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J L Carpentier

Publications and source records attributed to J L Carpentier.

At least 37 records · Page 2Linked to original sources
PubMed1996

Molecular and cellular mechanisms governing the ligand-specific and non-specific steps of insulin receptor internalization.

The surface events leading to insulin-induced internalization of its specific receptor can be subdivided in three major steps: the first step consists in the surface redistribution of the receptor from the villous to the non-villous region of the cell surface, it is ligand-specific, depends on kinase activation and phosphorylation of tyrosines 1146, 1150 and 1151, and consists in the relief of a constraint immobilizing the receptor on microvilli; the second step is characterized by the shift of the insulin-receptor complex in the plane of the membrane allowing it to get access to the nonvillous domain of the cell surface where internalization gates (clathrin-coated pits) are located; this stage is controlled, at least in part, by the transmembrane domain of the molecule and its flanking amino acids; the third step corresponds to the segregation of the insulin-receptor complex in clathrin-coated pits, this step is relatively non-specific and is governed by well defined signal sequences present in the juxtamembrane domain of the cytoplasmic segment of the b-subunit. These surface events are then automatically followed by the entry of the insulin receptor inside the cells through the formation of clathrin-coated vesicles, in its subsequent association with endosomes which acidic pH allows insulin dissociation from its receptor and the sorting of the receptor and the hormone in different directions: insulin is targetted to lysosomes to be degraded while the receptor is recycled back to the cell surface to be reused. This complex process does not seem to be involved in the transmission of the biological signal of the hormone. Nevertheless, it is initiated and controlled by insulin and results in the intracellular degradation of insulin and in the modulation of the number of surface insulin receptors. Thus, even if it does not directly participate in insulin signaling, insulin receptor internalization plays a crucial role in the control of insulin action.

Animals↗
PubMed1995

Insulin receptor kinase activation releases a constraint maintaining the receptor on microvilli.

To examine whether the surface redistribution of the insulin receptor from microvilli, where it sits in its unoccupied form, to the nonvillous domain, where it is internalized through clathrin-coated pits, is an active movement or a passive redistribution linked to the release of a restraint maintaining it on microvilli, we have generated a mutated insulin receptor with a truncation of exons 17-22 and tracked it biochemically and morphologically. Biochemical analysis indicates that this mutated receptor is constitutively internalized and recycled even in the absence of ligand. Quantitative electron microscope autoradiography analysis reveals that it does not preferentially associate with microvilli in its unoccupied form but is normally segregated in clathrin-coated pits through the preserved signal sequence(s) of exon 16. We conclude that (a) insulin receptor internalization initiated through receptor kinase activation and autophosphorylation, which free the receptor from constraints maintaining it on microvilli; (b) the signal sequences contained in exon 16 are entirely sufficient to promote clathrin-coated pit-mediated internalization of insulin receptors; (c) these sequences are not uncovered by kinase activation; and (d) the "code" maintaining the unoccupied receptors on microvilli is contained within exons 17-21 of the receptor.

Animals↗
PubMed1995

Role of the transmembrane domain and flanking amino acids in internalization and down-regulation of the insulin receptor.

We have characterized the internalization and down-regulation of the insulin receptor and nine receptors with mutations in the transmembrane (TM) domain and/or flanking charged amino acids to define the role of this domain in receptor cycling. When expressed in Chinese hamster ovary cells, all had normal tetrameric structure and normal insulin-stimulated autophosphorylation/kinase activity. Replacement of the TM domain with that of the platelet-derived growth factor receptor, insertion of 3 amino acids, and substitution of Asp for Val938 or of Ala for either Gly933 or Pro934 had no effect on internalization. Replacement of the TM domain with that of c-neu or conversion of the charged amino acids on the cytoplasmic flank to uncharged amino acids, on the other hand, resulted in a 40-60% decrease in insulin-dependent internalization rate constants. By contrast, substitution of Ala for both Gly933 and Pro934 increases lateral diffusion mobility and accelerates internalization rate. These changes in internalization were due to decreased or increased rates of redistribution of receptors from microvilli to the nonvillous cell surface. In all cases, receptor down-regulation and receptor-mediated insulin degradation paralleled the changes in internalization. Thus, the structure of the transmembrane domain of the insulin receptor and flanking amino acids are major determinants of receptor internalization, insulin degradation, and receptor down-regulation.

