Biomedical subjects

V Duronio

Publications and source records attributed to V Duronio.

65 records · Page 4Linked to original sources

PubMed1990

Characterization of platelet-activating factor receptors in porcine platelets.

Despite a large number of studies describing the properties and effects of platelet-activating factor (PAF), little is known about its receptor structure. The characterization of the PAF receptor from additional cell types and species is important for the design of strategies to purify and characterize the receptor molecule. Porcine platelets were shown to bind PAF with characteristics similar to several other species, based on receptor number, affinity, and the activity of PAF antagonists. We found that the affinity for binding was higher in porcine than in rabbit platelets (Kd = 0.68 +/- 0.13 nM for rabbit and 0.29 +/- 0.10 nM for porcine). Porcine platelets have approximately 281 +/- 158 receptors per cell compared with 689 +/- 229 receptors in rabbit platelets. Rabbit platelets respond to concentrations of PAF that are approximately 10(5)-fold lower than those required for aggregation of porcine platelets, but this difference is probably not due to the differences in receptor number alone. When binding was compared between purified membranes from these two cell types, porcine platelets had 20-fold fewer receptors per milligram of membrane protein, but this difference may have been due to an artifact of the membrane preparation procedure. Binding of PAF was severely hindered at cold temperatures. It was undetectable in whole cells on ice and greatly reduced with purified membranes. This study is the first to characterize PAF receptors in porcine platelets, which represent a potentially useful source of receptor for further biochemical characterization.

Animals↗

PubMed1990

The effect of protein kinase-C inhibition on insulin receptor phosphorylation.

The effect of protein kinase-C (PKC) inhibition on insulin receptor phosphorylation in HepG2 cells was analyzed by two-dimensional tryptic phosphopeptide maps. In basal cells, there was one major insulin receptor-derived tryptic phosphothreonine peptide and at least four phosphoserine peptides. Phorbol 12,13-dibutyrate (PDBU) stimulated phosphorylation of the phosphothreonine peptide, some of the basal phosphoserine peptides, and at least one phosphoserine peptide that was not detected in the basal state. Staurosporine completely inhibited the PDBU-mediated phosphorylation. Although staurosporine also inhibited basal phosphorylation of the phosphothreonine peptide, down-regulation of PKC did not, suggesting that PKC does not mediate basal insulin receptor phosphorylation. Insulin treatment resulted in the appearance of four phosphotyrosine peptides. It also stimulated the phosphorylation of at least two phosphoserine peptides. One of these may have been a complex of two or more distinct but poorly resolved phosphopeptides, which was seen in basal cells and a component of which seemed to be stimulated by PDBU. However, neither staurosporine nor down-regulation of PKC diminished insulin-stimulated serine phosphorylation of these peptides, indicating that insulin-stimulated receptor serine phosphorylation did not involve PKC activity. The addition of staurosporine to cells that had been incubated with PDBU resulted in the very rapid decay of phosphorylation of the phosphothreonine-containing peptide, indicating that this site of phosphorylation turns over very rapidly, while some of the other phosphoserine-containing peptides, including the major unique site of phosphorylation stimulated by PDBU, turned over more slowly. Thus, the insulin receptor contains several sites of serine/threonine phosphorylation, some of which are substrates for more than one protein kinase. This may permit complex modulation of insulin receptor functions in response to multiple signalling pathways.

Alkaloids↗

PubMed1989

Interleukin 3 stimulates phosphatidylcholine turnover in a mast/megakaryocyte cell line.

The hemopoietic growth factor, interleukin 3, has been shown to activate protein kinase C without causing hydrolysis of inositol phospholipids. The potential involvement of phosphatidylcholine hydrolysis as an alternative source of diacylglycerol was investigated in an interleukin 3-dependent murine mast/megakaryocyte cell line, R6-XE.4. Treatment of these cells with interleukin 3 rapidly stimulated both the release of water-soluble choline metabolites and the resynthesis of phosphatidylcholine. Therefore, a phosphatidylcholine cycle may operate as part of the signal transduction pathway in cells responding to interleukin 3.

Animals↗

PubMed1989

Insulin-like growth factor I receptor beta-subunit heterogeneity. Evidence for hybrid tetramers composed of insulin-like growth factor I and insulin receptor heterodimers.

