scholarly journals Biosynthesis of the insulin receptor in rat adipose cells. Intracellular processing of the Mr-190 000 pro-receptor

1985 ◽  
Vol 232 (1) ◽  
pp. 71-78 ◽  
Author(s):  
J A Hedo ◽  
I A Simpson
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Insulin Receptor ◽  
Golgi Complex ◽  
Microsomal Fraction ◽  
Primary Cultures ◽  
Sodium Dodecyl ◽  
High Density ◽  
Low Density ◽  
Adipose Cells

We investigated the biosynthesis of the insulin receptor in primary cultures of isolated rat adipose cells. Cells were pulse-chase-labelled with [3H]mannose, and at intervals samples were homogenized. Three subcellular membrane fractions were prepared by differential centrifugation: high-density microsomal (endoplasmic-reticulum-enriched), low-density microsomal (Golgi-enriched), and plasma membranes. After detergent solubilization, the insulin receptors were immunoprecipitated with anti-receptor antibodies and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography. After a 30 min pulse-label [3H]mannose first appeared in a band of Mr 190 000. More than 80% of the Mr-190 000 component was recovered in the microsomal fractions. Its intensity reached a maximum at 1 h in the high-density microsomal fraction and at 2 h in the low-density microsomal fraction, and thereafter declined rapidly (t 1/2 approx. 3 h) in both fractions. In the plasma-membrane fraction, the radioactivity in the major receptor subunits, of Mr 135 000 (alpha) and 95 000 (beta), rose steadily during the chase and reached a maximum at 6 h. The Mr-190 000 precursor could also be detected in the high-density microsomal fraction by affinity cross-linking to 125I-insulin. In the presence of monensin, a cationic ionophore that interferes with intracellular transport within the Golgi complex, the processing of the Mr-190 000 precursor into the alpha and beta subunits was completely inhibited. Our results suggest that the Mr-190 000 pro-receptor originates in the endoplasmic reticulum and is subsequently transferred to the Golgi complex. Maturation of the pro-receptor does not seem to be necessary for the expression of the insulin-binding site. Processing of the precursor into the mature receptor subunits appears to occur during the transfer of the pro-receptor from the Golgi complex to the plasma membrane.

scholarly journals Possible role for gp160 in constitutive but not insulin-stimulated GLUT4 trafficking: dissociation of gp160 and GLUT4 localization

1998 ◽  
Vol 330 (1) ◽  
pp. 405-411 ◽  
Author(s):  
Anthony FILIPPIS ◽  
Stella CLARK ◽  
Joseph PROIETTO
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
High Glucose ◽  
Marked Decrease ◽  
High Density ◽  
Major Protein ◽  
Low Density ◽  
Concomitant Increase ◽  
First Time

GLUT4-containing vesicles are constantly cycling in both basal and insulin-stimulated states. Our previous studies have shown that basal cycling of GLUT4 is impaired under conditions of high glucose or glucosamine and, as a consequence, GLUT4 is retained intracellularly in low-density microsomes [Filippis A., Clark, S., and Proietto, J. (1997) Biochem. J. 324, 981-985]. In addition to GLUT4 itself, a major protein component of GLUT4-containing vesicles is a glycoprotein of Mr 160000 (gp160). In all studies so far published gp160 has been co-localized with GLUT4 under all conditions. In this study, we show that retention of GLUT4 in low-density microsomes (enriched in Golgi apparatus) is associated with a decrease in gp160 levels in this compartment. A concomitant increase of gp160 in high-density microsomes (enriched in endoplasmic reticulum), demonstrates for the first time a dissociation in the localization of gp160 and GLUT4. Despite the marked decrease in gp160 levels in the GLUT4-containing compartment, insulin-stimulated translocation was normal, while little gp160 appeared in the plasma membrane in response to insulin. The retention of gp160 in the high-density microsomes is apparently not due to a change in the glycosylation state of gp160 as measured by [3H]mannose incorporation. It is concluded that, in rat adipocytes, gp160 is not required for insulin-stimulated translocation, but may be necessary for constitutive trafficking of the GLUT4-containing vesicle.

