A dual role of the N-terminal FQQI motif in GLUT4 trafficking

2009 ◽  
Vol 390 (9) ◽  
Author(s):  
Ulrike Bernhardt ◽  
Françoise Carlotti ◽  
Rob C. Hoeben ◽  
Hans-Georg Joost ◽  
Hadi Al-Hasani
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Dual Role ◽  
Endosomal Trafficking ◽  
Endosomal Sorting ◽  
Storage Compartment ◽  
Storage Vesicles ◽  
Intracellular Storage ◽  
Glucose Transporter Glut4

AbstractIn adipocytes, the glucose transporter GLUT4 recycles between intracellular storage vesicles and the plasma membrane. GLUT4 is internalized by a clathrin- and dynamin-dependent mechanism, and sorted into an insulin-sensitive storage compartment. Insulin stimulation leads to GLUT4 accumulation on the cell surface. The N-terminal F5QQI motif in GLUT4 has been shown previously to be required for sorting of the protein in the basal state. Here, we show that the FQQI motif is a binding site for the medium chain adaptin μ1, a subunit of the AP-1 adaptor complex that plays a role in post-Golgi/endosomal trafficking events. In order to investigate the role of AP-1 and AP-2 in GLUT4 trafficking, we generated 3T3-L1 adipocytes expressing HA-GLUT4-GFP and knocked down the AP-1 and AP-2 complex by RNAi, respectively. In AP-1 and AP-2 knockdown adipocytes, GLUT4 accumulates at the cell surface in the basal state, consistent with a role of AP-1 in post-endosomal sorting of GLUT4 to the insulin-sensitive storage compartment, and of AP-2 in clathrin-mediated endocytosis. Our data demonstrate a dual role of the F5QQI motif and support the conclusion that the AP complexes direct GLUT4 trafficking and endocytosis.

scholarly journals Characterization of Insulin-Responsive GLUT4 Storage Vesicles Isolated from 3T3-L1 Adipocytes

2000 ◽  
Vol 20 (1) ◽  
pp. 416-427 ◽  
Author(s):  
Mitsuru Hashiramoto ◽  
David E. James
Keyword(s):  
Glucose Transporter ◽  
Significant Proportion ◽  
Protein Composition ◽  
Cell Types ◽  
Storage Compartment ◽  
Storage Vesicles ◽  
Equilibrium Sedimentation ◽  
Insulin Responsiveness ◽  
Glucose Transporter Glut4 ◽  
Golgi Network

ABSTRACT Insulin regulates glucose transport in muscle and adipose tissue by triggering the translocation of a facilitative glucose transporter, GLUT4, from an intracellular compartment to the cell surface. It has previously been suggested that GLUT4 is segregated between endosomes, the trans-Golgi network (TGN), and a postendosomal storage compartment. The aim of the present study was to isolate the GLUT4 storage compartment in order to determine the relationship of this compartment to other organelles, its components, and its presence in different cell types. A crude intracellular membrane fraction was prepared from 3T3-L1 adipocytes and subjected to iodixanol equilibrium sedimentation analysis. Two distinct GLUT4-containing vesicle peaks were resolved by this procedure. The lighter of the two peaks (peak 2) was comprised of two overlapping peaks: peak 2b contained recycling endosomal markers such as the transferrin receptor (TfR), cellubrevin, and Rab4, and peak 2a was enriched in TGN markers (syntaxin 6, the cation-dependent mannose 6-phosphate receptor, sortilin, and sialyltransferase). Peak 1 contained a significant proportion of GLUT4 with a smaller but significant amount of cellubrevin and relatively little TfR. In agreement with these data, internalized transferrin (Tf) accumulated in peak 2 but not peak 1. There was a quantitatively greater loss of GLUT4 from peak 1 than from peak 2 in response to insulin stimulation. These data, combined with the observation that GLUT4 became more sensitive to ablation with Tf-horseradish peroxidase following insulin treatment, suggest that the vesicles enriched in peak 1 are highly insulin responsive. Iodixanol gradient analysis of membranes isolated from other cell types indicated that a substantial proportion of GLUT4 was targeted to peak 1 in skeletal muscle, whereas in CHO cells most of the GLUT4 was targeted to peak 2. These results indicate that in insulin-sensitive cells GLUT4 is targeted to a subpopulation of vesicles that appear, based on their protein composition, to be a derivative of the endosome. We suggest that the biogenesis of this compartment may mediate withdrawal of GLUT4 from the recycling system and provide the basis for the marked insulin responsiveness of GLUT4 that is unique to muscle and adipocytes.

scholarly journals Potential role of Rab4 in the regulation of subcellular localization of Glut4 in adipocytes.

