Sequence Determinants of GLUT1 Oligomerization: ANALYSIS BY HOMOLOGY-SCANNING MUTAGENESIS*

The human blood-brain barrier glucose transport protein (GLUT1) forms homodimers and homotetramers in detergent micelles and in cell membranes, where the GLUT1 oligomeric state determines GLUT1 transport behavior. GLUT1 and the neuronal glucose transporter GLUT3 do not form heterocomplexes in human embryonic kidney 293 (HEK293) cells as judged by co-immunoprecipitation assays. Using homology-scanning mutagenesis in which GLUT1 domains are substituted with equivalent GLUT3 domains and vice versa, we show that GLUT1 transmembrane helix 9 (TM9) is necessary for optimal association of GLUT1-GLUT3 chimeras with parental GLUT1 in HEK cells. GLUT1 TMs 2, 5, 8, and 11 also contribute to a less abundant heterocomplex. Cell surface GLUT1 and GLUT3 containing GLUT1 TM9 are 4-fold more catalytically active than GLUT3 and GLUT1 containing GLUT3 TM9. GLUT1 and GLUT3 display allosteric transport behavior. Size exclusion chromatography of detergent solubilized, purified GLUT1 resolves GLUT1/lipid/detergent micelles as 6- and 10-nm Stokes radius particles, which correspond to GLUT1 dimers and tetramers, respectively. Studies with GLUTs expressed in and solubilized from HEK cells show that HEK cell GLUT1 resolves as 6- and 10-nm Stokes radius particles, whereas GLUT3 resolves as a 6-nm particle. Substitution of GLUT3 TM9 with GLUT1 TM9 causes chimeric GLUT3 to resolve as 6- and 10-nm Stokes radius particles. Substitution of GLUT1 TM9 with GLUT3 TM9 causes chimeric GLUT1 to resolve as a mixture of 6- and 4-nm particles. We discuss these findings in the context of determinants of GLUT oligomeric structure and transport function.     

AS160 phosphotyrosine-binding domain constructs inhibit insulin-stimulated GLUT4 vesicle fusion with the plasma membrane

AS160 (TBC1D4) is a known Akt substrate that is phosphorylated downstream of insulin action and that leads to regulated traffic of GLUT4. As GLUT4 vesicle fusion with the plasma membrane is a highly regulated step in GLUT4 traffic, we investigated whether AS160 and 14-3-3 interactions are involved in this process. Fusion was inhibited by a human truncated AS160 variant that encompasses the first N-terminal phosphotyrosine-binding (PTB) domain, by either of the two N-terminal PTB domains, and by a tandem construct of both PTB domains of rat AS160. We also found that in vitro GLUT4 vesicle fusion was strongly inhibited by the 14-3-3-quenching inhibitors R18 and fusicoccin. To investigate the mode of interaction of AS160 and 14-3-3, we examined insulin-dependent increases in the levels of these proteins on GLUT4 vesicles. 14-3-3γ was enriched on insulin-stimulated vesicles, and its binding to AS160 on GLUT4 vesicles was inhibited by the AS160 tandem PTB domain construct. These data suggest a model for PTB domain action on GLUT4 vesicle fusion in which these constructs inhibit insulin-stimulated 14-3-3γ interaction with AS160 rather than AS160 phosphorylation.     

Isoform-selective Inhibition of Facilitative Glucose Transporters: ELUCIDATION OF THE MOLECULAR MECHANISM OF HIV PROTEASE INHIBITOR BINDING*

