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M Caron

Publications and source records attributed to M Caron.

At least 73 records · Page 4Linked to original sources
PubMed1994

Insulin receptor mutation at tyrosines 1162 and 1163 alters both receptor serine phosphorylation and desensitization.

Chinese hamster ovary (CHO) cells expressing human insulin receptor (hIR) of the wild-type (CHO R) or hIR mutated at tyrosines 1162 and 1163 (CHO Y2) were compared for agonist-induced receptor phosphorylation of serine/threonine residues and receptor desensitization. Relative to CHO R cells, CHO Y2 cells exhibited a marked decrease in their response to insulin and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) for hIR phosphorylation on serine residues. Moreover, the tyr1162,1163 mutant hIR could not be normally phosphorylated by purified protein kinase C (PKC) in vitro. Finally, in contrast to CHO R cells, CHO Y2 cells were refractory to PMA-induced IR desensitization for subsequent activation by insulin of exogenous tyrosine kinase and glycogen synthesis. These results strongly suggest that the replacement of tyrosines 1162 and 1163 by phenylalanine residues changes the IR beta-subunit conformation and thus impedes phosphorylation of the IR at crucial serine residues and prevents PMA-induced desensitization. This supports the hypothesis that IR serine phosphorylation and desensitization are related.

Animals↗
PubMed1994

Evidence for a role of protein kinase C in the activation of the pyruvate dehydrogenase complex by insulin in Zajdela hepatoma cells.

The signal transduction pathway involved in the activation of pyruvate dehydrogenase (PDH) by insulin is still unknown. In this study, we have examined the possible involvement of protein kinase C (PKC) in the process. In addressing this question, we examined (1) the insulin-like effects of the PKC activator 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) on the PDH complex, (2) the effects of various PKC inhibitors on the PDH activation by insulin, and (3) the response of PKC-depleted cells to insulin. We used as an experimental model Zajdela hepatoma cultured (ZHC) cells, which have been demonstrated to be responsive to physiological doses of insulin. Half-maximal and maximal stimulations of the PDH complex by insulin were observed at 0.05 and 5 nmol/L, respectively. Stimulation of PDH activity by insulin (5 nmol/L) occurred within 5 minutes of incubation and was maximal (+70%) at 7.5 minutes. In the presence of PMA (162 nmol/L), enzyme activity increased within 30 seconds, was maximal (+90%) at 5 minutes, and was no longer detectable after 10 minutes. Total PDH activity was unchanged by insulin or PMA treatment. The effects of PMA and insulin on basal PDH activity were not additive. Moreover, various inhibitors of PKC--staurosporine, sphingosine, acridine orange--completely blocked the stimulation of PDH activity induced by insulin or PMA. A 17-hour treatment of ZHC cells with 500 nmol/L PMA efficiently downregulated PKC, as attested by the marked decrease in the enzyme activity and the loss of phorbol 12,13-dibutyrate binding to intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1994

Insulin receptor autophosphorylation sites tyrosines 1162 and 1163 control both insulin-dependent and insulin-independent receptor internalization pathways.

We previously reported that Chinese hamster ovary (CHO) cell lines overexpressing mutated human insulin receptors (hIRs) in which the tyrosine residues 1162 and 1163 were replaced by phenylalanines (CHO-Y2) exhibited a marked defect in hormone-induced receptor internalization as compared to CHO transfectants overexpressing wild-type hIRs (CHO-R). These two cell lines are now used to compare the role of tyrosines 1162-1163 in basal and ligand-stimulated receptor internalization as well as in receptor turnover. We show here that (1) in CHO-Y2 cells, basal endocytosis, like insulin-induced internalization, was markedly altered despite normal receptor turnover and (2) in both CHO-R and CHO-Y2 cells, basal receptor endocytosis was altered by tunicamycin, an inhibitor of protein N-glycosylation, whereas insulin-induced internalization was not. These results support a role for tyrosines 1162-1163 of the IR beta-subunit major autophosphorylation domain in both basal and ligand-stimulated receptor endocytosis and provide evidence that the two processes follow distinct pathways.

Animals↗
PubMed1994

Phosphorylation and palmitoylation of the human D2L dopamine receptor in Sf9 cells.