1996 ◽  
Vol 16 (12) ◽  
pp. 6879-6886 ◽  
Author(s):  
M Cormont ◽  
M N Bortoluzzi ◽  
N Gautier ◽  
M Mari ◽  
E van Obberghen ◽  
...  
Keyword(s):  
Cell Surface ◽  
Subcellular Distribution ◽  
Potential Role ◽  
Glucose Transporter ◽  
Small Gtpase ◽  
Wild Type ◽  
Transfected Cells ◽  
Isolated Adipocytes ◽  
Glucose Transporter Glut4

A role for Rab4 in the translocation of the glucose transporter Glut4 induced by insulin has been recently proposed. To study more directly the role of this small GTPase, freshly isolated adipocytes were transiently transfected with the cDNAs of both an epitope-tagged Glut4-myc and Rab4, a system which allows direct measurement of the concentration of Glut4 molecules at the cell surface. When cells were cotransfected with Glut4-myc and Rab4, the concentration of Glut4-myc at the cell surface decreased in parallel with the increased expression of Rab4, suggesting that Rab4 participates in the intracellular retention of Glut4. In parallel, the amount of Rab4 associated with the Glut4-containing vesicles increased. When Rab4 was moderately overexpressed, the number of Glut4-myc molecules recruited to the cell surface in response to insulin was similar to that observed in mock-transfected cells, and thus the insulin efficiency was increased. When Rab4 was expressed at a higher level, the amount of Glut4-myc present at the cell surface in response to insulin decreased. Since the overexpressed protein was predominantly cytosolic, this suggests that the cytosolic Rab4 might complex some factor(s) necessary for insulin action. This hypothesis was strengthened by the fact that Rab4 deltaCT, a Rab4 mutant lacking the geranylgeranylation sites, inhibited insulin-induced recruitement of Glut4-myc to the cell surface, even when moderately overexpressed. Rab3D was without effect on Glut4-myc subcellular distribution in basal or insulin-stimulated conditions. While two mutated proteins unable to bind GTP did not decrease the number of Glut4-myc molecules in basal or insulin-stimulated conditions at the plasma membrane, the behavior of a mutated Rab4 protein without GTPase activity was similar to that of the wild-type Rab4 protein, indicating that GTP binding but not its hydrolysis was required for the observed effects. Altogether, our results suggest that Rab4, but not Rab3D, participates in the molecular mechanism involved in the subcellular distribution of the Glut4 molecules both in basal and in insulin-stimulated conditions in adipocytes.

scholarly journals GLUT4 Recycles via a trans-Golgi Network (TGN) Subdomain Enriched in Syntaxins 6 and 16 But Not TGN38: Involvement of an Acidic Targeting Motif

2003 ◽  
Vol 14 (3) ◽  
pp. 973-986 ◽  
Author(s):  
Annette M. Shewan ◽  
Ellen M. van Dam ◽  
Sally Martin ◽  
Tang Bor Luen ◽  
Wanjin Hong ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Adipocyte Differentiation ◽  
Attachment Protein ◽  
Trans Golgi Network ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Glucose Transporter Glut4 ◽  
Golgi Network ◽  
Endosomal System

Insulin stimulates glucose transport in fat and muscle cells by triggering exocytosis of the glucose transporter GLUT4. To define the intracellular trafficking of GLUT4, we have studied the internalization of an epitope-tagged version of GLUT4 from the cell surface. GLUT4 rapidly traversed the endosomal system en route to a perinuclear location. This perinuclear GLUT4 compartment did not colocalize with endosomal markers (endosomal antigen 1 protein, transferrin) or TGN38, but showed significant overlap with the TGN target (t)-solubleN-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Syntaxins 6 and 16. These results were confirmed by vesicle immunoisolation. Consistent with a role for Syntaxins 6 and 16 in GLUT4 trafficking we found that their expression was up-regulated significantly during adipocyte differentiation and insulin stimulated their movement to the cell surface. GLUT4 trafficking between endosomes and trans-Golgi network was regulated via an acidic targeting motif in the carboxy terminus of GLUT4, because a mutant lacking this motif was retained in endosomes. We conclude that GLUT4 is rapidly transported from the cell surface to a subdomain of thetrans-Golgi network that is enriched in the t-SNAREs Syntaxins 6 and 16 and that an acidic targeting motif in the C-terminal tail of GLUT4 plays an important role in this process.