Pharmacologic HIV protease inhibitors (PIs) and structurally related oligopeptides are known to reversibly bind and inactivate the insulin-responsive facilitative glucose transporter 4 (GLUT4). Several PIs exhibit isoform selectivity with little effect on GLUT1. The ability to target individual GLUT isoforms in an acute and reversible manner provides novel means both to investigate the contribution of individual GLUTs to health and disease and to develop targeted treatment of glucose-dependent diseases. To determine the molecular basis of transport inhibition, a series of chimeric proteins containing transmembrane and cytosolic domains from GLUT1 and GLUT4 and/or point mutations were generated and expressed in HEK293 cells. Structural integrity was confirmed via measurement of N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) labeling of the chimeric proteins in low density microsome fractions isolated from stably transfected 293 cells. Functional integrity was assessed via measurement of zero-trans 2-deoxyglucose (2-DOG) uptake. ATB-BMPA labeling studies and 2-DOG uptake revealed that transmembrane helices 1 and 5 contain amino acid residues that influence inhibitor access to the transporter binding domain. Substitution of Thr-30 and His-160 in GLUT1 to the corresponding positions in GLUT4 is sufficient to completely transform GLUT1 into GLUT4 with respect to indinavir inhibition of 2-DOG uptake and ATB-BMPA binding. These data provide a structural basis for the selectivity of PIs toward GLUT4 over GLUT1 that can be used in ongoing novel drug design.     

Rate and Regulation of Copper Transport by Human Copper Transporter 1 (hCTR1)

Human copper transporter 1 (hCTR1) is a homotrimer of a 190-amino acid monomer having three transmembrane domains believed to form a pore for copper permeation through the plasma membrane. The hCTR1-mediated copper transport mechanism is not well understood, nor has any measurement been made of the rate at which copper ions are transported by hCTR1. In this study, we estimated the rate of copper transport by the hCTR1 trimer in cultured cells using ⁶⁴Cu uptake assays and quantification of plasma membrane hCTR1. For endogenous hCTR1, we estimated a turnover number of about 10 ions/trimer/s. When overexpressed in HEK293 cells, a second transmembrane domain mutant of hCTR1 (H139R) had a 3-fold higher K_m value and a 4-fold higher turnover number than WT. Truncations of the intracellular C-terminal tail and an AAA substitution of the putative metal-binding HCH C-terminal tripeptide (thought to be required for transport) also exhibited elevated transport rates and K_m values when compared with WT hCTR1. Unlike WT hCTR1, H139R and the C-terminal mutants did not undergo regulatory endocytosis in elevated copper. hCTR1 mutants combining methionine substitutions that block transport (M150L,M154L) on the extracellular side of the pore and the high transport H139R or AAA intracellular side mutations exhibited the blocked transport of M150L,M154L, confirming that Cu⁺ first interacts with the methionines during permeation. Our results show that hCTR1 elements on the intracellular side of the hCTR1 pore, including the carboxyl tail, are not essential for permeation, but serve to regulate the rate of copper entry.     

Entry of newly synthesized GLUT4 into the insulin-responsive storage compartment is dependent upon both the amino terminus and the large cytoplasmic loop

We have recently reported that following initial biosynthesis, the GLUT4 protein exits the Golgi apparatus and directly enters the insulin-responsive compartment(s) without transiting the plasma membrane. To investigate the structural motifs involved in these initial sorting events, we have generated a variety of loss-of-function and gain-of-function GLUT4/GLUT1 chimera proteins. Substitution of the GLUT4 carboxyl-terminal domain with GLUT1 had no significant effect on the acquisition of insulin responsiveness. In contrast, substitution of either the GLUT4 amino-terminal domain or the large cytoplasmic loop between transmembrane domains 6 and 7 resulted in the rapid default of GLUT4 to the plasma membrane with blunted insulin response. Consistent with these findings, substitution of the amino-terminal, cytoplasmic loop, or carboxyl-terminal domains individually into GLUT1 backbone did not recapitulate normal GLUT4 trafficking. Similarly, dual substitutions of the GLUT1 amino and carboxyl termini with GLUT4 domains or the combination of the cytoplasmic loop plus the carboxyl terminus failed to display normal GLUT4 trafficking. However, the dual replacement of the amino terminus plus the cytoplasmic loop of GLUT4 in the GLUT1 backbone resulted in a complete restoration of normal GLUT4 trafficking. Alanine-scanning mutagenesis of the GLUT4 amino terminus demonstrated that Phe(5) and Ile(8) within the FQQI motif and, to a lesser extent, Asp(12)/Gly(13) were necessary for the appropriate initial trafficking following biosynthesis. In addition, amino acids 229-271 in the large intracellular loop between transmembrane domains 6 and 7 functionally cooperated with the amino-terminal domain. These data demonstrate that initial trafficking of GLUT4 from the Golgi to the insulin-responsive GLUT4 compartment requires the functional interaction of two distinct domains.     