We have expressed and biochemically characterized the human D2long (D2L) dopamine receptor isoform using the baculovirus/Sf9 cell system. The expressed receptor bound ligands with a pharmacological profile similar to that reported for neuronal and cloned D2L receptors expressed in mammalian cell lines. Dopamine binding to D2L receptor was sensitive to guanine nucleotides, indicating receptor coupling to endogenous G proteins. A D2L receptor-specific antibody identified two major protein species at approximately 44 kDa and at approximately 93 kDa in immunoblots, suggesting the presence of D2L receptor monomers and dimers. Both species were purified by immunoprecipitation from digitonin-solubilized preparation of cells expressing D2L receptor prelabeled with 32P(i) or [3H]-palmitate. These results constitute the first direct evidence for D2L receptor phosphorylation and palmitoylation.

Animals↗
PubMed1994

Normal qualitative and quantitative Tc-99m sestamibi myocardial SPECT: spectrum of intramyocardial distribution during exercise and at rest.

Exercise myocardial perfusion imaging with Tc-99m sestaMIBI is routinely used to detect underlying coronary stenoses. Ischemia is diagnosed in regions that display decreased tracer uptake during exercise as compared to rest. Tc-99m sestaMIBI SPECT images of 42 healthy volunteers were assessed both qualitatively (tomographic slices) and quantitatively (sectored polar map) for potential sources of misinterpretation. On the myocardial tomographic slices, the most common culprit artifacts were diaphragmatic attenuation and bowel interposition, which caused fixed or reversible "perfusion defects" in the inferior and posterior regions (in 19/35 abnormal segments), and artifacts related to the presence and shift of hot spots (observed in 11/28 men; in women, they were more difficult to demonstrate because of the overriding effect of breast attenuation). Hot spots shifts between exercise and rest usually resulted in pseudo-reversible defects in the anterolateral and lateral walls. The quantified polar map display of the myocardium showed a physiologic decrease in sestaMIBI activity in the basal anterolateral and basal posterolateral areas in men during exercise. There are many normal variants that may mimic coronary artery disease on tomographic sestaMIBI images. Before reporting an area of decreased activity as either a fixed or reversible perfusion defect, the interpreter should ensure that it does not represent an artifact or a normal variation in the intramyocardial distribution of sestaMIBI during exercise.

Adult↗
PubMed1993

Human serotonin1B receptor expression in Sf9 cells: phosphorylation, palmitoylation, and adenylyl cyclase inhibition.

Analysis of the primary protein structure of the human serotonin1B (5-HT1B) receptor reveals consensus sites for phosphorylation and a putative site for palmitoylation. To investigate these posttranslational modifications, we have expressed a c-myc epitope-tagged 5-HT1B (m5-HT1B) receptor in Sf9 cells. This strategy enabled receptors to be detected by immunoblot analysis and purified by immunoprecipitation using a monoclonal antibody, 9E10, specific for the c-myc epitope. Agonist radioligand [3H]5-HT binding studies showed that the expressed 5-HT1B and m5-HT1B receptors displayed the characteristic pharmacological profile of the neuronal 5-HT1B receptor. The expressed receptors displayed both high- and low-affinity states for [3H]5-HT, suggesting that the receptors were coupled to endogenous G-proteins. Indeed, agonist binding to the high-affinity receptor state was regulated in the presence of GTP gamma S, Gpp(NH)p, and pertussis toxin. [32P]ADP-ribosylation experiments identified a major approximately 41-kDa ADP-ribosylated protein present in Sf9 membranes that comigrated with partially purified bovine brain Gi alpha/G(o) alpha subunits. Measurements of adenylyl cyclase activity in membranes from cells expressing m5-HT1B receptors showed that serotonergic agonists mediated the inhibition of adenylyl cyclase activity with a rank order of potency comparable to their affinity constants. Immunoblot analysis of membranes prepared from cells expressing m5-HT1B receptors and photoaffinity labeling of the immunoprecipitated material revealed photolabeled species at approximately 95 and at approximately 42 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1993

Study of lectin-ganglioside interactions by high-performance liquid affinity chromatography.

A high-performance affinity column containing immobilized modified GM1 (lyso-GM1) was used to study the binding of an endogenous human brain lectin (HBL) in comparison with other carbohydrate-binding proteins. The proteins are previously converted into biotinylated derivatives. Detection of biotinylated proteins in the eluates by a microtitre plate assay ensures good sensitivity. The maximum binding capacity of the adsorbent for HBL is obtained in Tris buffer supplemented with beta-mercaptoethanol. The binding is inhibitable by specific sugar. It is concluded that the use of immobilized glycolipids in analytical high-performance liquid affinity chromatographic methods may serve as models in the study of interactions between gangliosides and carbohydrate-binding proteins.