scholarly journals RAB6 and microtubules restrict protein secretion to focal adhesions

2019 ◽  
Vol 218 (7) ◽  
pp. 2215-2231 ◽  
Author(s):  
Lou Fourriere ◽  
Amal Kasri ◽  
Nelly Gareil ◽  
Sabine Bardin ◽  
Hugo Bousquet ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Protein Secretion ◽  
Golgi Complex ◽  
Essential Role ◽  
Focal Adhesions ◽  
Secreted Proteins ◽  
Cell Compartments ◽  
Secretion Assay

To ensure their homeostasis and sustain differentiated functions, cells continuously transport diverse cargos to various cell compartments and in particular to the cell surface. Secreted proteins are transported along intracellular routes from the endoplasmic reticulum through the Golgi complex before reaching the plasma membrane along microtubule tracks. Using a synchronized secretion assay, we report here that exocytosis does not occur randomly at the cell surface but on localized hotspots juxtaposed to focal adhesions. Although microtubules are involved, the RAB6-dependent machinery plays an essential role. We observed that, irrespective of the transported cargos, most post-Golgi carriers are positive for RAB6 and that its inactivation leads to a broad reduction of protein secretion. RAB6 may thus be a general regulator of post-Golgi secretion.

scholarly journals Phosphatidylinositol 3-kinase acts at an intracellular membrane site to enhance GLUT4 exocytosis in 3T3-L1 cells

1996 ◽  
Vol 313 (1) ◽  
pp. 125-131 ◽  
Author(s):  
Jing YANG ◽  
James F. CLARKE ◽  
Catriona J. ESTER ◽  
Paul W. YOUNG ◽  
Masato KASUGA ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Receptor ◽  
Time Course ◽  
Glucose Transporter ◽  
Kinase Activity ◽  
Insulin Receptor Substrate ◽  
Low Density ◽  
Intracellular Membrane ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

Glucose transporters (GLUTs) are continuously recycled in 3T3-L1 cells and so insulin, through its action on phosphatidylinositol 3-kinase (PI 3-kinase), could potentially alter the distribution of these transporters by enhancing retention in the plasma membrane or acting intracellularly to increase exocytosis, either by stimulating a budding or a docking and fusion process. To examine the site of involvement of PI 3-kinase in the glucose transporter recycling pathway, we have determined the kinetics of recycling under conditions in which the PI 3-kinase activity is inhibited by wortmannin. Wortmannin addition to fully insulin-stimulated cells induces a net reduction of glucose transport activity with a time course that is consistent with a major effect on the return of internalized transporters to the plasma membrane. The exocytosis of GLUT1 and GLUT4 is reduced to very low levels in wortmannin-treated cells (≈ 0.009 min-1), but the endocytosis of these isoforms is not markedly perturbed and the rate constants are approx. 10-fold higher than for exocytosis (0.099 and 0.165 min-1, respectively). The slow reduction in basal activity following treatment with wortmannin is consistent with a wortmannin effect on constitutive recycling as well as insulin-regulated exocytosis. PI 3-kinase activity that is precipitated by anti-phosphotyrosine, anti-[insulin receptor substrate 1 (IRS1)] and anti-α-p85 antibodies show the same level of insulin-stimulated activity, ≈ 0.5 pmol/20 min per dish of 3T3-L1 cells. Since the activities precipitated by all three antibodies are similar, it seems unlikely that a second insulin receptor substrate, IRS2, contributes significantly to the insulin signalling observed in 3T3-L1 cells. To examine whether insulin targets PI 3-kinase to intracellular membranes we have carried out subcellular fractionation studies. These suggest that nearly all the insulin-stimulated PI 3-kinase activity is located on intracellular, low-density, membranes. In addition, the association of PI 3-kinase with IRS1 appears to partially deplete the cytoplasm of α-p85-precipitatable activity, suggesting that IRS1 may redistribute PI 3-kinase from the cytoplasm to the low-density microsome membranes. Taken together, the trafficking kinetic and PI 3-kinase distribution studies suggest an intracellular membrane site of action of the enzyme in enhancing glucose transporter exocytosis.