Domains responsible for the differential targeting of glucose transporter isoforms.

Facilitative glucose transporter isoforms, GLUT1 and GLUT4, have different intracellular distributions despite their very similar structure. In insulin-responsive tissues such as adipose tissues and muscle, GLUT4 protein resides mainly in the intracellular region in a basal condition and is translocated to the plasma membrane upon stimulation of insulin. In contrast, GLUT1 protein was distributed about equally between plasma membranes and low density microsomal membranes in 3T3-L1 adipocytes. Furthermore, GLUT1 and GLUT4 were reported to be differentially targeted to the plasma membrane and intracellular region, respectively, when expressed in Chinese hamster ovary cells and HepG2 cells. To elucidate the differential intracellular targeting mechanisms, several chimeric glucose transporters in which portions of GLUT4 are replaced with corresponding portions of GLUT1 have been stably expressed in Chinese hamster ovary cells. Immunofluorescence and immunoelectron microscopy as well as measurement of glucose transport activity revealed that two domains of GLUT4, which are not the NH2- or COOH-terminal domain, determine its targeting to the intracellular vesicles. The first domain contains the consensus sequence of the leucine zipper structure, suggesting that a dimer-forming structure of the glucose transporter might be required for its proper targeting. The other domain contains 28 amino acids, nine of which are different between GLUT1 and GLUT4. Immunoelectron microscopy revealed that the chimeric transporters containing both of these two domains of GLUT1, only the first domain of GLUT1, and none of the domains, exhibited a different cellular distribution with approximately 65, 30, and 15% of the transporters apparently on the plasma membrane, respectively. The addition of insulin did not alter the apparent cellular distributions of these chimeric transporters. These domains would be specifically recognized by intracellular targeting mechanisms in Chinese hamster ovary cells.     

The extended GLUT-family of sugar/polyol transport facilitators: nomenclature,sequence characteristics,and potential function of its novel members

During the last 2 years, several novel genes that encode glucose transporter-like proteins have been identified and characterized. Because of their sequence similarity with GLUT1, these genes appear to belong to the family of solute carriers 2A ( SLC2A, protein symbol GLUT). Sequence comparisons of all 13 family members allow the definition of characteristic sugar/polyol transporter signatures: (1) the presence of 12 membrane-spanning helices, (2) seven conserved glycine residues in the helices, (3) several basic and acidic residues at the intracellular surface of the proteins, (4) two conserved tryptophan residues, and (5) two conserved tyrosine residues. On the basis of sequence similarities and characteristic elements, the extended GLUTfamily can be divided intothree subfamilies, namely class I (the previously known glucose transporters GLUT1-4), class II (the previously known fructose transporter GLUT5, the GLUT7, GLUT9 and GLUT11), and class III (GLUT6, 8, 10, 12, and the myoinositol transporter HMIT1). Functional characteristics have been reported for some of the novel GLUTs. Like GLUT1-4, they exhibit a tissue/cell-specific expression (GLUT6, leukocytes, brain; GLUT8, testis, blastocysts, brain, muscle, adipocytes; GLUT9, liver, kidney; GLUT10, liver, pancreas; GLUT11, heart, skeletal muscle). GLUT6 and GLUT8 appear to be regulated by sub-cellular redistribution, because they are targeted to intracellular compartments by dileucine motifs in a dynamin dependent manner. Sugar transport has been reported for GLUT6, 8, and 11; HMIT1 has been shown to be a H + /myo-inositol co-transporter. Thus, the members of the extended GLUT family exhibit a surprisingly diverse substrate specificity, and the definition of sequence elements determining this substrate specificity will require a full functional characterization of all members.     