Amino Acid Sequence↗
PubMed1995

Transmembrane domain inversion blocks ER release and insulin receptor signaling.

Activation of the insulin receptor, like other tyrosine kinase receptors, appears to require dimerization. We have shown previously that, even in the absence of insulin, full receptor activation can be induced by changes in the receptor transmembrane domain (TMD), suggesting that TMD dimerization is sufficient for receptor activation. To further understand the importance of the TMD in insulin receptor activation, we have inverted the entire TMD sequence including flanking basic amino acids, residue-for-residue. This mutation was predicted to alter the ability of a TMD alpha-helix to form homodimers and higher level aggregates. Despite apparently normal protein folding on either side of the membrane, this mutation caused ER retention and, for those receptors that reached the cell surface, blockade of insulin-stimulated kinase signal transmission. However, the signaling blockade could be overcome by proteolytic activation with trypsin. In contrast, shifting only the basic cytoplasmic residues to the opposite side of the TMD or mutation to neutral residues had no detectable effect on assembly, biosynthesis, topology, or signaling. These findings extend our previous observations to suggest that TMD interactions within the membrane are not only sufficient for receptor activation, but may be required. TMD interactions also appear to be necessary for oligomeric assembly and biosynthetic maturation of the insulin receptor.

Amino Acid Sequence↗
PubMed1994

Redistribution of intracellular Ca2+ stores during phagocytosis in human neutrophils.

Subcellular gradients of cytosolic free Ca2+ concentration, [Ca2+]i, are thought to be critical for the localization of functional responses within a cell. A potential but previously unexplored mechanism for the generation of gradients of [Ca2+]i is the accumulation of Ca2+ stores at the site of Ca2+ action. The distribution of the Ca2+ store markers Ca(2+)-dependent adenosine triphosphatase and calreticulin was investigated in resting and phagocytosing human neutrophils. Both proteins showed an evenly distributed fine granular pattern in nonphagocytosing cells, but became markedly concentrated in the filamentous actin-rich cytoplasmic area around the ingested particle during phagocytosis. This redistribution began at early stages of phagocytosis and did not depend on an increase in [Ca2+]i. Thus, accumulation of Ca2+ stores in a restricted area of the cell may contribute to the generation of localized increases in [Ca2+]i.

Actins↗
PubMed1994

The functions of the human insulin receptor are affected in different ways by mutation of each of the four N-glycosylation sites in the beta subunit.

The functional role of the oligosaccharide chains linked to the insulin receptor (IR) beta subunit was investigated by site-directed mutagenesis of each of the 4 acceptor asparagines (N1 to N4 from the amino to the carboxyl terminus) and stable expression of the receptors in CHO cells. All mutant receptors are expressed normally at the cell surface, bind insulin with similar affinity, but have a beta subunit of smaller molecular mass, and a defect in ligand-induced internalization as compared to wild type receptor. In terms of receptor activation and signal transduction, the N1 and N2 mutants function normally, whereas the N4 mutant exhibits major alterations in in vitro tyrosine kinase activity and autophosphorylation and is unable to transduce the signal for either glycogen or DNA synthesis. By contrast, in vivo autophosphorylation and IRS-1 phosphorylation appear quantitatively normal, and only partial alterations of phosphatidylinositol 3-kinase and mitogen-activated protein kinase activation are observed. Mutation of the N3 site results in partial defect of IR activation. These data provide evidence for (i) glycosylation of each N-linked glycosylation site of the IR beta subunit, (ii) absence of correlation between internalization and transmembrane signaling, and (iii) a major role for oligosaccharide side chain(s) located close to the cell membrane in IR activation and transmembrane signaling.

Animals↗
PubMed1994

Identification of membrane-bound CR1 (CD35) in human urine: evidence for its release by glomerular podocytes.