In both NIH3T3 cells and HepG2 cells, insulin-like growth factor I (IGF-I) receptors possess two beta-subunits that display different electrophoretic mobilities. Increasing concentrations of IGF-I stimulated the phosphorylation of both beta-subunits to a similar extent, whereas insulin stimulated the phosphorylation of both subunits only at elevated concentrations. Both beta-subunits were immunoprecipitated with p5, an insulin receptor-specific anti-peptide antibody, or with A410, a polyclonal anti-insulin receptor antisera. However, if the tetrameric IGF-I receptor was first dissociated into alpha-beta heterodimers with 1 mM dithiothreitol, only the lower molecular weight beta-subunit was immunoprecipitated. These results suggested that p5 and A410 specifically recognized the lower molecular weight beta-subunit but immunoprecipitated the higher molecular weight beta-subunit because it was present in the same disulfide linked tetramer. Similarly, alpha-IR-3, an antibody specific for the alpha-subunit of the IGF-I receptor, immunoprecipitated both types of beta-subunit from the intact tetramer but only the higher molecular weight beta-subunit from the dissociated heterodimers, suggesting that there are two types of alpha-subunits in the same tetramer and that the alpha-subunit recognized by alpha-IR-3 is only associated with the higher molecular weight beta-subunit. Tryptic phosphopeptide maps of the lower molecular weight beta-subunit of IGF-I receptor were different from the higher molecular weight beta-subunit, but were similar to those of the insulin receptor beta-subunit. Thus, by immunochemical cross-reactivity and structural criteria, the lower molecular weight beta-subunit of the IGF-I receptor was similar to the beta-subunit of insulin receptor. These data suggest that there exists a species of IGF-I receptor that is a hybrid composed of an insulin receptor alpha-beta heterodimer and an IGF-I receptor alpha-beta heterodimer. The existence of such a hybrid receptor could have important functional consequences.

Animals↗

PubMed1988

Effects of inhibitors of N-linked oligosaccharide processing on the biosynthesis and function of insulin and insulin-like growth factor-I receptors.

We have used specific inhibitors of oligosaccharide processing enzymes as probes to determine the involvement of oligosaccharide residues in the biosynthesis and function of insulin and insulin-like growth factor-I receptors. In a previous study (Duronio, V., Jacobs, S., and Cuatrecasas, P. (1986) J. Biol. Chem. 261, 970-975) swainsonine was used to inhibit mannosidase II, resulting in the production of receptors containing only hybrid-type oligosaccharides. These receptors had a slightly lower molecular weight and were much more sensitive to endoglycosidase H, but otherwise behaved identically to normal receptors. In this study, we used two compounds that inhibit oligosaccharide processing at earlier steps: (i) N-methyl-1-deoxynojirimycin (MedJN), which inhibits glucosidases I and II and yields glucosylated, high mannose oligosaccharides, and (ii) manno-1-deoxynojirimycin (MandJN), which inhibits mannosidase I and yields high mannose oligosaccharides. In the presence of MandJN, HepG2 cells synthesized receptors of lower molecular weight, which were cleaved into alpha and beta subunits and were able to bind hormone and autophosphorylate. These receptors were as sensitive to endoglycosidase H as receptors made in the presence of swainsonine. In the presence of MedJN, receptors of only slightly lower molecular weight than normal were synthesized and were shown to contain some glucosylated high mannose oligosaccharides. These receptors were able to bind hormone and retained hormone-sensitive autophosphorylation activity. In both cases, the incompletely processed receptors could be detected at the cell surface by cross-linking of iodinated hormone and susceptibility to trypsin digestion, although less receptor was present in cells treated with MedJN. Studies of receptor synthesis using pulse-chase labeling showed that the receptor precursors synthesized in the presence of MedJN were cleaved into alpha and beta subunits at a slower rate than normal receptors or those made in the presence of MandJN. Inhibition of oligosaccharide processing had no effect on the association of the receptor subunits into disulfide-linked oligomeric complexes.

Animals↗

PubMed1986

Stimulation of hexose transport in L6 rat myoblasts by antibody and by glucose starvation.

Treatment of glucose-grown L6 rat myoblasts with rabbit or sheep anti-(L6-rat myoblast) antibody for 35 min or glucose starvation for at least 8 h results in a 2-fold increase in the Vmax. of 2-deoxy-D-glucose (dGlc) and 3-O-methyl-D-glucose uptake. In both cases, apparent transport affinities were not affected. Furthermore, once stimulation has occurred, further increases in hexose uptake could not be produced. Assays of antibody binding to whole cells suggested that the antibody is not internalized but remains bound on the cell surface. To elucidate the site and mechanism of antibody action, plasma-membrane vesicles from L6 cells were prepared. Anti-L6 antibody was found to cause a time- and dosage-dependent stimulation of dGlc transport in these vesicles. Maximum activation was achieved after 30 min exposure. This antibody-mediated activation could be inhibited by treatment of vesicles with various proteinase inhibitors. Treatment of vesicles with trypsin was also found to activate dGlc transport to levels observed with antibody. These results are virtually identical with those obtained with whole cells and suggest that antibody-mediated activation of hexose transport results from interaction of antibody with a specific membrane component(s).

