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

1995 ◽  
Vol 130 (5) ◽  
pp. 1071-1079 ◽  
Author(s):  
K J Verhey ◽  
J I Yeh ◽  
M J Birnbaum
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Transporter ◽  
Storage Site ◽  
Dileucine Motif ◽  
Pool Model ◽  
Intracellular Storage ◽  
Intracellular Sequestration ◽  
Hormone Responsiveness

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 NH2 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 intracellular 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.

scholarly journals The efficient intracellular sequestration of the insulin-regulatable glucose transporter (GLUT-4) is conferred by the NH2 terminus

1992 ◽  
Vol 117 (4) ◽  
pp. 729-743 ◽  
Author(s):  
RC Piper ◽  
C Tai ◽  
JW Slot ◽  
CS Hahn ◽  
CM Rice ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Transport ◽  
Cho Cells ◽  
Sindbis Virus ◽  
Glucose Transporter ◽  
Intracellular Compartment ◽  
Glut 1 ◽  
Glut 4 ◽  
Intracellular Sequestration

GLUT-4 is the major facilitative glucose transporter isoform in tissues that exhibit insulin-stimulated glucose transport. Insulin regulates glucose transport by the rapid translocation of GLUT-4 from an intracellular compartment to the plasma membrane. A critical feature of this process is the efficient exclusion of GLUT-4 from the plasma membrane in the absence of insulin. To identify the amino acid domains of GLUT-4 which confer intracellular sequestration, we analyzed the subcellular distribution of chimeric glucose transporters comprised of GLUT-4 and a homologous isoform, GLUT-1, which is found predominantly at the cell surface. These chimeric transporters were transiently expressed in CHO cells using a double subgenomic recombinant Sindbis virus vector. We have found that wild-type GLUT-4 is targeted to an intracellular compartment in CHO cells which is morphologically similar to that observed in adipocytes and muscle cells. Sindbis virus-produced GLUT-1 was predominantly expressed at the cell surface. Substitution of the GLUT-4 amino-terminal region with that of GLUT-1 abolished the efficient intracellular sequestration of GLUT-4. Conversely, substitution of the NH2 terminus of GLUT-1 with that of GLUT-4 resulted in marked intracellular sequestration of GLUT-1. These data indicate that the NH2-terminus of GLUT-4 is both necessary and sufficient for intracellular sequestration.

scholarly journals GLUT-4 NH2 terminus contains a phenylalanine-based targeting motif that regulates intracellular sequestration.

1993 ◽  
Vol 121 (6) ◽  
pp. 1221-1232 ◽  
Author(s):  
R C Piper ◽  
C Tai ◽  
P Kulesza ◽  
S Pang ◽  
D Warnock ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Cho Cells ◽  
Sindbis Virus ◽  
Glucose Transporter ◽  
Expression System ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Glut 4 ◽  
Intracellular Sequestration

Expression of chimeras, composed of portions of two different glucose transporter isoforms (GLUT-1 and GLUT-4), in CHO cells had indicated that the cytoplasmic NH2 terminus of GLUT-4 contains important targeting information that mediates intracellular sequestration of this isoform (Piper, R. C., C. Tai, J. W. Slot, C. S. Hahn, C. M. Rice, H. Huang, D. E. James. 1992. J. Cell Biol. 117:729-743). In the present studies, the amino acid constituents of the GLUT-4 NH2-terminal targeting domain have been identified. GLUT-4 constructs containing NH2-terminal deletions or alanine substitutions within the NH2 terminus were expressed in CHO cells using a Sindbis virus expression system. Deletion of eight amino acids from the GLUT-4 NH2 terminus or substituting alanine for phenylalanine at position 5 in GLUT-4 resulted in a marked accumulation of the transporter at the plasma membrane. Mutations at other amino acids surrounding Phe5 also caused increased cell surface expression of GLUT-4 but not to the same extent as the Phe5 mutation. GLUT-4 was also localized to clathrin lattices and this colocalization was abolished when either the first 13 amino acids were deleted or when Phe5 was changed to alanine. To ascertain whether the targeting information within the GLUT-4 NH2-terminal targeting domain could function independently of the glucose transporter structure this domain was inserted into the cytoplasmic tail of the H1 subunit of the asialoglycoprotein receptor. H1 with the GLUT-4 NH2 terminus was predominantly localized to an intracellular compartment similar to GLUT-4 and was sequestered more from the cell surface than was the wild-type H1 protein. It is concluded that the NH2 terminus of GLUT-4 contains a phenylalanine-based targeting motif that mediates intracellular sequestration at least in part by facilitating interaction of the transporter with endocytic machinery located at the cell surface.