Complement receptor 1 (CR1) is present on erythrocytes (E-CR1), various leucocytes, and renal glomerular epithelial cells (podocytes). In addition, plasma contains a soluble form of CR1 (sCR1). By using a specific ELISA, CR1 was detected in the urine (uCR1) of normal individuals (excretion rate in 12 subjects, 3.12 +/- 1.15 micrograms/24 h). Contrary to sCR1, uCR1 was pelleted by centrifugation at 200,000 g for 60 min. Analysis by sucrose density gradient ultracentrifugation showed that uCR1 was sedimenting in fractions larger than 19 S, whereas sCR1 was found as expected in fractions smaller than 19 S. The addition of detergents reduced the apparent size of uCR1 to that of sCR1. After gel filtration on Sephacryl-300 of normal urine, the fractions containing uCR1 were found to be enriched in cholesterol and phospholipids. The membrane-association of uCR1 was demonstrated by analyzing immunoaffinity purified uCR1 by electron microscopy which revealed membrane-bound vesicles. The apparent molecular mass of uCR1 was 15 kD larger than E-CR1 and sCR1 when assessed by SDS-PAGE and immunoblotting. This difference in size could not be explained on the basis of glycosylation only, since pretreatment with N-glycosidase F reduced the size of all forms of CR1; however, the difference in regular molecular mass was not abrogated. The structural alleles described for E-CR1 were also found for uCR1. The urine of patients who had undergone renal transplantation contained alleles of uCR1 which were discordant with E-CR1 in 7 of 11 individuals, indicating that uCR1 originated from the kidney. uCR1 was shown to bind C3b-coated immune complexes, suggesting that the function of CR1 was not destroyed in urine. A decrease in uCR1 excretion was observed in 3 of 10 patients with systemic lupus erythematosus, corresponding to the three who had severe proliferative nephritis, and in three of three patients with focal sclerosis, but not in six other patients with proteinuria. Taken together, these data suggest that glomerular podocytes release CR1-coated vesicles into the urine. The function of this release remains to be defined, but it may be used as a marker for podocyte injury.

Adult↗
PubMed1994

Epidermal growth factor secretion by submandibular glands is not perturbed in the early phase of streptozotocin-induced diabetes in mice.

In rodents, diabetes mellitus is accompanied by a decreased concentration of epidermal growth factor (EGF) in plasma and urine and by a reduced number of EGF receptors on the surface of target cells. A combination of these two abnormalities may reduce the effects of EGF and could be implicated in some complications of diabetes. The aim of the present work was to investigate the possible implication of the major source of EGF in the organism, the submandibular glands, upon the reduced EGF concentration in blood and urine. Firstly, we measured the content of mice submandibular gland EGF by radioimmunoassay and by morphometric determinations of the relative volume occupied by granular convoluted tubules in the gland and by the EGF-containing granules in the cells at the light and electron microscopical levels respectively. We found no differences in the EGF content of submandibular glands of streptozotocin-treated diabetic animals compared to control ones. Secondly, we estimated stimulus-secretion coupling by EGF-secreting cells, present in an enriched preparation of granular convoluted tubules (GCT) perifused in thermoregulated chambers. Phenylephrine was the only agent tested that was capable of stimulating EGF secretion. The stimulation was dose-dependent and similar in streptozotocin-diabetic and control mice. Thus, the EGF deficiency observed in diabetic mice is related neither to a defect of EGF content in the submandibular glands nor to an abnormal EGF secretion by the glands.

Animals↗
PubMed1994

Insulin receptor internalization: molecular mechanisms and physiopathological implications.