Growth hormone-induced insulin resistance: role of the insulin receptor, IRS-1, GLUT-1, and GLUT-4

1997 ◽  
Vol 272 (6) ◽  
pp. E1071-E1079 ◽  
Author(s):  
T. R. Smith ◽  
J. S. Elmendorf ◽  
T. S. David ◽  
J. Turinsky
Keyword(s):  
Insulin Resistance ◽  
Growth Hormone ◽  
Plasma Membrane ◽  
Insulin Receptor ◽  
Insulin Response ◽  
Low Density ◽  
Glut 1 ◽  
Glut 4 ◽  
Fasting Plasma ◽  
Receptor Beta

Treatment of rats with growth hormone (GH; 1 mg/kg sc) twice daily over 2.5 days did not alter fasting plasma glucose or glucose tolerance but increased fasting plasma insulin levels 65% and peak insulin response to a glucose load 35% over controls, indicating the development of insulin resistance. Studies on partially purified insulin receptors from soleus muscles showed that GH increased the abundance of insulin receptor beta-subunits by 48% as measured by immunoblotting. Despite this increase, GH abolished the increase in autophosphorylation of the insulin receptor beta-subunit in response to physiological hyperinsulinemia and diminished by 28% the response to supraphysiological hyperinsulinemia. Similarly, insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) was decreased 25% by GH, but the abundance of IRS-1 was not affected. Studies on rats pretreated with streptozotocin suggested that the effects of GH are direct and not secondary to GH-induced hyperinsulinemia. GH decreased basal GLUT-1 abundance in the low-density microsome and plasma membrane fractions of epididymal adipocytes by 50 and 42%, respectively, but decreased basal GLUT-4 abundance only in the low-density microsome fraction by 24%. Despite these alterations, the abundance of both transporters in the plasma membrane fraction of adipocytes incubated with 0.1 U insulin/ml was not diminished by GH.

Raft Localization and Nucleotidase Activity of CD39 Depends on Internal Proteolytic Cleavage

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3925-3925
Author(s):  
Kim E Olson ◽  
Joan HF Drosopoulos ◽  
Ashley E Olson ◽  
Marinus Johan Broekman ◽  
Aaron J Marcus
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Sucrose Gradient ◽  
Proteolytic Cleavage ◽  
High Density ◽  
Full Length ◽  
Low Density ◽  
293 Cells ◽  
Discontinuous Sucrose Gradient

