Exofacial epitope-tagged glucose transporter chimeras reveal COOH-terminal sequences governing cellular localization.

Czech, Michael P.; Chawla, Anil; Woon, Chee-Wai; Buxton, Joanne M.; Armoni, Michal; Tang, Wei; Joly, Marguerite; Corvera, Silvia

doi:10.1083/jcb.123.1.127

Cited by 78 publications

(69 citation statements)

References 32 publications

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“…This observation extends, therefore, recent results showing the presence of Daxx in the cytoplasm (2, 14 -16). The punctate staining of the cytoplasm of 3T3-L1 fibroblasts and 3T3-L1 adipocytes incubated with antibodies against Daxx agrees with the localization of Daxx in LDM, the cellular fraction that enriched in endosomes contains a sizable part of GLUT4 (30). The localization of Daxx to endosomes and the identification of endosomes carrying ligand-receptor complexes as the sites where signal transduction is often initiated (31) suggests that the interaction of Daxx with proteins such as Fas receptor T␤RII could also occur at endosomes.…”

Section: Discussionmentioning

confidence: 63%

The Insulin-sensitive Glucose Transporter, GLUT4, Interacts Physically with Daxx

Lalioti

Vergarajaúregui²,

Pulido

et al. 2002

Journal of Biological Chemistry

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In this study we have used the yeast two-hybrid system to identify proteins that interact with the carboxylcytoplasmic domain (residues 464 -509) of the insulinsensitive glucose transporter GLUT4 (C-GLUT4). Using as bait C-GLUT4, we have isolated the carboxyl domain of Daxx (C-Daxx), the adaptor protein associated with the Fas and the type II TGF-␤ (T␤RII) receptors (1, 2). The two-hybrid interaction between C-GLUT4 and CDaxx is validated by the ability of in vitro translated C-GLUT4 to interact with in vitro translated full-length Daxx and C-Daxx. C-Daxx does not interact with the C-cytoplasmic domain of GLUT1, the ubiquitous glucose transporter homologous to GLUT4. Replacement of alanine and serine for the dileucine pair (Leu 489 -Leu 490 ) critical for targeting GLUT4 from the trans-Golgi network to the perinuclear intracellular store as well as for its surface internalization by endocytosis inhibits 2-fold the interaction of C-GLUT4 with Daxx. Daxx is pulled down with GLUT4 immunoprecipitated from lysates of 3T3-L1 fibroblasts stably transfected with GLUT4 and 3T3-L1 adipocytes expressing physiological levels of the two proteins. Similarly, GLUT4 is recovered with antiDaxx immunoprecipitates. Using an established cell fractionation procedure we present evidence for the existence of two distinct intracellular Daxx pools in the nucleus and low density microsomes. Confocal immunofluorescence microscopy studies localize Daxx to promyelocytic leukemia nuclear bodies and punctate cytoplasmic structures, often organized in strings and underneath the plasma membrane. Daxx and GLUT4 are SUMOlated as shown by their reaction with an anti-SUMO1 antibody and by the ability of this antibody to pull down Daxx and GLUT4.The cytoplasmic domain of membrane proteins plays important roles in their transport, signal transduction, organization of protein scaffolds, and regulation of their turnover. Trafficking of GLUT4 in adipose and skeletal muscle cells is regulated by insulin and muscle contraction and is critical for the control of glucose levels in blood. Upon increase in insulin levels and muscle contraction the GLUT4 retained in intracellular stores is translocated to the plasma membrane, where it facilitates glucose transport (3). Trafficking of GLUT4 is mediated by motifs localized to the amino and carboxyl-cytoplasmic domains of the protein, though their characterization and the identification of the factors involved in their reading is incomplete.SUMO (also called sentrin, PIC1, and GMP1), a 101-amino acid ubiquitin-like modifier protein that is highly conserved from yeast to human, appears to control protein turnover and compartmentalization (4). Three members of the SUMO family have been described in vertebrates. They show major structural differences in the sequences of their N-extensions, which are absent in ubiquitin. It has been shown recently that Ubc9, the only E2-type SUMO1-conjugating enzyme described in vertebrates, interacts with the carboxyl-cytoplasmic domain of GLUT4 as part of a mechanism that slows its...