Properties of the human erythrocyte glucose transport protein are determined by cellular context

Human erythrocyte hexose transfer is mediated by the glucose transport protein GLUT1 and is characterized by a complexity that is unexplained by available hypotheses for carrier-mediated sugar transport [Cloherty, E. K., Heard, K. S., and Carruthers, A. (1996) Biochemistry 35, 10411-10421]. The study presented here examines the possibility that the operational properties of GLUT1 are determined by host cell environment. A glucose transport-null strain of Saccharomyces cerevisiae (RE700A) was transfected with the p426 GPD yeast expression vector containing DNA encoding the wild-type human glucose transport protein (GLUT1), mutant GLUT1 (GLUT1(338)(-)(A3)), or carboxy-terminal hemagglutinin-polyHis-tagged GLUT1 (GLUT1-HA-H6). GLUT1 and GLUT1-HA-H6 are expressed at the yeast cell membrane and restore 2-deoxy-d-glucose, 3-O-methylglucose, and d-glucose transport capacity to RE700A. GLUT1-HA-H6 confers GLUT1-specific sugar transport characteristics to transfected RE700A, including inhibition by cytochalasin B and high-affinity transport of the nonmetabolized sugar 3-O-methylglucose. GLUT1(338)(-)(A3), a catalytically inactive GLUT1 mutant, is expressed but fails to restore RE700A sugar uptake capacity or growth on glucose. In contrast to transport in human red cells, K(m(app)) for 2-deoxy-d-glucose uptake equals K(i(app)) for 2-deoxy-d-glucose inhibition of 3-O-methylglucose uptake. Unlike transport in human red cells or transport in human embryonic kidney cells transfected with GLUT1-HA-H6, unidirectional sugar uptake in RE700A-GLUT1-HA-H6 is not inhibited by reductant and is not stimulated by intracellular sugar. Net uptake of subsaturating 3-O-methylglucose by RE700A-GLUT1-HA-H6 is a simple, first-order process. These findings support the hypothesis that red cell sugar transport complexity is host cell-specific.     

Protein kinase WNK1 promotes cell surface expression of glucose transporter GLUT1 by regulating a Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4)-Rab8A complex

One mechanism by which mammalian cells regulate the uptake of glucose is the number of glucose transporter proteins (GLUT) present at the plasma membrane. In insulin-responsive cells types, GLUT4 is released from intracellular stores through inactivation of the Rab GTPase activating protein Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4) (also known as AS160). Here we describe that TBC1D4 forms a protein complex with protein kinase WNK1 in human embryonic kidney (HEK293) cells. We show that WNK1 phosphorylates TBC1D4 in vitro and that the expression levels of WNK1 in these cells regulate surface expression of the constitutive glucose transporter GLUT1. WNK1 was found to increase the binding of TBC1D4 to regulatory 14-3-3 proteins while reducing its interaction with the exocytic small GTPase Rab8A. These effects were dependent on the catalytic activity because expression of a kinase-dead WNK1 mutant had no effect on binding of 14-3-3 and Rab8A, or on surface GLUT1 levels. Together, the data describe a pathway regulating constitutive glucose uptake via GLUT1, the expression level of which is related to several human diseases.     

A Glucose Transporter Can Mediate Ribose Uptake: DEFINITION OF RESIDUES THAT CONFER SUBSTRATE SPECIFICITY IN A SUGAR TRANSPORTER*

Sugars, the major energy source for many organisms, must be transported across biological membranes. Glucose is the most abundant sugar in human plasma and in many other biological systems and has been the primary focus of sugar transporter studies in eukaryotes. We have previously cloned and characterized a family of glucose transporter genes from the protozoan parasite Leishmania. These transporters, called LmGT1, LmGT2, and LmGT3, are homologous to the well characterized glucose transporter (GLUT) family of mammalian glucose transporters. We have demonstrated that LmGT proteins are important for parasite viability. Here we show that one of these transporters, LmGT2, is a more effective carrier of the pentose sugar d-ribose than LmGT3, which has a 6-fold lower relative specificity (V_max/K_m) for ribose. A pair of threonine residues, located in the putative extracellular loops joining transmembrane helices 3 to 4 and 7 to 8, define a filter that limits ribose approaching the exofacial substrate binding pocket in LmGT3. When these threonines are substituted by alanine residues, as found in LmGT2, the LmGT3 permease acquires ribose permease activity that is similar to that of LmGT2. The location of these residues in hydrophilic loops supports recent suggestions that substrate recognition is separated from substrate binding and translocation in this important group of transporters.     