scholarly journals Increased intracellular sequestration of the insulin-regulated aminopeptidase upon differentiation of 3T3-L1 cells

1998 ◽  
Vol 330 (2) ◽  
pp. 1003-1008 ◽  
Author(s):  
A. Stuart ROSS ◽  
R. Susanna KELLER ◽  
E. Gustav LIENHARD
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Transporter ◽  
Fold Increase ◽  
Membrane Receptors ◽  
Cell Surface Biotinylation ◽  
A Cell ◽  
Plasma Membrane Receptors ◽  
Glucose Transporter Glut4 ◽  
Intracellular Sequestration

In fat and muscle cells, the glucose transporter GLUT4 is sequestered in an intracellular compartment under basal conditions and redistributes markedly to the plasma membrane in response to insulin. Recently, we characterized a membrane aminopeptidase, designated IRAP (insulin-regulated aminopeptidase), that colocalizes with intracellular GLUT4 and similarly redistributes markedly to the plasma membrane in response to insulin in adipocytes. In contrast to GLUT4, IRAP is also expressed in 3T3-L1 fibroblasts, and this finding provided an opportunity to compare its subcellular distribution in fibroblasts and adipocytes. The relative amount of IRAP at the cell surface was measured by a cell surface biotinylation method. The portion of total IRAP at the cell surface in unstimulated adipocytes was 30% of that in unstimulated fibroblasts. Upon insulin treatment the portion of IRAP at the cell surface was the same in fibroblasts and adipocytes, and was increased 1.8-fold in fibroblasts and 8-fold in adipocytes. A similar analysis of the distribution of the transferrin receptor (TfR), the paradigm for recycling plasma membrane receptors, revealed that the portions of the TfR at the cell surface in both the basal and insulin-treated states were almost unchanged upon differentiation, and that insulin caused an increase of about 1.6-fold in the amount of TfR at the cell surface. These results show that enhanced intracellular sequestration of IRAP occurs during adipogenesis, and that this effect underlies the larger insulin-elicited fold increase of IRAP at the cell surface in adipocytes.

scholarly journals Molecular regulation of GLUT-4 targeting in 3T3-L1 adipocytes.

1995 ◽  
Vol 130 (5) ◽  
pp. 1081-1091 ◽  
Author(s):  
B J Marsh ◽  
R A Alm ◽  
S R McIntosh ◽  
D E James
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Subcellular Fractionation ◽  
Molecular Regulation ◽  
Surface Distribution ◽  
Amino Terminal ◽  
Glut 4 ◽  
Dileucine Motif ◽  
A Cell ◽  
Insulin Dependent