The initial interaction between insulin and its receptor on target cell surface is followed by a series of surface and intracellular steps which participate in the control of insulin action. Abnormalities of any of these steps could result in mishandling of the receptor leading to defective modulation of receptor number on the cell surface and to inappropriate cell sensitivity to the hormone. Thus, the identification of each of these steps as well as understanding the mechanisms governing them is obligatory to unravel some aspects of the pathogenesis of insulin resistance states. This was the goal of the studies we have carried out during recent years using combined molecular and cellular biology as well as biochemical techniques. These studies allowed us to propose the following ordered sequence of events: 1) insulin binds to receptors preferentially associated with microvilli on the cell surface; 2) insulin triggers receptor kinase activation and autophosphorylation which not only results in initiation of the various biological signals leading to insulin action but also in redistribution of the hormone-receptor complex in the plane of the membrane; 3) on the non-villous domain of the cell surface, insulin receptors anchor to clathrin-coated pits through specific "internalization sequences" present in their cytoplasmic juxtamembrane domain; 4) insulin-receptor complexes are internalized together with other receptors present in the same clathrin-coated pits through the formation of clathrin-coated vesicles; 5) the complexes are delivered to endosomes, the acidic pH of which induces the dissociation of insulin molecules from insulin receptors and their sorting in different directions; 6) insulin molecules are targetted to late endosomes and lysosomes where they are degraded; 7) receptors are recycled back to the cell surface in order to be reused.

Animals↗
PubMed1993

Clathrin-coated pit-mediated receptor internalization. Role of internalization signals and receptor mobility.

Most signals controlling receptor-mediated endocytosis have been identified by alteration of sequences present in receptors normally internalized via clathrin-coated pits. In the present work we have reconsidered the factors that control internalization the other way around: i.e. by introducing potential internalization sequences in complement receptor 1 (CR1) which does not preferentially associate with clathrin-coated pits. The analysis of the internalization efficiency of NPxY related motifs generated by substituting His2010 and/or Glu2015 by either Phe or Tyr indicates that FxNPxY is the stronger promoter of endocytosis and that the signal efficiency depends on the presence of aromatic residues (including a tyrosine) at both ends of the -xNPx- motif. Moreover, CR1-tyr (substitution of Glu2015 for Tyr) internalization was superposable to that of a receptor composed of the extracellular and transmembrane domains of CR1 fused to the intracytoplasmic tail of the low density lipoprotein (LDL) receptor (including the FxNPxY motif) (CR1-LDL). When analyzed by fluorescence recovery after photobleaching, the surface mobility of CR1-LDL was decreased as compared with that of either CR1-tyr or CR1-wt, despite a similar association with clathrin-coated pits. The role of receptor mobility in internalization was confirmed by the observation that CR1-tl, with a deletion of the cytoplasmic tail, was more mobile and more efficiently internalized than CR1-wt.

Amino Acid Sequence↗
PubMed1993

Specific glycosylation site mutations of the insulin receptor alpha subunit impair intracellular transport.

The insulin receptor is a transmembrane protein found on multiple cell types. This receptor is synthesized as a 190-kDa proreceptor which is cleaved to produce mature alpha and beta subunits. The proreceptor contains 18 potential sites for N-linked glycosylation: 14 on the alpha subunit and 4 on the beta subunit. The codons for asparagine in the first four sites at the amino terminus of the alpha subunit were mutated to code for glutamine. This mutant receptor cDNA was stably transfected into NIH 3T3 cells. The insulin receptor produced in these cells remained in the proreceptor form; no mature alpha and beta subunits were produced. The proreceptor was slightly smaller on SDS-PAGE gels than the wild-type proreceptor and contained four less oligosaccharide chains by tryptic peptide mapping. The carbohydrate chains on the mutant proreceptor remained endoglycosidase H sensitive. However, in the presence of brefeldin A, these oligosaccharide chains could be processed to endoglycosidase H resistant chains. By immunofluorescence, the mutant proreceptor was shown to be localized to the endoplasmic reticulum. No insulin receptors could be found on the cell-surface either with cell surface labeling with biotin or with 125I-insulin binding. Thus, glycosylation of the first four N-linked glycosylation sites of the insulin receptor is necessary for the proper processing and intracellular transport of the receptor. This is in contrast to glycosylation at the four sites on the beta subunit which appear not to be important for processing but necessary for signal transduction. Therefore, N-linked glycosylation of the insulin receptor at specific sites has multiple distinctive roles.