Transmembrane Topology and Oligomeric Structure of the High-affinity Choline Transporter

The high-affinity choline transporter CHT1 mediates choline uptake essential for acetylcholine synthesis in cholinergic nerve terminals. CHT1 belongs to the Na⁺/glucose cotransporter family (SLC5), which is postulated to have a common 13-transmembrane domain core; however, no direct experimental evidence for CHT1 transmembrane topology has yet been reported. We examined the transmembrane topology of human CHT1 using cysteine-scanning analysis. Single cysteine residues were introduced into the putative extra- and intracellular loops and probed for external accessibility for labeling with a membrane-impermeable, sulfhydryl-specific biotinylating reagent in intact cells expressing these mutants. The results provide experimental evidence for a topological model of a 13-transmembrane domain protein with an extracellular amino terminus and an intracellular carboxyl terminus. We also constructed a three-dimensional homology model of CHT1 based on the crystal structure of the bacterial Na⁺/galactose cotransporter, which supports our conclusion of CHT1 transmembrane topology. Furthermore, we examined whether CHT1 exists as a monomer or oligomer. Chemical cross-linking induces the formation of a higher molecular weight form of CHT1 on the cell surface in HEK293 cells. Two different epitope-tagged CHT1 proteins expressed in the same cells can be co-immunoprecipitated. Moreover, co-expression of an inactive mutant I89A with the wild type induces a dominant-negative effect on the overall choline uptake activity. These results indicate that CHT1 forms a homo-oligomer on the cell surface in cultured cells.     

Trafficking of glucose transporters-signals and mechanisms

The uptake of glucose into mammalian cells, catalysed by members of the GLUT family of glucose transporters, is regulated by a variety of hormones, growth factors and other agents. In adipocytes, skeletal muscle and heart the principal regulator is the hormone insulin, which rapidly stimulates glucose uptake by bringing about the translocation of the GLUT4 glucose transporter isoform from an intracellular vesicular compartment to the cell surface. Recent studies have implicated theC-terminal hydrophilic region of this protein as being primarily responsible for its insulin-regulated trafficking. In an attempt to identify the protein machinery involved in this trafficking, we have used glutathione S-transferase fusion proteins bearing hydrophilic domains of various GLUT transporters in affinity purification experiments on detergent-solubilized extracts of 3T3-L1 adipocyte intracellular membranes. TheC-terminal region of GLUT4 was found specifically to bind a number of polypeptides in these extracts, which are therefore candidates for components of the trafficking machinery. Although these proteins did not bind to the corresponding region of the more widely-distributed GLUT1 glucose transporter isoform, regulation of this transporter also appears to be of physiological importance in some cell types. To study such regulation we have used as a model system the interleukin-3 (IL-3)-dependent haemopoietic cell line IC.DP. These cells express a temperature-sensitive mutane of thev-abl tyrosine kinase, whose activation at the permissive temperature permits cell survival in the absence of IL-3 by suppression of apoptosis, although the growth factor is still required for proliferation. Both IL-3 and activation of the kinase were found to stimulate glucose transport by promoting the translocation of GLUT1 to the cell surface. Moreover, inhibition of glucose uptake by addition of transport inhibitors markedly increased the rate of apoptosis, an effect which could be reversed by the provision of alternative energy sources. These observations suggest that the trafficking of GLUT1, regulated by growth factors or oncogenes, may play an important role in the suppression of apoptosis in haemopoietic cells.     