Insulin stimulates glucose transport in muscle and adipose tissue by triggering the movement of the glucose transporter GLUT-4 from an intracellular compartment to the cell surface. Fundamental to this process is the intracellular sequestration of GLUT-4 in nonstimulated cells. Two distinct targeting motifs in the amino and carboxy termini of GLUT-4 have been previously identified by expressing chimeras comprised of portions of GLUT-4 and GLUT-1, a transporter isoform that is constitutively targeted to the cell surface, in heterologous cells. These motifs-FQQI in the NH2 terminus and LL in the COOH terminus-resemble endocytic signals that have been described in other proteins. In the present study we have investigated the roles of these motifs in GLUT-4 targeting in insulin-sensitive cells. Epitope-tagged GLUT-4 constructs engineered to differentiate between endogenous and transfected GLUT-4 were stably expressed in 3T3-L1 adipocytes. Targeting was assessed in cells incubated in the presence or absence of insulin by subcellular fractionation. The targeting of epitope-tagged GLUT-4 was indistinguishable from endogenous GLUT-4. Mutation of the FQQI motif (F5 to A5) caused GLUT-4 to constitutively accumulate at the cell surface regardless of expression level. Mutation of the dileucine motif (L489L490 to A489A490) caused an increase in cell surface distribution only at higher levels of expression, but the overall cells surface distribution of this mutant was less than that of the amino-terminal mutants. Both NH2- and COOH-terminal mutants retained insulin-dependent movement from an intracellular to a cell surface locale, suggesting that neither of these motifs is involved in the insulin-dependent redistribution of GLUT-4. We conclude that the phenylalanine-based NH2-terminal and the dileucine-based COOH-terminal motifs play important and distinct roles in GLUT-4 targeting in 3T3-L1 adipocytes.

Transmembrane domain length determines intracellular membrane compartment localization of syntaxins 3, 4, and 5

2001 ◽  
Vol 281 (1) ◽  
pp. C215-C223 ◽  
Author(s):  
Robert T. Watson ◽  
Jeffrey E. Pessin
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Glucose Transporter ◽  
Transmembrane Domain ◽  
Transmembrane Domains ◽  
Snare Protein ◽  
Glut 4 ◽  
Golgi Localization ◽  
Syntaxin 3

Insulin recruits glucose transporter 4 (GLUT-4) vesicles from intracellular stores to the plasma membrane in muscle and adipose tissue by specific interactions between the vesicle membrane-soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) protein VAMP-2 and the target membrane SNARE protein syntaxin 4. Although GLUT-4 vesicle trafficking has been intensely studied, few have focused on the mechanism by which the SNAREs themselves localize to specific membrane compartments. We therefore set out to identify the molecular determinants for localizing several syntaxin isoforms, including syntaxins 3, 4, and 5, to their respective intracellular compartments (plasma membrane for syntaxins 3 and 4; cis-Golgi for syntaxin 5). Analysis of a series of deletion and chimeric syntaxin constructs revealed that the 17-amino acid transmembrane domain of syntaxin 5 was sufficient to direct the cis-Golgi localization of several heterologous reporter constructs. In contrast, the longer 25-amino acid transmembrane domain of syntaxin 3 was sufficient to localize reporter constructs to the plasma membrane. Furthermore, truncation of the syntaxin 3 transmembrane domain to 17 amino acids resulted in a complete conversion to cis-Golgi compartmentalization that was indistinguishable from syntaxin 5. These data support a model wherein short transmembrane domains (≤17 amino acids) direct the cis-Golgi localization of syntaxins, whereas long transmembrane domains (≥23 amino acids) direct plasma membrane localization.

scholarly journals The Glucose Transporter 4 FQQI Motif Is Necessary for Akt Substrate of 160-Kilodalton-Dependent Plasma Membrane Translocation But Not Golgi-Localized γ-Ear-Containing Arf-Binding Protein-Dependent Entry into the Insulin-Responsive Storage Compartment

2007 ◽  
Vol 21 (12) ◽  
pp. 3087-3099 ◽  
Author(s):  
Encarnación Capilla ◽  
Naoko Suzuki ◽  
Jeffrey E. Pessin ◽  
June Chunqiu Hou
Keyword(s):  
Plasma Membrane ◽  
Point Mutation ◽  
Glucose Transporter ◽  
Binding Protein ◽  
Amino Terminus ◽  
Glucose Transporter 4 ◽  
Wild Type ◽  
Intracellular Loop ◽  
Storage Compartment ◽  
Intracellular Sequestration