scholarly journals Characterisation of GLUT4 trafficking in HeLa cells: comparable kinetics and orthologous trafficking mechanisms to 3T3-L1 adipocytes

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8751 ◽  
Author(s):  
Silke Morris ◽  
Niall D. Geoghegan ◽  
Jessica B.A. Sadler ◽  
Anna M. Koester ◽  
Hannah L. Black ◽  
...  
Keyword(s):  
Cell Surface ◽  
Hela Cells ◽  
Glucose Transporter ◽  
Characteristic Property ◽  
Cell Types ◽  
Model System ◽  
Human Model ◽  
Small Scale ◽  
Glucose Transporter Glut4 ◽  
Surface Fusion

Insulin-stimulated glucose transport is a characteristic property of adipocytes and muscle cells and involves the regulated delivery of glucose transporter (GLUT4)-containing vesicles from intracellular stores to the cell surface. Fusion of these vesicles results in increased numbers of GLUT4 molecules at the cell surface. In an attempt to overcome some of the limitations associated with both primary and cultured adipocytes, we expressed an epitope- and GFP-tagged version of GLUT4 (HA–GLUT4–GFP) in HeLa cells. Here we report the characterisation of this system compared to 3T3-L1 adipocytes. We show that insulin promotes translocation of HA–GLUT4–GFP to the surface of both cell types with similar kinetics using orthologous trafficking machinery. While the magnitude of the insulin-stimulated translocation of GLUT4 is smaller than mouse 3T3-L1 adipocytes, HeLa cells offer a useful, experimentally tractable, human model system. Here, we exemplify their utility through a small-scale siRNA screen to identify GOSR1 and YKT6 as potential novel regulators of GLUT4 trafficking in human cells.

scholarly journals Adipsin and the Glucose Transporter GLUT4 Traffic to the Cell Surface via Independent Pathways in Adipocytes

Traffic ◽  
2000 ◽  
Vol 1 (2) ◽  
pp. 141-151 ◽  
Author(s):  
Caroline A. Millar ◽  
Timo Meerloo ◽  
Sally Martin ◽  
Gilles R.X. Hickson ◽  
Neil J. Shimwell ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Glucose Transporter Glut4

scholarly journals Sorting of GLUT4 into its insulin-sensitive store requires the Sec1/Munc18 protein mVps45

2013 ◽  
Vol 24 (15) ◽  
pp. 2389-2397 ◽  
Author(s):  
Jennifer Roccisana ◽  
Jessica B. A. Sadler ◽  
Nia J. Bryant ◽  
Gwyn W. Gould
Keyword(s):  
Glucose Transport ◽  
Glucose Transporter ◽  
Vitro Assay ◽  
Protein Receptor ◽  
Storage Vesicles ◽  
Storage Vesicle ◽  
Impaired Insulin ◽  
Endosomal Pathway

Insulin stimulates glucose transport in fat and muscle cells by regulating delivery of the facilitative glucose transporter, glucose transporter isoform 4 (GLUT4), to the plasma membrane. In the absence of insulin, GLUT4 is sequestered away from the general recycling endosomal pathway into specialized vesicles, referred to as GLUT4-storage vesicles. Understanding the sorting of GLUT4 into this store is a major challenge. Here we examine the role of the Sec1/Munc18 protein mVps45 in GLUT4 trafficking. We show that mVps45 is up-regulated upon differentiation of 3T3-L1 fibroblasts into adipocytes and is expressed at stoichiometric levels with its cognate target–soluble N-ethylmaleimide–sensitive factor attachment protein receptor, syntaxin 16. Depletion of mVps45 in 3T3-L1 adipocytes results in decreased GLUT4 levels and impaired insulin-stimulated glucose transport. Using sub­cellular fractionation and an in vitro assay for GLUT4-storage vesicle formation, we show that mVps45 is required to correctly traffic GLUT4 into this compartment. Collectively our data reveal a crucial role for mVps45 in the delivery of GLUT4 into its specialized, insulin-regulated compartment.