3T3 Cells↗
PubMed1993

Co-regulation of the mitogen-activated protein kinase, extracellular signal-regulated kinase 1, and the 90-kDa ribosomal S6 kinase in PC12 cells. Distinct effects of the neurotrophic factor, nerve growth factor, and the mitogenic factor, epidermal growth factor.

We recently characterized the association of the 44-kDa mitogen-activated protein kinase, also known as extracellular-regulated kinase 1 (ERK1), with the 90-kDa ribosomal S6 kinase (pp90rsk), one of its putative substrates in intact PC12 cells. Using antibodies to ERK1 that precipitate a functional ERK1.pp90rsk phosphoprotein complex, we demonstrate here the regulation of both kinases by various stimuli. In mouse fibroblasts expressing human insulin receptors, insulin and vanadate swiftly stimulated ERK1 activity within 5 min. While the hormonal effect was short-lived, vanadate led to a first peak followed by a progressively increasing second phase. In PC12 cells, epidermal growth factor, which is a growth promoting factor, provokes a rapid but evanescent activation of ERK1. In contrast, nerve growth factor (NGF), which acts as a neuronal differentiation factor for PC12 cells, induced a swift monophasic response followed by a sustained second phase. This strikingly different pattern of ERK1 stimulation by NGF and epidermal growth factor was associated to a contrasting effect on ERK1 cellular translocation. Thus, NGF induced a nuclear translocation of ERK1, while epidermal growth factor was without noticeable effect on ERK1 localization. In both cell systems all effectors tested stimulated ERK1 phosphorylation on both threonine and tyrosine residues in an 1:1 ratio. During ERK1 inactivation, phosphothreonine and phosphotyrosine were dephosphorylated in a similar fashion. Concurrent with ERK1 activation was the de novo appearance of phosphothreonine and an increase in phosphoserine on pp90rsk. The pp90rsk phosphothreonine content paralleled the ERK1 activity more closely than the phosphoserine level. These results provide compelling evidence that in fibroblasts and PC12 cells ERK1 plays a direct role in the phosphorylation of pp90rsk and that pp90rsk represents a physiologically relevant substrate of extracellular-regulated kinases. Finally, we would like to suggest that the differentiating action of NGF in PC12 cells might be due, at least in part, to the conjunction of its sustained and robust stimulation of ERK1 and pp90rsk, and of its induction of ERK1 nuclear translocation.

Amino Acids↗
PubMed1993

Robert Feulgen Prize Lecture 1993. The journey of the insulin receptor into the cell: from cellular biology to pathophysiology.

The data that we have reviewed indicate that insulin binds to a specific cell-surface receptor. The complex then becomes involved in a series of steps which lead the insulin-receptor complex to be internalized and rapidly delivered to endosomes. From this sorting station, the hormone is targeted to lysosomes to be degraded while the receptor is recycled back to the cell surface. This sequence of events presents two degrees of ligand specificity: (a) The first step is ligand-dependent and requires insulin-induced receptor phosphorylation of specific tyrosine residues. It consists in the surface redistribution of the receptor from microvilli where it preferentially localizes in its unoccupied form. (b) The second step is more general and consists in the association with clathrin-coated pits which represents the internalization gate common to many receptors. This sequence of events participates in the regulation of the biological action of the hormone and can thus be implicated in the pathophysiology of diabetes mellitus and various extreme insulin resistance syndromes, including type A extreme insulin resistance, leprechaunism, and Rabson-Mendehall syndrome. Alterations of the internalization process can result either from intrinsic abnormalities of the receptor or from more general alteration of the plasma membrane or of the cell metabolism. Type I diabetes is an example of the latter possibility, since general impairment of endocytosis could contribute to extracellular matrix accumulation and to an increase in blood cholesterol. Thus, better characterization of the molecular and cellular biology of the insulin receptor and of its journey inside the cell definitely leads to better understanding of disease states, including diabetes.

Biological Transport↗
PubMed1993

Two steps of insulin receptor internalization depend on different domains of the beta-subunit.