PACSIN3 overexpression increases adipocyte glucose transport through GLUT1

PACSIN family members regulate intracellular vesicle trafficking via their ability to regulate cytoskeletal rearrangement. These processes are known to be involved in trafficking of GLUT1 and GLUT4 in adipocytes. In this study, PACSIN3 was observed to be the only PACSIN isoform that increases in expression during 3T3-L1 adipocyte differentiation. Overexpression of PACSIN3 in 3T3-L1 adipocytes caused an elevation of glucose uptake. Subcellular fractionation revealed that PACSIN3 overexpression elevated GLUT1 plasma membrane localization without effecting GLUT4 distribution. In agreement with this result, examination of GLUT exofacial presentation at the cell surface by photoaffinity labeling revealed significantly increased GLUT1, but not GLUT4, after overexpression of PACSIN3. These results establish a role for PACSIN3 in regulating glucose uptake in adipocytes via its preferential participation in GLUT1 trafficking. They are consistent with the proposal, which is supported by a recent study, that GLUT1, but not GLUT4, is predominantly endocytosed via the coated pit pathway in unstimulated 3T3-L1 adipocytes.     

Kinetic Evidence for Unique Regulation of GLUT4 Trafficking by Insulin and AMP-activated Protein Kinase Activators in L6 Myotubes

In L6 myotubes, redistribution of a hemagglutinin (HA) epitope-tagged GLUT4 (HA-GLUT4) to the cell surface occurs rapidly in response to insulin stimulation and AMP-activated protein kinase (AMPK) activation. We have examined whether these separate signaling pathways have a convergent mechanism that leads to GLUT4 mobilization and to changes in GLUT4 recycling. HA antibody uptake on GLUT4 in the basal steady state reached a final equilibrium level that was only 81% of the insulin-stimulated level. AMPK activators (5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and A-769662) led to a similar level of antibody uptake to that found in insulin-stimulated cells. However, the combined responses to insulin stimulation and AMPK activation led to an antibody uptake level of ∼20% above the insulin level. Increases in antibody uptake due to insulin, but not AICAR or A-769662, treatment were reduced by both wortmannin and Akt inhibitor. The GLUT4 internalization rate constant in the basal steady state was very rapid (0.43 min⁻¹) and was decreased during the steady-state responses to insulin (0.18 min⁻¹), AICAR (0.16 min⁻¹), and A-769662 (0.24 min⁻¹). This study has revealed a nonconvergent mobilization of GLUT4 in response to activation of Akt and AMPK signaling. Furthermore, GLUT4 trafficking in L6 muscle cells is very reliant on regulated endocytosis for control of cell surface GLUT4 levels.     

Acetylation of TUG Protein Promotes the Accumulation of GLUT4 Glucose Transporters in an Insulin-responsive Intracellular Compartment

Insulin causes the exocytic translocation of GLUT4 glucose transporters to stimulate glucose uptake in fat and muscle. Previous results support a model in which TUG traps GLUT4 in intracellular, insulin-responsive vesicles termed GLUT4 storage vesicles (GSVs). Insulin triggers TUG cleavage to release the GSVs; GLUT4 then recycles through endosomes during ongoing insulin exposure. The TUG C terminus binds a GSV anchoring site comprising Golgin-160 and possibly other proteins. Here, we report that the TUG C terminus is acetylated. The TUG C-terminal peptide bound the Golgin-160-associated protein, ACBD3 (acyl-CoA-binding domain-containing 3), and acetylation reduced binding of TUG to ACBD3 but not to Golgin-160. Mutation of the acetylated residues impaired insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes. ACBD3 overexpression enhanced the translocation of GSV cargos, GLUT4 and insulin-regulated aminopeptidase (IRAP), and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. Sirtuin 2 (SIRT2), a NAD⁺-dependent deacetylase, bound TUG and deacetylated the TUG peptide. SIRT2 overexpression reduced TUG acetylation and redistributed GLUT4 and IRAP to the plasma membrane in 3T3-L1 adipocytes. Mutation of the acetylated residues in TUG abrogated these effects. In mice, SIRT2 deletion increased TUG acetylation and proteolytic processing. During glucose tolerance tests, glucose disposal was enhanced in SIRT2 knock-out mice, compared with wild type controls, without any effect on insulin concentrations. Together, these data support a model in which TUG acetylation modulates its interaction with Golgi matrix proteins and is regulated by SIRT2. Moreover, acetylation of TUG enhances its function to trap GSVs within unstimulated cells and enhances insulin-stimulated glucose uptake.     