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Section: Discussionmentioning

confidence: 63%

The Insulin-sensitive Glucose Transporter, GLUT4, Interacts Physically with Daxx

Lalioti

Vergarajaúregui²,

Pulido

et al. 2002

Journal of Biological Chemistry

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“…The chimeric transporter 4HB1, which contains the NH2-terminal 183 amino acids of GLUT4, also showed some intracellular localization in NIH3T3 and 3T3-L1 fibroblasts (43; and Fig. 3), though other investigators have found this chimeric transporter located primarily at the cell surface in COS cells and Xenopus oocytes (11,27). In 3T3-L1 adipocytes, the chimeric transporter 4HB1 was detected primarily in intracellular compartments in the basal state, and only slightly redistributed to the cell surface in response to insulin (Figs.…”

Section: Discussionmentioning

confidence: 99%

“…Experimental strategies based on the construction of chimeric transporter proteins have led to identification of several structural domains implicated as critical to the intracellular sequestration of GLUT4 (11,27,29,30,43). However, a major limitation of the previous studies has been that none of the cells into which the transporters have been introduced represents a physiologically meaningful model system as defined by two criteria: expression of significant quantities of the endogenous GLUT4 glucose transporter and an insulin-stimulatable augmentation in hexose uptake comparable to the in vivo target tissue.…”

Section: Discussionmentioning

confidence: 99%

“…Piper et al have found the NH 2 terminus of GLUT4 necessary for the intracellular sequestration of this isoform in Chinese hamster ovary cells, and a Phe critical, presumably by affecting the internalization of GLUT4 from the cell surface (29,30). However, several laboratories have concluded that the primary targeting information resides in the COOHterminal 30 amino acids of the transporters (11,27,43). In particular, a dileucine motif within this region is necessary for the intracellular sequestration of GLUT4 in fibroblasts (10,42).…”

Section: Lucose Uptake Into Mammalian Cells Is Accom-mentioning

confidence: 99%

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Distinct signals in the GLUT4 glucose transporter for internalization and for targeting to an insulin-responsive compartment.

Verhey

Yeh

Birnbaum

1995

The Journal of Cell Biology

108

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Abstract. In adipose and muscle cells, insulin stimulates a rapid and dramatic increase in glucose uptake, primarily by promoting the redistribution of the GLUT4 glucose transporter from its intracellular storage site to the plasma membrane. In contrast, the more ubiquitously expressed isoform GLUT1 is localized at the cell surface in the basal state, and shows a less dramatic translocation in response to insulin. To identify sequences involved in the differential subcellular localization and hormone-responsiveness of these isoforms, chimeric GLUT1/GLUT4 transporters were stably expressed in mouse 3T3-L1 adipocytes. The NH 2 terminus of GLUT4 contains sequences capable of sequestering the transporter inside the cell, although not in an insulin-sensitive pool. In contrast, the COOH-terminal 30 amino acids of GLUT4 are sufficient for its correct localization to an intraceUular storage pool which translocates to the cell surface in response to insulin. The dileucine motif within this domain, which is required for intracellular sequestration of chimeric transporters in fibroblasts, is not critical for targeting to the hormone-responsive compartment in adipocytes. Analysis of rates of internalization of chimeric transporter after the removal of insulin from cells, as well as the subcellular distribution of transporters in cells unexposed to or treated with insulin, leads to a three-pool model which can account for the data. LUCOSE uptake into mammalian cells is accom-plished by a family of proteins, the facilitative glucose transporters, which differ in their tissue distribution and physiological roles. Two of the isoforms identified to date, GLUT1 and GLUT4, are expressed in adipose and muscle, those tissues which exhibit a marked increase in glucose uptake in response to insulin (5). The differential localization of these isoforms within the cell may contribute to their distinct functions. GLUT1, which is expressed in virtually all tissues, is distributed to both the plasma membrane and the interior of the cell in the basal state. Insulin causes a two-to fivefold increase in the amount of GLUT1 present at the cell surface, a recruitment similar to other recycling membrane proteins such as the insulin-like growth factor II and transferrin receptors (39). GLUT4, which is expressed exclusively in insulinresponsive cell types, is excluded from the plasma membrane and instead localizes to an intracellular storage pool in the basal state. Insulin stimulates the specific recruitment of these vesicles to the cell surface, increasing by 10-to 40-fold the amount of GLUT4 present at the cell surface, and thereby dramatically increasing the hexose uptake capacity of the cell (8,9,21,24,34,(36)(37)(38)49 The mechanisms responsible for the insulin-regulated movement of GLUT4 in adipose and muscle may be similar to those utilized by other cell types for regulated secretion. This is supported by the ability of GLUT4 to segregate to large dense core vesicles when expressed in the neuroendocrine cell line PC12 (23). The involvement ...

