosphorylation via catalysis involving the kinase itself. These findings raise the possibility of a novel mechanism for termination of the insulin receptor signal.

Animals↗
PubMed1992

Structural organization of the human insulin receptor ectodomain.

To provide an experimental system amenable to a detailed biochemical and structural investigation of the extracellular (ligand binding) domain of the insulin receptor, we developed a mammalian heterologous cell expression system from which tens of milligrams of the soluble secreted ectodomain (the IR921 protein) can be routinely purified using methods that do not require harsh elution conditions. The purified IR921 protein has a Stokes radius of 6.8 nm and a sedimentation coefficient of 9.8 S, from which we calculate a hydro-dynamic mass of 281 kDa. Electron microscopic images, using both rotary shadowing and negative staining techniques, demonstrate a characteristic substructure for the IR921 protein consisting of two elongated arms, with a globular domain at each end, connected to each other at a point somewhat off-center to form a Y structure. Analysis using circular dichroism and fluorescence spectroscopy illustrate that insulin binding results in conformational changes in the ectodomain. Furthermore, fluorescence anisotropy decay data reveal segmental mobility within the IR921 protein that is successively frozen as a result of insulin binding, in contrast to results obtained in a previous study of the epidermal growth factor receptor ectodomain. This result suggests a divergence in hormone-induced signaling mechanisms used by the insulin and epidermal growth factor receptors.

Amino Acid Sequence↗
PubMed1992

An assessment of human insulin receptor phosphorylation and exogenous kinase activity following deletion of 69 residues from the carboxyl-terminus of the receptor beta-subunit.

A mutant human insulin receptor with a carboxyl-terminal deletion of 69 amino acids (proreceptor residues 1287-1355) is expressed as a stable protein in transiently transfected COS cells. We find that in intact cells this mutant is phosphorylated in an insulin-dependent manner on core tyrosines 1158, 1163 and 1163. As expected, the carboxyl-terminal beta-subunit phosphorylation sites (serines 1305/6, tyrosines 1328/34 and threonine 1348) are absent from this mutant. However, the two major insulin-stimulated serine phosphopeptides remain. In intact cells, insulin stimulates exogenous substrate phosphorylation by the truncated receptor only approximately 1.9-fold (cf. approximately 9-fold for the wild-type receptor in these cells), a consequence of a approximately 4.8-fold elevation in basal insulin-independent kinase activity.

Amino Acid Sequence↗
PubMed1992

Characterization of insulin-stimulated protein serine/threonine kinases in CHO cells expressing human insulin receptors with point and deletion mutations.

The activation of insulin-stimulated protein-serine/threonine kinases has been investigated in CHO cell lines transfected with cDNAs encoding either wild-type or mutant human insulin receptors. (1) Insulin treatment of CHO cells over-expressing wild-type insulin receptors resulted in the rapid and substantial (5-10-fold) activation of cytosolic protein kinases which phosphorylated myelin basic protein, Kemptide and two peptide substrates based on sites phosphorylated on ribosomal protein S6 in vivo. (2) Further fractionation of cytosolic extracts by MonoQ chromatography revealed two peaks of insulin-stimulated myelin basic protein kinase activity which were highly related to the previously described mitogen-activated protein (MAP) kinases ERK1 and ERK2. In addition, at least two major peaks of S6 kinase activity were resolved, which exhibited properties similar to the 70 kDa and 90 kDa S6 kinases described by others; the predominant effect of insulin was on the activity of the 90 kDa enzyme and was in excess of 10-fold. (3) MonoQ fractionation of extracts from parental CHO cells, or cells expressing kinase-deficient receptors, showed all insulin-stimulated peaks of activity to be almost completely absent. (4) Further studies demonstrated that substitution of tyrosine residues 1162 and 1163 (or 1162 alone) with phenylalanine led to a substantial reduction in the ability of insulin to stimulate these protein kinase activities when assayed in cytosolic extracts. In contrast, deletion of 69 amino acids from the C-terminus of the insulin receptor beta-subunit caused a leftward shift in the insulin dose-response curve of the MAP kinase activity, but apparently not in that of the 90 kDa S6 kinase activity.

Amino Acid Sequence↗
PubMed1992

A developmentally regulated plasmalemmal antigen present in synaptosomes but not in growth cones.