Abstract We have previously shown that CD39 undergoes limited cleavage and that inhibition of proteolysis results in a decrease in ATPase activity. The reduction in enzymatic activity correlated with a decrease in the fraction of full-length CD39 present in active membrane raft-localized oligomeric complexes. We exploited N-and C-terminal VP16-and V5-tagged CD39, both transiently and stably expressed in 293 cells, to further elucidate the role of cleavage in the regulation of CD39 processing and activity. To characterize the complexes generated by cross-linking, N-terminal VP16-tagged and C-terminal V5-tagged CD39 were co-expressed in 293 cells. Following crosslinking of membranes with DTSSP and immunoprecipitation with anti-V5, DTT-cleaved species were visualized by Western Blot using VP16 antibody. Interestingly, both VP16-tagged full-length and N-terminal fragments (30 kDa) were immunoprecipitated by anti-V5. This indicates that both full-length CD39 and the N-terminal cleavage fragment are present in raft-localized complexes. The composition of raft-localized CD39 complexes was studied by separating membrane fractions on a discontinuous sucrose gradient using a non-detergent method. When overexpressed, CD39 and its C-terminal fragment distribute across the gradient as visualized by Western with anti-VP16. Importantly, specific activity (expressed as ATPase activity divided by total CD39 content) was 8 times greater in low-density raft-enriched fractions than in high density raft-free fractions. In addition, relative ADPase activity was higher in fractions containing a higher proportion of C-terminal CD39 relative to full-length CD39. Thus, CD39 forms oligomeric complexes and possesses optimal enzyme activity in lipid rafts. The relationship between CD39 cleavage, ATPase activity and raft localization was further studied in 293 cells transfected with C-or N-terminal VP16-tagged CD39. Subcellular fractionation on a discontinuous sucrose gradient yielded membrane fractions enriched in endoplasmic reticulum (ER), early endosomes (EE) and plasma membrane/Golgi (PM-Golgi). Importantly, the EE fraction contained both full-length and C-terminal (or N-terminal) CD39 at the same level as seen in the PM-Golgi fraction, suggesting that near 50% of CD39 resides in the EE compartment. Furthermore, EE-expressed CD39 exhibited an ATPase and ADPase activity equivalent to that seen in Golgi-PM fractions. This led us to examine effects of NH4Cl and bafilomycin (which block acidification of EE), and chloroquine (blocks EE maturation) on CD39 cleavage, activity and raft localization. Each treatment inhibited CD39 cleavage and correspondingly decreased ATPase activity. A shift of ~50% of full-length CD39 from raft fractions to high density membrane fractions was observed upon sucrose gradient fractionation following chloroquine treatment of cells transfected with N-terminal VP16 tagged CD39. This redistribution of CD39 in the membrane correlated with a 40% decrease in ATPase activity and a striking inhibition of CD39 cleavage. Here, at a lower level of expression than cited above, ATPase activity in low-density raft fractions was ~100-fold greater than in high density fractions. Thus, cleavage of a portion of CD39 molecules is required for both raft localization of full-length CD39 and optimal enzyme activity. Regulated proteolytic cleavage of CD39 would allow for rapid upregulation of CD39 activity in response to alterations in cell environment. This would occur via cycling of CD39 between plasma membrane and endosomal compartments, the proposed site of CD39 cleavage and assembly of fully active oligomeric complexes.

scholarly journals Novel adipocyte aminopeptidases are selectively upregulated by insulin in healthy and obese rats

2015 ◽  
Vol 228 (2) ◽  
pp. 97-104 ◽  
Author(s):  
Rafaela Fadoni Alponti ◽  
Patricia Lucio Alves ◽  
Paulo Flavio Silveira
Keyword(s):  
Plasma Membrane ◽  
Energy Metabolism ◽  
Enzyme Activities ◽  
Metabolic Pathways ◽  
Membrane Fraction ◽  
Microsomal Fraction ◽  
Low Density ◽  
Plasma Membrane Fraction ◽  
Catalytic Activities ◽  
Obese Rats

The lack of a complete assembly of the sensitivity of subcellular aminopeptidase (AP) activities to insulin in different pathophysiological conditions has hampered the complete view of the adipocyte metabolic pathways and its implications in these conditions. Here we investigated the influence of insulin on basic AP (APB), neutral puromycin-sensitive AP (PSA), and neutral puromycin-insensitive AP (APM) in high and low density microsomal and plasma membrane fractions from adipocytes of healthy and obese rats. Catalytic activities of these enzymes were fluorometrically monitoring in these fractions with or without insulin stimulus. Canonical traffic such as insulin-regulated AP was not detected for these novel adipocyte APs in healthy and obese rats. However, insulin increased APM in low density microsomal and plasma membrane fractions from healthy rats, APB in high density microsomal fraction from obese rats and PSA in plasma membrane fraction from healthy rats. A new concept of intracellular compartment-dependent upregulation of AP enzyme activities by insulin emerges from these data. This relatively selective regulation has pathophysiological significance, since these enzymes are well known to act as catalysts and receptor of peptides directly related to energy metabolism. Overall, the regulation of each one of these enzyme activities reflects certain dysfunction in obese individuals.

scholarly journals Assembly of influenza hemagglutinin trimers and its role in intracellular transport.