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“…After washing the coverslips were mounted in 90% glycerol containing 2.5% diazabicyclo[2.2.2]octane. To examine Myc-GLUT4-EGFP translocation in adipocytes transfected with plasmid DNAs, cells were washed, fixed, and immunostained with a set of antibodies using procedure described previously (36). Briefly, first the cell surface Myc-GLUT4-GFP was detected with anti-myc monoclonal antibody and rhodamine B-labeled goat anti-mouse IgG.…”

Section: Methodsmentioning

confidence: 99%

A Phosphatidylinositol 3-Kinase-independent Insulin Signaling Pathway to N-WASP/Arp2/3/F-actin Required for GLUT4 Glucose Transporter Recycling

Jiang

Chawla

Bose

et al. 2002

Journal of Biological Chemistry

137

151

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Recruitment of intracellular glucose transporter 4 (GLUT4) to the plasma membrane of fat and muscle cells in response to insulin requires phosphatidylinositol (PI) 3-kinase as well as a proposed PI 3-kinase-independent pathway leading to activation of the small GTPase TC10. Here we show that in cultured adipocytes insulin causes acute cortical localization of the actin-regulatory neural Wiskott-Aldrich syndrome protein (N-WASP) and actinrelated protein-3 (Arp3) as well as cortical F-actin polymerization by a mechanism that is insensitive to the PI 3-kinase inhibitor wortmannin. Expression of the dominant inhibitory N-WASP-⌬WA protein lacking the Arp and actin binding regions attenuates the cortical F-actin rearrangements by insulin in these cells. Remarkably, the N-WASP-⌬WA protein also inhibits insulin action on GLUT4 translocation, indicating dependence of GLUT4 recycling on N-WASP-directed cortical F-actin assembly. TC10 exhibits sequence similarity to Cdc42 and has been reported to bind N-WASP. We show the inhibitory TC10 (T31N) mutant, which abrogates insulin-stimulated GLUT4 translocation and glucose transport, also inhibits both cortical localization of N-WASP and F-actin formation in response to insulin. These findings reveal that N-WASP likely functions downstream of TC10 in a PI 3-kinase-independent insulin signaling pathway to mobilize cortical F-actin, which in turn promotes GLUT4 responsiveness to insulin.Insulin stimulates glucose uptake by skeletal muscle and adipose tissues primarily through regulation of the subcellular distribution of the glucose transporter 4 (GLUT4) 1 (1, 2). In response to insulin, a fraction of GLUT4 present in intracellular membranes is redistributed to the plasma membrane, resulting in an increase of GLUT4 on the cell surface and enhanced glucose uptake by these cells. This effect of insulin is important in maintaining glucose homeostasis in humans, and impaired insulin action can contribute to the pathogenesis of type 2 diabetes (3). The precise mechanism by which insulin directs exocytosis of GLUT4-containing membrane vesicles remains obscure. However, it is established that activation of the insulin receptor tyrosine kinase catalyzes tyrosine phosphorylation of insulin receptor substrate proteins that bind to Srchomology 2 domain-containing molecules, including the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (4, 5). This results in activation of the p110 catalytic subunit of the kinase, which then phosphorylates cellular polyphosphoinositides at the D-3 position, forming signaling molecules such as phosphatidylinositol 3,4,5-trisphosphate. Multiple studies using various pharmacologic inhibitors, overexpression of constitutively active or dominant negative mutants, and microinjection of blocking antibodies have suggested a necessary role of the p85/p110-type PI 3-kinase in insulin-stimulated GLUT4 translocation and glucose transport (1, 2, 6 -8).On the other hand, several lines of evidence suggest the requirement of PI 3-kinase-independent pathway(s) in G...

show abstract

Exofacial epitope-tagged glucose transporter chimeras reveal COOH-terminal sequences governing cellular localization.

Cited by 78 publications

References 32 publications

The Insulin-sensitive Glucose Transporter, GLUT4, Interacts Physically with Daxx

The Insulin-sensitive Glucose Transporter, GLUT4, Interacts Physically with Daxx

Distinct signals in the GLUT4 glucose transporter for internalization and for targeting to an insulin-responsive compartment.

A Phosphatidylinositol 3-Kinase-independent Insulin Signaling Pathway to N-WASP/Arp2/3/F-actin Required for GLUT4 Glucose Transporter Recycling

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