Monoclonal antibody 2L4 was generated against rat synaptosomes but does not cross-react with nerve growth cones. Expression of the 2L4 antigen is developmentally regulated in a manner that is, in part, the opposite of the expression of the 5B4-CAM antigen, a marker of neuronal outgrowth belonging to the N-CAM family. While 5B4-CAM appears and increases during sprouting and then decreases to reach low levels in the adult, the 2L4 antigen appears only late in development, when neuronal outgrowth ceases, at or around the time of synaptogenesis. Once expressed, the antigen is found on the entire plasmalemmal surface of the neuron, but seems to be enriched at synaptic endings, at least of some neuron types. Biochemical analyses involving blotting of non-denaturing gels and immunoaffinity chromatography identify the antigen as a pair of polypeptides with similar, basic isoelectric points. These polypeptides form a somewhat diffuse, probably glycosylated band at 67 kDa and may be part of a hetero-oligomeric complex. The localization, biochemical, and developmental results suggest that the 2L4 antigen is a plasmalemmal marker of maturing and/or mature neurons whose expression may be triggered by synaptogenesis.

Aging↗
PubMed1992

Detection of a new hormone contact site within the insulin receptor ectodomain by the use of a novel photoreactive insulin.

We have used a preparation of soluble human insulin receptor ectodomain and a novel photoreactive, biotinylated derivative of insulin (4-azidosalicyloyl(B1-biocytinyl-B2-lysine)-insulin) to identify a new hormone contact site within the extracellular domain of the insulin receptor. The ectodomain was photoaffinity-labeled and digested to completion with trypsin, and the resulting tryptic fragment was purified by either HPLC or by streptavidin-affinity chromatography. The amino terminus of the fragment was identified as Gly390 within the alpha-subunit. These results suggest that residues that are carboxyl-terminal to the cysteine-rich domain, in addition to previously identified regions within the amino terminus of the alpha-subunit, contribute to the insulin binding site. The implications of these results for the de novo folding of the insulin receptor to constitute the hormone binding site are discussed.

Affinity Labels↗
PubMed1992

Racial/ethnic variations in male testosterone levels: a probable contributor to group differences in health.

Racial and ethnic variations in serum testosterone levels were investigated among a large sample of male Vietnam era veterans. Based on geometric means, significant average differences were found between 3,654 non-Hispanic white and 525 black individuals. The geometric mean for testosterone levels among 200 Hispanic individuals was similar to that of non-Hispanic white individuals. Regarding two other racial/ethnic groups (Asian/Pacific Islanders and Native Americans), no significant differences were found, due perhaps to small sample sizes. Results were interpreted as having considerable potential for explaining some of the race differences in the incidences of cardiovascular diseases, hypertension, and prostate cancer.

Adult↗
PubMed1992

Bentiromide test using liquid-chromatographic measurement of p-aminobenzoic acid and its metabolites for diagnosing pancreatic insufficiency in childhood.

We assessed the diagnostic capability of the bentiromide test using a high-pressure liquid-chromatography method to analyze p-aminobenzoic acid and its metabolites in plasma as an indirect measure of exocrine pancreatic function. Mean total amine concentration in pancreatic-insufficient subjects was significantly lower than in control subjects. There were 3 of 15 false-negative results and no false-positive results. We conclude that this chromatographic method is an effective means of analyzing p-aminobenzoic acid and its metabolites after ingestion of bentiromide.

4-Aminobenzoic Acid↗
PubMed1992

Differential regulation of multiple hepatic protein tyrosine phosphatases in alloxan diabetic rats.

The involvement of tyrosine phosphorylation in insulin action led us to hypothesize that increased activity of protein tyrosine phosphatases (PTPases) might contribute to insulin resistance in alloxan diabetes in the rat. Hepatic PTPase activity was measured using two artificial substrates phosphorylated on tyrosine: reduced, carboxyamidomethylated, and maleylated lysozyme (P-Tyr-RCML) and myelin basic protein (P-Tyr-MBP), as well as an autophosphorylated 48-kD insulin receptor tyrosine kinase domain (P-Tyr-IRKD). Rats that were made alloxan diabetic exhibited a significant increase in hepatic membrane (detergent-soluble) PTPase activity measured with P-Tyr-MBP, without a change in activity measured with P-Tyr-RCML or the P-Tyr-IRKD. The PTPase active with P-Tyr-MBP behaved as a high molecular weight peak during gel filtration chromatography. Characterization of this enzyme indicated it shared properties with CD45, the prototype for a class of transmembrane, receptor-like PTPases. Our results indicate that alloxan diabetes in the rat is associated with an increase in the activity of a large, membrane-associated PTPase which accounts for only a small proportion of insulin receptor tyrosine dephosphorylation. Nonetheless, increased activity of this PTPase may oppose tyrosine kinase-mediated insulin signal transmission, thus contributing to insulin resistance.

Alloxan↗
PubMed1991

Insulin-stimulated serine and threonine phosphorylation of the human insulin receptor. An assessment of the role of serines 1305/1306 and threonine 1348 by their replacement with neutral or negatively charged amino acids.