Biotin↗
PubMed1992

Agonist-modulated palmitoylation of beta 2-adrenergic receptor in Sf9 cells.

The palmitoylation of the human beta 2-adrenergic receptor (beta 2-AR) was studied in recombinant baculovirus-infected insect Sf9 cells. At 48 h post-infection, a high level expression of an epitope-tagged beta 2-AR (10-25 pmol/mg protein) was detected by [125I]iodocyanopindolol ([125I]CYP) binding assays. The identity of the receptor was confirmed both by photoaffinity labeling and immunoblotting. The fusion receptor displayed typical beta 2-AR pharmacological properties and conferred a beta-adrenergic sensitive adenylyl cyclase activity to the Sf9 cells. Moreover, exposure of the Sf9 cells to the beta-adrenergic agonist isoproterenol induced a rapid desensitization of the receptor-stimulated adenylyl cyclase activity. Purification of the epitope-tagged beta 2-AR by immunoprecipitation as well as by alprenolol-Sepharose affinity chromatography revealed that the receptor is covalently modified with palmitic acid in the insect cells as is observed in mammalian cells. In addition, short-term incubation of the cells with isoproterenol led to a specific increase in the incorporation of [3H]palmitate in the receptor, consistent with a rapid agonist-modulated turnover of the beta 2-AR-attached palmitic acid. These results suggest that agonist-mediated regulation of beta 2-AR post-translational palmitoylation could represent an other regulatory process for G protein-coupled receptors.

Affinity Labels↗
PubMed1992

Differential role of insulin receptor autophosphorylation sites 1162 and 1163 in the long-term insulin stimulation of glucose transport, glycogenesis, and protein synthesis.

The long-term regulatory effect of insulin on glucose transport activity and glucose transporter expression was examined in Chinese hamster ovary (CHO) transfectants that overexpress either human insulin receptors of the wild type (CHO-R cells) or human insulin receptors mutated at two major autophosphorylation sites, Tyr1162 and Tyr1163 (CHO-Y2 cells). Previous studies showed that, when acutely stimulated by insulin, CHO-Y2 cells exhibit decreased receptor kinase activity along with decreased signaling of several pathways, including that for glucose transport, as compared with CHO-R cells. We now report the following. (i) When treated for 24 h with insulin (10(-10) to 10(-6) M), CHO-R and CHO-Y2 cells displayed closely similar concentration-dependent increases in 2-deoxyglucose uptake. In both transfectants, the maximal insulin-induced increase (approximately 3.5-fold) in uptake was cycloheximide-sensitive and was paralleled by equivalent increases in the levels of GLUT-1 immunoreactive protein and mRNA. (ii) By contrast, under similar conditions, CHO-Y2 cells exhibited a marked decrease in their response to insulin for [U-14C]glucose incorporation into glycogen (decreased sensitivity and maximal responsiveness) and for [U-14C]leucine incorporation into protein (decreased sensitivity) as compared with CHO-R cells. (iii) After a 24-h treatment with 10(-7) M insulin, CHO-R (but not CHO-Y2) cells showed a decreased ability to respond to a subsequent acute insulin stimulation of either receptor exogenous kinase activity or 2-deoxyglucose uptake as compared with respective untreated controls. These results indicate that (i) insulin receptors mutated at Tyr1162 and Tyr1163 retain normal signaling of the long-term stimulatory effect of insulin on glucose transport activity and GLUT-1 expression, but not on glycogenesis and overall protein synthesis; (ii) these three insulin signaling pathways may be triggered by distinct domains of the insulin receptor beta-subunit; and (iii) wild-type (but not twin-tyrosine mutant) receptors undergo negative regulation by chronic insulin treatment for subsequent signaling of acute biological actions of insulin.

Animals↗
PubMed1992

Baculovirus expression of mammalian G protein alpha subunits.