1986 ◽  
Vol 103 (4) ◽  
pp. 1179-1191 ◽  
Author(s):  
C S Copeland ◽  
R W Doms ◽  
E M Bolzau ◽  
R G Webster ◽  
A Helenius
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Quaternary Structure ◽  
Subunit Interactions ◽  
Oligomer Formation ◽  
Influenza Hemagglutinin ◽  
Triton X

The hemagglutinin (HA) of influenza virus is a homotrimeric integral membrane glycoprotein. It is cotranslationally inserted into the endoplasmic reticulum as a precursor called HA0 and transported to the cell surface via the Golgi complex. We have, in this study, investigated the kinetics and cellular location of the assembly reaction that results in HA0 trimerization. Three independent criteria were used for determining the formation of quaternary structure: the appearance of an epitope recognized by trimer-specific monoclonal antibodies; the acquisition of trypsin resistance, a characteristic of trimers; and the formation of stable complexes which cosedimented with the mature HA0 trimer (9S20,w) in sucrose gradients containing Triton X-100. The results showed that oligomer formation is a posttranslational event, occurring with a half time of approximately 7.5 min after completion of synthesis. Assembly occurs in the endoplasmic reticulum, followed almost immediately by transport to the Golgi complex. A stabilization event in trimer structure occurs when HA0 leaves the Golgi complex or reaches the plasma membrane. Approximately 10% of the newly synthesized HA0 formed aberrant trimers which were not transported from the endoplasmic reticulum to the Golgi complex or the plasma membrane. Taken together the results suggested that formation of correctly folded quaternary structure constitutes a key event regulating the transport of the protein out of the endoplasmic reticulum. Further changes in subunit interactions occur as the trimers move along the secretory pathway.

scholarly journals Rab 3D in rat adipose cells and its overexpression in genetic obesity (Zucker fatty rat)

1997 ◽  
Vol 321 (1) ◽  
pp. 89-94 ◽  
Author(s):  
Michèle GUERRE-MILLO ◽  
Giulia BALDINI ◽  
Harvey F. LODISH ◽  
Marcelle LAVAU ◽  
Samuel W. CUSHMAN
Keyword(s):  
Molecular Basis ◽  
Plasma Membranes ◽  
Microsomal Fraction ◽  
Zucker Rats ◽  
Low Density ◽  
Secretory Function ◽  
Gtp Binding ◽  
Specific Manner ◽  
Intracellular Vesicles ◽  
Adipose Cells

Members of the Rab 3 subfamily of low-molecular-mass GTP-binding proteins have been functionally implicated in regulated exocytosis. The aim of the present study was to examine the subcellular distribution of a member of this family, Rab 3D, in rat adipose cells, given the hypothesis that this protein might be involved in insulin-stimulated GLUT4 exocytosis. We show that Rab 3D immunoreactivity is associated predominantly with the high-density microsomal fraction, where the signal intensity is 3-and 7-fold greater than that in plasma membranes and low-density microsomes respectively. Rab 3D does not co-localize with GLUT4 on immuno-isolated intracellular vesicles and, unlike GLUT4, it is not redistributed in response to insulin. Thus, if Rab 3D plays a role in GLUT4 trafficking, it relies on mechanisms independent of relocation. We observed that Rab 3D is overexpressed in adipose cells of obese (fa/fa) Zucker rats, in a tissue- and isoform-specific manner. The pathophysiological significance of this defect remains elusive. This could form the molecular basis for altered adipose secretory function in obesity.

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