Abstract Newly synthesized glucose transporter 4 (GLUT4) enters into the insulin-responsive storage compartment in a process that is Golgi-localized γ-ear-containing Arf-binding protein (GGA) dependent, whereas insulin-stimulated translocation is regulated by Akt substrate of 160 kDa (AS160). In the present study, using a variety of GLUT4/GLUT1 chimeras, we have analyzed the specific motifs of GLUT4 that are important for GGA and AS160 regulation of GLUT4 trafficking. Substitution of the amino terminus and the large intracellular loop of GLUT4 into GLUT1 (chimera 1-441) fully recapitulated the basal state retention, insulin-stimulated translocation, and GGA and AS160 sensitivity of wild-type GLUT4 (GLUT4-WT). GLUT4 point mutation (GLUT4-F5A) resulted in loss of GLUT4 intracellular retention in the basal state when coexpressed with both wild-type GGA and AS160. Nevertheless, similar to GLUT4-WT, the insulin-stimulated plasma membrane localization of GLUT4-F5A was significantly inhibited by coexpression of dominant-interfering GGA. In addition, coexpression with a dominant-interfering AS160 (AS160-4P) abolished insulin-stimulated GLUT4-WT but not GLUT4-F5A translocation. GLUT4 endocytosis and intracellular sequestration also required both the amino terminus and large cytoplasmic loop of GLUT4. Furthermore, both the FQQI and the SLL motifs participate in the initial endocytosis from the plasma membrane; however, once internalized, unlike the FQQI motif, the SLL motif is not responsible for intracellular recycling of GLUT4 back to the specialized compartment. Together, we have demonstrated that the FQQI motif within the amino terminus of GLUT4 is essential for GLUT4 endocytosis and AS160-dependent intracellular retention but not for the GGA-dependent sorting of GLUT4 into the insulin-responsive storage compartment.

scholarly journals Fate of plasma membrane during endocytosis. II. Evidence for recycling (shuttle) of plasma membrane constituents

1979 ◽  
Vol 82 (2) ◽  
pp. 466-474 ◽  
Author(s):  
Y Schneider ◽  
P Tulkens ◽  
C de Duve ◽  
A Trouet
Keyword(s):  
Plasma Membrane ◽  
Horseradish Peroxidase ◽  
Immune Complexes ◽  
Alternative Explanation ◽  
Storage Site ◽  
Substantial Portion ◽  
Rat Embryo ◽  
Soluble Immune Complexes ◽  
Intracellular Storage ◽  
Coated Membrane

Cultured rat embryo fibroblasts were first allowed to store for 24 h fluorescein-labeled goat immunoglobulins directed against rabbit immunoglobulins (F anti-R IgG), and were subsequently exposed for 24 h to [(3)H]acetylated rabbit immunoglobulins known to bind to the cell membrane either specifically (anti-plasma membrane IgG: A anti-PM IgG) or unspecifically (contol IgG: AC IgG). As a result of immunological interaction between the two antibodies (no effect was found if the cells had been preloaded with control goat FC IgG), a substantial portion of the stored F anti-R IgG was unloaded from its intracellular storage site, appearing in the medium in the form of soluble immune complexes with rabbit A IgG. Part of the unloaded F anti-R IgG also was recovered in association with the plasma membrane, but only when A anti-PM IgG was used. In addition, significant reverse translocation of AC IgG from plasma membrane to lysosomes or some related intracellular storage compartment was also observed. With A anti-PM IgG, this translocation was less marked and affecte at the same time the plasma membrane marker 5'- nucleotidase. Cells that had stored horseradish peroxidase (HRP) simultaneously with F anti-R IgG did not unload HRP when exposed to A anti-PM IgG. These results support strongly, though not unequivocally, the concept that plasma membrane patches interiorized by endocytosis are recycled, or shuttled, back to the cell surface. In the framework of this concept, recycling antibody-coated membrane is taken to serve as vehicle for the selective intracellular capture and extracellular discharge of immunologically bound F anti-R IgG. The alternative explanation of regurgitation triggered off by immune complexes is considered less likely in view of the lack of HRP unloading.