Complementary DNAs encoding three subtypes of the alpha subunit (alpha i-1, alpha o and alpha s) of rat guanyl nucleotide regulatory proteins were used to construct recombinant baculoviruses which direct high-level expression of the corresponding proteins in cultured Sf9 insect cells. The expressed proteins were recognized by polyclonal antisera specific for the different alpha chains, and co-migrated with the native proteins from rat brain membranes in immunoblotting analyses. Soluble and particulate forms of all three immunoreactive alpha chains were observed following ultracentrifugation of cell lysates. Biosynthetic radiolabelling of infected cells with [35S]methionine or [3H]myristate showed that both soluble and particulate forms of alpha i-1 and alpha o were myristoylated; in contrast, alpha s did not incorporate myristate. The soluble fractions from cells expressing alpha chains showed high levels of GTP-binding activity over that observed in uninfected cells, or in cells infected with wild-type virus. The peak expression levels observed at 72 h post-infection were highest for alpha o at ca. 400 pmol of GTP-gamma-35S/mg protein, or roughly 2% of the total soluble protein. The results of this work show that the baculovirus system can be employed for high-level production of mammalian G protein alpha chains which retain GTP-binding activity and are appropriately modified by myristoylation.

Animals↗
PubMed1992

Electrophoretic study of conformational changes of a human soluble beta-D-galactoside-binding lectin upon storage.

Human brain lectin (HBL), a beta-galactoside specific soluble lectin, was purified by affinity chromatography. An alkylated derivative of this lectin was also prepared. Both native and modified molecules were conserved at -20 degrees C in the presence or absence of beta-mercaptoethanol, a reducing agent which was described to maintain the lectin activity in vitro or in the presence of beta-mercaptoethanol and lactose. The impact of storage conditions, over one year, on the native and derivated lectins, was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing and titration curve, using the PhastSystem (Pharmacia). Western-blot analysis using an anti-HBL antibody and size-exclusion high performance liquid chromatography were used to complete the study. The subunit M(r)s were estimated before freezing (T0) and after three and twelve months (T3, T12). They were comparable for all preparations. In all samples tested, isoelectric focusing demonstrated the existence of at least three acidic proteins, with the pI ranging between 4.7-4.9. Titration curves clearly showed pH-dependent conformational changes, resulting in a panel of differently charged molecular species, some of which may be related to different oxidative states of the cysteine residues. We concluded that lectin can be stored at -20 degrees C for at least one year before use as a reagent since the modifications revealed by electrophoretic analysis do not alter the hemagglutination activity and carbohydrate binding properties. The immunoreactivity also remained unchanged.

Blotting, Western↗
PubMed1992

Development of screening methods for detection of carbohydrate-binding proteins by use of soluble glycosylated polyacrylamide-based copolymers.

Mammalian endogenous carbohydrate-binding proteins (lectins) play fundamental roles in a variety of mechanisms of interactions both at the molecular and cellular levels. We have investigated the binding of one of them (human brain lectin) to soluble acrylamide copolymerized with derivatives of either lactose (O-beta-lactosyloxyallylallylaminoacrylamide copolymer) or D-mannose (D-alpha-mannosyloxyallylallylaminoacrylamide copolymer) in direct enzyme affinoassays, in an attempt to develop simple procedures for detection and estimation of its carbohydrate-binding activity. Biotinylated plant lectins were utilized as reference standards. Affinoassays employed the polymer dotted on nitrocellulose and the polymer coated on microtiter plates as well as detection of bound biotinylated lectin by streptavidin/horseradish peroxidase reagent. Both assays provided reproducible binding, inhibitable by specific sugars. The microtiter plate assay is well suited to sensitive detection of the negative endogenous lectin by competition with biotinylated brain lectin. We conclude that the use of derivatized acrylamide in dotting and microtiter plate assays may prove practical for detection of endogenous lectins and that such polymers may serve as model substances in the study of biological partners of these carbohydrate-binding proteins.

Acrylic Resins↗
PubMed1992

Use of an immobilized human endogenous lectin for the purification of complementary ligands.

1. A galactoside-specific endogenous lectin isolated from human brain was covalently immobilized on divinylsulfone-activated agarose. This highly selective affinity adsorbent proved to be useful in purifying soluble protein ligands. 2. The maximum binding capacity of the adsorbent for complementary proteins was calculated to be 618 micrograms per g of gel (wet resin). 3. Sequential elutions using 0.1 M lactose, 0.3 M lactose and 0.5 M NaCl, and competition assays using incorporation in the presence 0.1 M lactose revealed differences in lectin-ligand interactions.

Brain Chemistry↗
PubMed1992

Human brain lectin: a soluble lectin that binds actin.