Amines↗

PubMed1986

Complete glycosylation of the insulin and insulin-like growth factor I receptors is not necessary for their biosynthesis and function. Use of swainsonine as an inhibitor in IM-9 cells.

Swainsonine, an indolizidine alkaloid which is a potent inhibitor of the Golgi enzyme, mannosidase II, leads to the production of incompletely processed glycoproteins lacking complex type oligosaccharides. This inhibitor has been used to examine the importance of terminal sugar groups in the biosynthesis and function of both the insulin receptor and the insulin-like growth factor I receptor. IM-9 cells were metabolically labeled using [35S]methionine and the two receptors were independently immunoprecipitated using specific monoclonal antibodies. The incompletely processed receptors have slightly lower molecular weights and contain hybrid rather than complex type oligosaccharides as indicated by their sensitivity to endoglycosidase H and neuraminidase. Both receptors made in the presence of swainsonine are still autophosphorylated in the presence of the respective hormone. The insulin receptor made in the presence of the inhibitor can be affinity labeled at the cell surface using 125I-insulin and disuccinimidyl suberate cross-linking; there is also no significant difference in its affinity for insulin. These results suggest that for the insulin and insulin-like growth factor I receptors to be synthesized, processed, and function normally, they do not require all of the sugars which are normally added in the terminal stages of glycosylation.

Affinity Labels↗

PubMed1986

Isolation and characterization of hexose transport mutants in L6 rat myoblasts.

A method for the selection and isolation of hexose transport mutants in undifferentiated rat myoblast L6 cells is reported; 2-deoxy-D-glucose (2-DOG)-and 2-deoxy-2-fluoro-D-glucose (2FG)-resistant mutants were selected after mutagenization of L6 cells with ethyl methanesulfonate. Of these, D18 and D23 (selected with 0.1 mM 2-DOG) and F72 and F76 (selected with 0.1 mM 2FG) exhibited the lowest hexose transport activity. Uptake of 0.06 mM 2-DOG, 2FG, or 3-O-methyl-D-glucose (3-OMG) by mutants grown in fructose medium supplemented with 0.05 mM 2FG was about four- to five-fold lower than the parental L6 cells. These mutants contain normal levels of ATP and glycolytic enzyme activities. They also exhibit normal transport activities for alpha-aminoisobutyric acid and fructose. Furthermore, hexose transport was observed to be decreased in plasma membrane vesicles prepared from these mutants. Kinetic analysis of 2-DOG and 3-OMG transport in mutant F72 demonstrated that the Vmax for 2-DOG uptake was significantly reduced, whereas the Vmax for 3-OMG transport was not affected. In all cases, the affinity for these hexose analogues was unaffected. In addition mutant F72 was found to be only slightly affected by treatment with various energy inhibitors and sulfhydryl reagents. The results suggest that this mutant is defective in, or has low levels of, a plasma membrane component(s) involved in the high-affinity hexose transport system.

3-O-Methylglucose↗

PubMed1986

Rapid formation of diacylglycerol from phosphatidylcholine: a pathway for generation of a second messenger.