Endoproteolytic cleavage of TUG protein regulates GLUT4 glucose transporter translocation

To promote glucose uptake into fat and muscle cells, insulin causes the translocation of GLUT4 glucose transporters from intracellular vesicles to the cell surface. Previous data support a model in which TUG traps GLUT4-containing vesicles and tethers them intracellularly in unstimulated cells and in which insulin mobilizes this pool of vesicles by releasing this tether. Here we show that TUG undergoes site-specific endoproteolytic cleavage, which separates a GLUT4-binding, N-terminal region of TUG from a C-terminal region previously suggested to bind an intracellular anchor. Cleavage is accelerated by insulin stimulation in 3T3-L1 adipocytes and is highly dependent upon adipocyte differentiation. The N-terminal TUG cleavage product has properties of a novel 18-kDa ubiquitin-like modifier, which we call TUGUL. The C-terminal product is observed at the expected size of 42 kDa and also as a 54-kDa form that is released from membranes into the cytosol. In transfected cells, intact TUG links GLUT4 to PIST and also binds Golgin-160 through its C-terminal region. PIST is an effector of TC10α, a GTPase previously shown to transmit an insulin signal required for GLUT4 translocation, and we show using RNAi that TC10α is required for TUG proteolytic processing. Finally, we demonstrate that a cleavage-resistant form of TUG does not support highly insulin-responsive GLUT4 translocation or glucose uptake in 3T3-L1 adipocytes. Together with previous results, these data support a model whereby insulin stimulates TUG cleavage to liberate GLUT4 storage vesicles from the Golgi matrix, which promotes GLUT4 translocation to the cell surface and enhances glucose uptake.     

Enhanced Fasting Glucose Turnover in Mice with Disrupted Action of TUG Protein in Skeletal Muscle

Insulin stimulates glucose uptake in 3T3-L1 adipocytes in part by causing endoproteolytic cleavage of TUG (tether containing a ubiquitin regulatory X (UBX) domain for glucose transporter 4 (GLUT4)). Cleavage liberates intracellularly sequestered GLUT4 glucose transporters for translocation to the cell surface. To test the role of this regulation in muscle, we used mice with muscle-specific transgenic expression of a truncated TUG fragment, UBX-Cter. This fragment causes GLUT4 translocation in unstimulated 3T3-L1 adipocytes. We predicted that transgenic mice would have GLUT4 translocation in muscle during fasting. UBX-Cter expression caused depletion of PIST (PDZ domain protein interacting specifically with TC10), which transmits an insulin signal to TUG. Whereas insulin stimulated TUG proteolysis in control muscles, proteolysis was constitutive in transgenic muscles. Fasting transgenic mice had decreased plasma glucose and insulin concentrations compared with controls. Whole-body glucose turnover was increased during fasting but not during hyperinsulinemic clamp studies. In muscles with the greatest UBX-Cter expression, 2-deoxyglucose uptake during fasting was similar to that in control muscles during hyperinsulinemic clamp studies. Fasting transgenic mice had increased muscle glycogen, and GLUT4 targeting to T-tubule fractions was increased 5.7-fold. Whole-body oxygen consumption (VO₂), carbon dioxide production (VCO₂), and energy expenditure were increased by 12–13%. After 3 weeks on a high fat diet, the decreased fasting plasma glucose in transgenic mice compared with controls was more marked, and increased glucose turnover was not observed; the transgenic mice continued to have an increased metabolic rate. We conclude that insulin stimulates TUG proteolysis to translocate GLUT4 in muscle, that this pathway impacts systemic glucose homeostasis and energy metabolism, and that the effects of activating this pathway are maintained during high fat diet-induced insulin resistance in mice.     