Insulin promotes insulin receptor beta-subunit phosphorylation on tyrosine, serine, and threonine residues in a variety of cells, including simian COS cells which transiently express human insulin receptors following transfection with a cDNA encoding the wild-type receptor protein. To examine the potential roles of serines 1305 and 1306 and threonine 1348 as sites of insulin-stimulated phosphorylation in these cells, these residues (i.e. either serines 1305 and 1306, or threonine 1348) were replaced with neutral (alanine) or negatively charged (aspartate) amino acids. Following transient expression of each of these mutant receptors in COS cells, two-dimensional phosphopeptide mapping reveals that threonine 1348 is the major, if not the only, insulin-stimulated threonine phosphorylation site. In contrast, while serines 1305 and/or 1306 are phosphorylated in an insulin-dependent manner, these sites comprise only a minor proportion of insulin receptor serine phosphorylation in these cells. Substitution of either serines 1305 and 1306 or threonine 1348 with neutral or negatively charged amino acids has no effect on insulin-stimulated tyrosine autophosphorylation of these mutant receptors in intact cells. Furthermore, insulin-stimulated exogenous protein-tyrosine kinase activity of the mutant receptors is unaffected, as assessed following either phosphorylation of receptors in intact cells or following immunopurification of receptors and their autophosphorylation in vitro.

Amino Acid Sequence↗
PubMed1991

Evidence for hybrid rodent and human insulin receptors in transfected cells.

Chinese hamster ovary cells and NIH 3T3 cells overexpressing mutant human insulin receptors were examined for the presence of hybrid receptors composed of human and rodent insulin receptors. In the present studies, most of the endogenous rodent receptors were found to be immunoprecipitated from the transfected cells but not the parental cells with a monoclonal antibody specific for human receptor. These data indicate that in these transfected cells, most of the endogenous rodent receptors are in a hybrid complex with the overexpressed human receptor. These results together with the in vitro studies of Treadway et al. (Treadway, J.L., Morrison, B.D., Soos, M.A., Siddle, K., Olefsky, J., Ullrich, A., McClain, D.A., and Pessin, J.E. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 214-218) showing that hybrid receptors exhibit transdominant inhibition explain the prior finding indicating that overexpression of defective insulin receptors interferes with the normal signaling of endogenous receptors.

Animals↗
PubMed1991

Deletion of approximately 10 kDa from the carboxyl terminus of a soluble approximately 48-kDa insulin receptor protein-tyrosine kinase results in slower rates of diphosphorylation of a series of dodecapeptide substrates. An assessment by 1H NMR.

Autophosphorylation of a soluble approximately 48-kDa derivative of the insulin receptor protein-tyrosine kinase is accompanied by an increase in its specific activity towards exogenous substrates. In the present study, we have utilized 1H NMR to compare the order and rate of mono- and diphosphorylation of multiple tyrosine residues in a series of synthetic dodecapeptide substrates (based on the receptor sequence, which includes major sites of autophosphorylation (RRDIYETDYYRK), with substitution(s) at positions 6 and/or 7 based on residue size and/or charge) by the approximately 48-kDa enzyme and by a approximately 38-kDa enzyme generated by tryptic deletion of approximately 10 kDa from the carboxyl terminus of the approximately 48-kDa protein. Both enzymes exhibit a marked order and progression of phosphorylation of peptide tyrosine residues; for each peptide, phosphorylation initiates and proceeds to completion first on tyrosine 9, followed by phosphorylation on tyrosine 10. Although removal of the carboxyl terminus does not affect the rate of monophosphorylation of these peptides on tyrosine 9, the smaller enzyme exhibits a slower rate of diphosphorylation (at tyrosine 10), as compared with the approximately 48-kDa enzyme.

Amino Acid Sequence↗
PubMed1991

Biosynthesis of the precursor of a soluble human insulin receptor ectodomain in insect Sf9 cells infected with a recombinant baculovirus.

In contrast to transfected mammalian cells, insect Sf9 cells infected with a recombinant Baculovirus inefficiently process and secrete a soluble derivative of the extracellular domain of the human insulin receptor. The high-mannose form of the receptor precursor that accumulates intracellularly is not grossly aberrant or malfolded, as its interaction with a diverse panel of monoclonal antibodies are comparable to secreted precursor and proteolytically processed receptor, both of which bear partially trimmed oligosaccharide chains. Thus the inefficient step in the biosynthesis of this protein in Sf9 cells is either at, or just preceding, the trimming of its high-mannose oligosaccharide chains.

Animals↗
PubMed1991

A soluble insulin receptor kinase catalyzes ordered phosphorylation at multiple tyrosines of dodecapeptide substrates.