The classic pathway for agonist-induced generation of diacylglycerol is via activation of a phospholipase C-mediated hydrolysis of the "phosphoinositides." We now report findings from a variety of cell types, which indicate that tumor-promoting phorbol diesters, serum, and platelet-derived growth factor activate within seconds the hydrolysis of phosphatidylcholine, as detected by the formation of diacylglycerol and phosphocholine. It is known that phorbol diesters do not stimulate hydrolysis of the phosphoinositides. Yet, in cells prelabeled with either [14C]oleate or [32P]orthophosphate, addition of the tumor promoter phorbol dibutyrate (PBt2) resulted in the rapid generation of both diacylglycerol and phosphatidate in a time- and dose-dependent manner. The fatty acid composition of the phosphatidate most resembled the fatty acid profile of phosphatidylcholine from the same cell type. Taken together, these findings suggested a role for protein kinase C in the generation of diacylglycerol (and phosphatidate) from phosphatidylcholine. To define further the pathways involved, the metabolism of cellular phosphatidylcholine was studied. In cells prelabeled with [3H]choline, addition of PBt2, but not 4 alpha-phorbol, stimulated the formation of intracellular phosphocholine within 45 sec. Furthermore, addition of platelet-derived growth factor (PDGF) or serum to "serum-starved" cells prelabeled with [3H]choline resulted in increased levels of intracellular phosphocholine within 15-30 sec. Thus, the data suggest that agonists that stimulate protein kinase C either directly (e.g., PBt2) or indirectly via activation of phosphoinositide hydrolysis (e.g., PDGF and serum) may stimulate degradation of phosphatidylcholine by phospholipase C in intact cells. However, prior down-regulation of protein kinase C by prolonged pretreatment of cells with PBt2 almost totally abolished subsequent stimulation of phosphatidylcholine degradation by PBt2 but only partially attenuated subsequent stimulation by PDGF and serum. These observations suggest that PDGF and serum act, at least partially, through a protein kinase C-independent mechanism. Lastly, the size of the cellular choline and CDP-choline pools were shown to be small and relatively insensitive to agonist addition, as compared to the size and behavior of the phosphocholine pool. Thus, the rapidly increased levels of phosphocholine (and diacylglycerol) arising in response to agonist addition appear to be derived directly from phosphatidylcholine by a phospholipase C-mediated mechanism.

Cells, Cultured↗

PubMed1984

Activation of hexose transport by antibody.

In our attempts to study alterations in cell behaviour by membrane perturbations, the effects of specific antisera on membrane functions were examined. The present communication reports the nature and kinetics of the changes in hexose transport as a result of treatment of L6 rat myoblast cells with specific antibodies. Exposure to complement-inactivated rabbit anti-myoblast antiserum resulted in an almost immediate two- to three-fold increase in hexose transport; morphological changes were observed only after prolonged incubation with the antiserum. Similar stimulation of hexose transport was observed upon exposure of cells to immunoglobulin G (IgG) from both sheep anti-myoblast and rabbit anti-myoblast plasma membrane sera. It seems that the interaction of specific IgG with cell surface components is sufficient to elicit this response. We have established that over 90% of 2-deoxyglucose is phosphorylated upon entrance into rat myoblasts and that exposure of cells to specific antibody does not cause changes in cell volume or general leakiness of the cell membrane. The antibody-stimulated hexose transport system resembles its normal counterpart in its substrate affinity and specificity; it differs from that of the control cells only in the transport capacity. This increase in hexose capacity is brought about by processes independent of protein synthesis and is not likely the result of extensive reorganization of membrane components.

Animals↗

PubMed1984

Mechanism of antibody stimulation of hexose transport in rat myoblasts.

We have recently demonstrated that exposure of rat myoblasts to anti-rat myoblast antiserum results in two- to three-fold activation of hexose transport. The present communication reports the possible mechanism(s) by which specific antibody can bring about such activation. Studies with Fab and Fc fragments indicate that the binding of Fab to specific cell surface component(s) is not sufficient to trigger activation of hexose transport; the immunoglobulin G (IgG) mediated dimerization of membrane components is required for this process. Although cytochalasin D has no effect on hexose transport in control and antibody-treated cells, pretreatment of cells with this inhibitor prevents antibody-mediated activation of hexose transport. It may be inferred from this observation that proper disposition of membrane components is required for the dimerization of membrane receptors. Since this activation of hexose transport is an irreversible process, it is possible that covalent modification of membrane components may have occurred as a result of antibody treatment. Pretreatment of cells with ammonium chloride or methylamine is found to abolish the antibody-mediated activation of hexose transport, even though these inhibitors have no effect on hexose transport in control and antibody-treated cells. These inhibitors may be acting on transglutaminase and (or) on some other proteins involved in the activation process. Several lines of evidence suggest that limited proteolytic cleavage of membrane components may be involved in the antibody-mediated activation of hexose transport. First, pretreatment with several protease inhibitors prevents activation of hexose transport. Second, several cell surface proteins are missing in antibody-treated cells. Third, limited proteolysis of cell surface proteins with trypsin can also bring about activation of hexose transport. In view of the fact that proteolytic activity cannot be detected in various IgG and serum preparations, it seems likely that endogenous membrane associated proteases may be involved in this activation process.

Ammonium Chloride↗