Replacement of intracellular C-terminal domain of GLUT1 glucose transporter with that of GLUT2 increases Vmax and Km of transport activity.

The intracellular C-terminal domain is diverse in size and amino acid sequence among facilitative glucose transporter isoforms. The characteristics of glucose transport are also divergent, and GLUT2 has far higher Km and Vmax values compared with GLUT1. To investigate the role of the intracellular C-terminal domain in glucose transport, we expressed in Chinese hamster ovary cells the mutated GLUT1 protein whose intracellular C-terminal domain was replaced with that of GLUT2 by means of engineering the chimeric cDNA. Cytochalasin B, for which GLUT2 protein has much lower affinity, bound to this chimeric protein in a fashion similar to GLUT1. In contrast, greater transport activity was observed in this chimeric glucose transporter compared with the wild-type GLUT1 at 10 mM 2-deoxy-D-glucose concentration. The kinetic studies on 2-deoxy-D-glucose uptake revealed a 3.8-fold increase in Km and a 4.3-fold increase in Vmax in this chimeric glucose transporter compared with the wild-type GLUT1. Thus, replacement of the intracellular C-terminal domain confers the GLUT2-like property on the glucose transporter. These results strongly suggest that the diversity of intracellular C-terminal domain contributes to the diversity of glucose transport characteristics among isoforms.     

Fatty acid-binding proteins transport N-acylethanolamines to nuclear receptors and are targets of endocannabinoid transport inhibitors

N-acylethanolamines (NAEs) are bioactive lipids that engage diverse receptor systems. Recently, we identified fatty acid-binding proteins (FABPs) as intracellular NAE carriers. Here, we provide two new functions for FABPs in NAE signaling. We demonstrate that FABPs mediate the nuclear translocation of the NAE oleoylethanolamide, an agonist of nuclear peroxisome proliferator-activated receptor α (PPARα). Antagonism of FABP function through chemical inhibition, dominant-negative approaches, or shRNA-mediated knockdown reduced PPARα activation, confirming a requisite role for FABPs in this process. In addition, we show that NAE analogs, traditionally employed as inhibitors of the putative endocannabinoid transmembrane transporter, target FABPs. Support for the existence of the putative membrane transporter stems primarily from pharmacological inhibition of endocannabinoid uptake by such transport inhibitors, which are widely employed in endocannabinoid research despite lacking a known cellular target(s). Our approach adapted FABP-mediated PPARα signaling and employed in vitro binding, arachidonoyl-[1-(14)C]ethanolamide ([(14)C]AEA) uptake, and FABP knockdown to demonstrate that transport inhibitors exert their effects through inhibition of FABPs, thereby providing a molecular rationale for the underlying physiological effects of these compounds. Identification of FABPs as targets of transport inhibitors undermines the central pharmacological support for the existence of an endocannabinoid transmembrane transporter.     

DPP6 Domains Responsible for Its Localization and Function

Dipeptidyl peptidase-like protein 6 (DPP6) is an auxiliary subunit of the Kv4 family of voltage-gated K⁺ channels known to enhance channel surface expression and potently accelerate their kinetics. DPP6 is a single transmembrane protein, which is structurally remarkable for its large extracellular domain. Included in this domain is a cysteine-rich motif, the function of which is unknown. Here we show that this cysteine-rich domain of DPP6 is required for its export from the ER and expression on the cell surface. Disulfide bridges formed at C349/C356 and C465/C468 of the cysteine-rich domain are necessary for the enhancement of Kv4.2 channel surface expression but not its interaction with Kv4.2 subunits. The short intracellular N-terminal and transmembrane domains of DPP6 associates with and accelerates the recovery from inactivation of Kv4.2, but the entire extracellular domain is necessary to enhance Kv4.2 surface expression and stabilization. Our findings show that the cysteine-rich domain of DPP6 plays an important role in protein folding of DPP6 that is required for transport of DPP6/Kv4.2 complexes out of the ER.