scholarly journals Roles of the N- and C-termini of GLUT4 in endocytosis

2002 ◽  
Vol 115 (1) ◽  
pp. 131-140 ◽  
Author(s):  
Hadi Al-Hasani ◽  
Raghu K. Kunamneni ◽  
Kevin Dawson ◽  
Cynthia S. Hinck ◽  
Dirk Müller-Wieland ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Surface Expression ◽  
Dominant Negative ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Dileucine Motif ◽  
Intracellular Compartments ◽  
Targeting Signal

In insulin target cells, the predominantly expressed glucose transporter isoform GLUT4 recycles between distinct intracellular compartments and the plasma membrane. To characterize putative targeting signals within GLUT4 in a physiologically relevant cell type, we have analyzed the trafficking of hemagglutinin (HA)-epitope-tagged GLUT4 mutants in transiently transfected primary rat adipose cells. Mutation of the C-terminal dileucine motif (LL489/90) did not affect the cell-surface expression of HA-GLUT4. However, mutation of the N-terminal phenylalanine-based targeting sequence (F5) resulted in substantial increases, whereas deletion of 37 or 28 of the 44 C-terminal residues led to substantial decreases in cell-surface HA-GLUT4 in both the basal and insulin-stimulated states. Studies with wortmannin and coexpression of a dominant-negative dynamin GTPase mutant indicate that these effects appear to be primarily due to decreases and increases, respectively, in the rate of endocytosis. Yeast two-hybrid analyses revealed that the N-terminal phenylalanine-based targeting signal in GLUT4 constitutes a binding site for medium chain adaptins μ1, μ2, and μ3A, implicating a role of this motif in the targeting of GLUT4 to clathrin-coated vesicles.

scholarly journals Dual role of ERK5 in the regulation of T cell receptor expression at the T cell surface

2015 ◽  
Vol 99 (1) ◽  
pp. 143-152 ◽  
Author(s):  
Xavier Rovira-Clavé ◽  
Maria Angulo-Ibáñez ◽  
Cathy Tournier ◽  
Manuel Reina ◽  
Enric Espel
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Dual Role ◽  
Receptor Expression

scholarly journals The exocyst complex regulates insulin-stimulated glucose uptake of skeletal muscle cells

2019 ◽  
Vol 317 (6) ◽  
pp. E957-E972
Author(s):  
Brent A. Fujimoto ◽  
Madison Young ◽  
Lamar Carter ◽  
Alina P. S. Pang ◽  
Michael J. Corley ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Cell Surface Receptor ◽  
Exocyst Complex

Skeletal muscle handles ~80–90% of the insulin-induced glucose uptake. In skeletal muscle, insulin binding to its cell surface receptor triggers redistribution of intracellular glucose transporter GLUT4 protein to the cell surface, enabling facilitated glucose uptake. In adipocytes, the eight-protein exocyst complex is an indispensable constituent in insulin-induced glucose uptake, as it is responsible for the targeted trafficking and plasma membrane-delivery of GLUT4. However, the role of the exocyst in skeletal muscle glucose uptake has never been investigated. Here we demonstrate that the exocyst is a necessary factor in insulin-induced glucose uptake in skeletal muscle cells as well. The exocyst complex colocalizes with GLUT4 storage vesicles in L6-GLUT4myc myoblasts at a basal state and associates with these vesicles during their translocation to the plasma membrane after insulin signaling. Moreover, we show that the exocyst inhibitor endosidin-2 and a heterozygous knockout of Exoc5 in skeletal myoblast cells both lead to impaired GLUT4 trafficking to the plasma membrane and hinder glucose uptake in response to an insulin stimulus. Our research is the first to establish that the exocyst complex regulates insulin-induced GLUT4 exocytosis and glucose metabolism in muscle cells. A deeper knowledge of the role of the exocyst complex in skeletal muscle tissue may help our understanding of insulin resistance in type 2 diabetes.

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