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 Truncations of the C-terminal cytoplasmic domain of MG160, a medial Golgi sialoglycoprotein, result in its partial transport to the plasma membrane and filopodia

1998 ◽  
Vol 111 (2) ◽  
pp. 249-260 ◽  
Author(s):  
J.O. Gonatas ◽  
Y.J. Chen ◽  
A. Stieber ◽  
Z. Mourelatos ◽  
N.K. Gonatas
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Growth Factor Receptor ◽  
Cytoplasmic Domain ◽  
Type I ◽  
Ovary Cells

MG160, a type I cysteine-rich membrane sialoglycoprotein residing in the medial cisternae of the rat Golgi apparatus, is highly homologous to CFR, a fibroblast growth factor receptor, and ESL-1, an E-selectin ligand located at the cell surface of mouse myeloid cells and recently detected in the Golgi apparatus as well. The mechanism for the transport of MG160 from the Golgi apparatus to the cell surface is unknown. In this study we found that differential processing of the carboxy-terminal cytoplasmic domain (CD), consisting of amino acids Arg1159 Ile Thr Lys Arg Val Thr Arg Glu Leu Lys Asp Arg1171, resulted in the partial transport of the protein to the plasma membrane and filopodia. In Chinese hamster ovary cells (CHO), stably transfected with the entire cDNA encoding MG160, the protein was localized in the Golgi apparatus. However, when the terminal Arg1171 or up to nine distal amino acids were deleted, the protein was distributed to the plasma membrane and filopodia as well as the Golgi apparatus. This report shows that the CD of an endogenous type I Golgi protein is important for its efficient retention and identifies a unique residue preference in this process. Cleavage within the CD of MG160 may constitute a regulatory mechanism for the partial export of the protein from the Golgi apparatus to the plasma membrane and filopodia.

scholarly journals Effects of noradrenaline on the cell-surface glucose transporters in cultured brown adipocytes: novel mechanism for selective activation of GLUT1 glucose transporters

1998 ◽  
Vol 330 (1) ◽  
pp. 397-403 ◽  
Author(s):  
Yasutake SHIMIZU ◽  
Shinobu SATOH ◽  
Hajime YANO ◽  
Yasuhiko MINOKOSHI ◽  
W. Samuel CUSHMAN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Glucose Transporters ◽  
Brown Adipocytes ◽  
Photoaffinity Labelling ◽  
Glucose Binding ◽  
Glut1 Protein ◽  
Dependent Mechanism

Glucose transport into rat brown adipocytes has been shown to be stimulated directly by the sympathetic neurotransmitter, noradrenaline, without a significant increase in the protein content of either GLUT1 or GLUT4 glucose transporter in the plasma membrane [Shimizu, Kielar, Minokoshi and Shimazu (1996) Biochem. J.314, 485-490]. In the present study, we labelled the exofacial glucose-binding sites of GLUT1 and GLUT4 with a membrane-impermeant photoaffinity reagent, 2-N-[4-(1-azitrifluoroethyl)benzoyl]-[2-3H]1,3-bis-(D-mannos-4-yloxy)-2-propylamine (ATB-[3H]BMPA), to determine which isoform is responsible for the noradrenaline-induced increase in glucose transport into intact brown adipocytes in culture. Insulin stimulated the rate of hexose transport by increasing ATB-[3H]BMPA-labelled cell-surface GLUT4. In contrast, the noradrenaline-induced increase in glucose transport was not accompanied by an increased ATB-[3H]BMPA labelling of GLUT4, nor with an increased amount of GLUT4 in the plasma membrane fraction as assessed by Western blotting, indicating that noradrenaline does not promote the translocation of GLUT4. However, noradrenaline induced an increase in photoaffinity labelling of cell-surface GLUT1 without an apparent increase in the immunoreactive GLUT1 protein in the plasma membrane. This is suggestive of an increased affinity of GLUT1 for the ligand. In fact, the Ki value of non-radioactive ATB-BMPA for 2-deoxy-d-glucose uptake was significantly decreased after treatment of the cells with noradrenaline. The increased photoaffinity labelling of GLUT1 and increased glucose transport caused by noradrenaline were inhibited by a cAMP antagonist, cAMP-S Rp-isomer. These results demonstrate that noradrenaline stimulates glucose transport in brown adipocytes by enhancing the functional activity of GLUT1 through a cAMP-dependent mechanism.

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