At present, the requirements for efficient phosphorylation of exogenous substrates by protein-tyrosine kinases are largely unknown. The proton resonances of each of the 3 tyrosines of the dodecapeptide substrate RRDIYETDYYRK are well resolved in the aromatic region of the 1H NMR spectra: thus, it is feasible to directly monitor phosphorylation at each site. A soluble approximately 48-kDa derivative of the human insulin receptor cytoplasmic protein-tyrosine kinase domain phosphorylates this peptide at all 3 tyrosine sites and does so in a highly ordered and progressive manner (Y9, then Y10 and finally Y5), proceeding to full stoichiometry at each site before phosphorylating the next. This experimental system now provides an approach by which to follow the stereochemical requirements and dynamics of substrate phosphorylation by a protein-tyrosine kinase in solution in real time.

Amino Acid Sequence↗
PubMed1991

Molecular basis of the activation of the tumorigenic potential of Gag-insulin receptor chimeras.

A previous study showed that the human insulin receptor (IR) could be activated by insertion of a 3' portion of the cDNA encoding the beta subunit into a retrovirus genome to form a Gag-IR fusion protein. While capable of transforming cells in culture, this IR cDNA-containing virus, called UIR, was not able to induce tumors in animals. Subsequently, we isolated a spontaneous sarcomagenic variant called UIR19t from the parental UIR. UIR19t was molecularly cloned, sequenced, and found to harbor two mutations. A 44-amino acid deletion immediately upstream from the transmembrane domain of the Gag-IR fusion protein removes all the extracellular sequence of the IR remaining in the original UIR construct. In addition, a single nucleotide deletion at the 3' end results in truncation and replacement of the carboxyl-terminal 12 amino acids by 4 new amino acids. The specific kinase activity of UIR19t is 4- to 5-fold higher than that of the parental UIR. However, no new cellular substrates were detected in UIR19t-transformed cells as compared to UIR cells. Viruses containing either the 5' or the 3' deletion mutation were constructed and assessed for their biological function. Our data indicate that the 5' deletion alone is sufficient to confer tumorigenic ability. We conclude that sequence immediately upstream from the transmembrane domain imposes a negative effect on the transforming and tumorigenic potential of the Gag-IR fusion protein.

Amino Acid Sequence↗
PubMed1991

Phosphorylation of tyrosines 1158, 1162 and 1163 on a synthetic dodecapeptide by the insulin receptor protein-tyrosine kinase.

To investigate the mechanism of tyrosine phosphorylation by the insulin receptor protein-tyrosine kinase, we utilized a synthetic dodecapeptide substrate (RRDIYETDYYRK; amino acids 1155-1165) containing the three major insulin receptor autophosphorylation sites. (1) We show that all three tyrosines on this peptide are rapidly phosphorylated and that phosphorylation is probably initiated at tyrosine 9. This peptide thus serves as a useful tool to study the mechanism of transphosphorylation by the insulin receptor. (2) A proteolytic activity was detected in purified receptor preparations that removed basic residues from the peptide and prevented it binding to phosphocellulose paper. Such activity could pose a serious problem when using peptide substrates to assay for protein kinases in other acellular systems.

Amino Acid Sequence↗
PubMed1991

Two-dimensional phosphopeptide analysis of the autophosphorylation cascade of a soluble insulin receptor tyrosine kinase. The tyrosines phosphorylated are typical of those observed following phosphorylation of the heterotetrameric insulin receptor in intact cells.

A soluble derivative of the human insulin receptor cytoplasmic domain, as expressed in insect cells via a Baculovirus vector, is an active protein-tyrosine kinase. In the present study, we find that three forms of the enzyme (48, 43, and 38 kDa) can be partially purified by MonoQ fast protein liquid chromatography. Two-dimensional thin layer phosphopeptide mapping reveals that the 48-kDa enzyme undergoes a rapid autophosphorylation on the same tyrosines (residues 1158, 1162, 1163, 1328, and 1334) that have previously been shown to be major autophosphorylation sites on the native insulin receptor beta-subunit in intact cells. Furthermore, the 48- and 43-kDa proteins are phosphorylated on serine residues by a serine kinase(s) that copurifies through MonoQ fast protein liquid chromatography. Tyrosine autophosphorylation sites 1328 and 1334 and virtually all serine phosphorylation sites are absent in the 38-kDa kinase. Partial tryptic proteolysis of the 48-kDa kinase generates a core 38-kDa enzyme that undergoes autophosphorylation almost exclusively on tyrosines 1158, 1162, and 1163. Phosphorylation of these tyrosine residues occurs in a cascade manner analogous to that found in the intact insulin receptor beta-subunit.

Amino Acid Sequence↗