The internalization of signaling receptors such as the insulin receptor is a complex, multi-step process. The aim of the present work was to determine the various steps in internalization of the insulin receptor and to establish which receptor domains are implicated in each of these by the use of receptors possessing in vitro mutations. We find that kinase activation and autophosphorylation of all three regulatory tyrosines 1146, 1150, and 1151, but not tyrosines 1316 and 1322 in the COOH-terminal domain, are required for the ligand-specific stage of the internalization process; i.e., the surface redistribution of the receptor from microvilli where initial binding occurs to the nonvillous domain of the cell. Early intracellular steps in insulin signal transduction involving the activation of phosphatidylinositol 3'-kinase are not required for this redistribution. The second step of internalization consists in the anchoring of the receptors in clathrin-coated pits. In contrast to the first ligand specific step, this step is common to many receptors including those for transport proteins and occurs in the absence of kinase activation and receptor autophosphorylation, but requires a juxta-membrane cytoplasmic segment of the beta-subunit of the receptor including a NPXY sequence. Thus, there are two independent mechanisms controlling insulin receptor internalization which depend on different domains of the beta-subunit.

Amino Acid Sequence↗
PubMed1993

Insulin increases the Na(+)-K(+)-ATPase alpha 2-subunit in the surface of rat skeletal muscle: morphological evidence.

The cellular localization of the alpha 2-subunit of the Na(+)-K(+)-ATPase was defined by immunoelectron microscopy, and the effect of insulin on the amount of alpha 2-immunoreactive subunits on the cell surface was quantitated. Two protocols were used for tissue fixation and immunolocalization. Protocol 1 was characterized by fixation with 2% paraformaldehyde, use of a monoclonal antibody, and detection with 3-nm-diameter gold-labeled Fab fragments or 10-nm gold-labeled immunoglobulin G. Protocol 2 was characterized by fixation with 4% paraformaldehyde plus 0.1% glutaraldehyde, use of a polyclonal antibody, and detection with 10-nm gold-labeled protein A. In control muscle, the alpha 2-subunit of the Na(+)-K(+)-ATPase was present at the plasma membrane and in intracellular tubular and vesicular structures located in subsarcolemmal and triadic regions. Acute insulin stimulation increased the number of immunolabeled alpha 2-subunits in the plasma membrane after both fixation protocols. The gain in the plasma membrane ranged from 1.5- to 3.7-fold and was significant at the level of P < 0.005. These results provide morphological quantitative evidence that the alpha 2-subunit of the Na(+)-K(+)-ATPase is present both at the plasma membrane and intracellularly in mammalian skeletal muscle and that insulin acutely increases its abundance in the muscle surface.

Animals↗
PubMed1993

Endocytosis is inhibited in hepatocytes from diabetic rats.

The consequences of type I diabetes on cellular endocytosis were investigated by comparing [125I]insulin, [125I]alpha 2-macroglobulin, and Lucifer yellow uptake in hepatocytes freshly isolated from control and STZ-induced diabetic rats. In addition to the previously described reversible inhibition of ligand-induced internalization of the insulin receptor, we report a decrease in the constitutive receptor-mediated endocytosis of alpha 2-macroglobulin and a near abolition of fluid-phase endocytosis of Lucifer yellow in cells from diabetic animals. Despite decreased receptor autophosphorylation and internalization, the ligand-induced surface redistribution of the insulin receptor was normal in the diabetic cell population. By contrast, the insulin receptor association with clathrin-coated pits was impaired in diabetic cells as a result of a decreased concentration of these specialized invaginations on the nonvillous cell surface. The morphology and diameter of clathrin-coated pits were similar in both conditions under study. These results demonstrate a general impairment of endocytosis in hypoinsulinemic diabetes: receptor-mediated endocytosis was less affected than fluid-phase endocytosis. Impaired endocytosis of specific ligands or other macromolecules could be an important mechanism underlying the accumulation of extracellular matrix or even blood cholesterol removal in diabetes.

Animals↗