A biotinylated probe was used for detection of endogenous ligands of a human brain lectin on blotted human brain soluble proteins. Of the various proteins from brain extract resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, five reacted with the biotinylated probe. After elimination of saccharidic moieties by periodic treatment of the same extract, a single band with Mr approximately 43,000 was recognized by the lectin. This band was identified as actin using an anti-actin antibody. These results were confirmed by binding of biotinylated lectin to purified actin.

Actins↗
PubMed1992

Regulation of a beta-galactoside-binding lectin and potential ligands during postnatal maturation of rat brain.

Variations in expression and activity of a beta-galactoside-specific lectin were quantified during postnatal maturation of the rat brain. Lectin expression, estimated from enzymatic immunoassay data, and lectin activity, evaluated by rabbit erythrocyte agglutination, are higher in the brains of young animals (5 days after birth) than in older ones (2 months of postnatal life). Concurrently, modifications in glycosylation during cellular differentiation were detected by affinoblotting using a biotinylated derivative of a beta-galactoside-specific brain lectin. This study shows that membrane and cytosolic glycoconjugates of the rat brain bear appropriate beta-galactoside moieties which may be recognized by an endogenous lectin expressed in the tissue itself in relation to brain maturation. The observed variations in the expression of lectin and complementary glycoconjugates can be correlated with significant events of rat brain development.

Aging↗
PubMed1991

Human brain lectin immunoreactive material in cerebrospinal fluids determined by enzyme immunoassay (EIA).

A sensitive enzyme immunoassay (EIA) micromethod is described which can measure levels of a 14 kDa human brain lectin (HBL) in the cerebrospinal fluid (CSF) of patients submitted to CSF examination. The assay is based on the use of a polyclonal antibody to HBL and the simultaneous application of biotinylated and unlabeled HBL. Biotin was then reacted with a streptavidin-peroxidase (Strep-HRP) conjugate and the bound enzyme quantified with the substrate orthophenylenediamine (OPD). The assay requires only 50 microliters of CSF and is very sensitive: as little as 6 ng/ml of HBL 14 can be detected. In a blind-test screening, the mean (+/- SEM) concentration of the HBL immunoreactive material (HIM) in CSF was determined to be 72.4 +/- 6.6 ng/ml. Our results indicate that EIA measurement of HIM levels in the CSF may find useful applications in elucidating the involvement of HBL in the physiopathology of human nervous system (NS).

Antibodies↗
PubMed1991

Increased insulin action in cultured hepatocytes from rats with diabetes induced by neonatal streptozotocin.

Previous studies have shown that Wistar rats injected at birth (n0) with STZ (n0-STZ) develop as adults a noninsulin-dependent diabetic state characterized by a lack of insulin response to glucose in vivo, a mild basal hyperglycemia, and an impaired glucose tolerance. Our former in vivo studies using the insulin-glucose clamp technique revealed an increased insulin action upon hepatic glucose production in these animals. We have now cultured hepatocytes from these mildly diabetic rats in parallel with hepatocytes from control rats, to examine more closely basal and insulin-regulated glucose production and glucose incorporation into glycogen. In addition, we extended our investigation to other hepatic functions such as lipid synthesis and amino acid transport, which could not be studied in vivo. Although glucose production from glycogenolysis or gluconeogenesis in absence or presence of glucagon was identical in the two cell populations, glucagon-stimulated glycogenolysis was more sensitive to insulin action in diabetic hepatocytes. Similarly, insulin action on glucose incorporation into glycogen, lipogenesis, and amino acid transport were enhanced in diabetic hepatocytes. The hormone effect was manifested by an increase in the sensitivity and/or in the responsiveness, reflecting the multiplicity of the pathways whereby the insulin signal is transduced through the insulin receptor to multiple postreceptor sites. To gain insight into the possible mechanism of these disturbances, we evaluated the initial insulin receptor interaction and the kinase activity of the receptor beta-subunit. In accordance with our previous study on intact livers, we found no alteration in either of these parameters in n0-STZ rat hepatocytes. Thus, the present study clearly demonstrates that these diabetic rats exhibit a postreceptor hyperresponsiveness to insulin at the cellular level. It strengthens the notion that a beta-cell deficiency with glucose intolerance does not necessarily lead to a hepatic insulin resistance.

Aminoisobutyric Acids↗