A double leucine within the GLUT4 glucose transporter COOH-terminal domain functions as an endocytosis signal [published erratum appears in J Cell Biol 1994 Sep;126(6):1625]

The unique COOH-terminal 30-amino acid region of the adipocyte/skeletal muscle glucose transporter (GLUT4) appears to be a major structural determinant of this protein's perinuclear localization, from where it is redistributed to the cell surface in response to insulin. To test whether an underlying mechanism of this domain's function involves glucose transporter endocytosis rates, transfected cells were generated expressing exofacial hemagglutinin epitope (HA)-tagged erythrocyte/brain glucose transporter (GLUT1) or a chimera containing the COOH-terminal 30 amino acids of GLUT4 substituted onto this GLUT1 construct. Incubation of COS-7 or CHO cells expressing the HA-tagged chimera with anti-HA antibody at 37 degrees resulted in an increased rate of antibody internalization compared to cells expressing similar levels of HA-tagged GLUT1, which displays a cell surface disposition. Colocalization of the internalized anti-HA antibody in vesicular structures with internalized transferrin and with total transporters was established by digital imaging microscopy, suggesting the total cellular pool of transporters are continuously recycling through the coated pit endocytosis pathway. Mutation of the unique double leucines 489 and 490 in the rat GLUT4 COOH-terminal domain to alanines caused the HA-tagged chimera to revert to the slow endocytosis rate and steady- state cell surface display characteristic of GLUT1. These results support the hypothesis that the double leucine motif in the GLUT4 COOH terminus operates as a rapid endocytosis and retention signal in the GLUT4 transporter, causing its localization to intracellular compartments in the absence of insulin.     

Insulin-sensitive targeting of the GLUT4 glucose transporter in L6 myoblasts is conferred by its COOH-terminal cytoplasmic tail

The GLUT4 glucose transporter appears to be targeted to a unique insulin-sensitive intracellular membrane compartment in fat and muscle cells. Insulin stimulates glucose transport in these cell types by mediating the partial redistribution of GLUT4 from this intracellular compartment to the plasma membrane. The structural basis for the unique targeting behavior of GLUT4 was investigated in the insulin-sensitive L6 myoblast cell line. Analysis of immunogold-labeled cells of independent clonal lines by electron microscopy indicated that 51-53% of GLUT1 was present in the plasma membrane in the basal state. Insulin did not significantly affect this distribution. In contrast, only 4.2- 6.1% of GLUT4 was present in the plasma membrane of basal L6 cells and insulin increased this percentage by 3.7-6.1-fold. Under basal conditions and after insulin treatment, GLUT4 was detected in tubulovesicular structures, often clustered near Golgi stacks, and in endosome-like vesicles. Analysis of 25 chimeric transporters consisting of reciprocal domains of GLUT1 and GLUT4 by confocal immunofluorescence microscopy indicated that only the final 25 amino acids of the COOH- terminal cytoplasmic tail of GLUT4 were both necessary and sufficient for the targeting pattern observed for GLUT4. A dileucine motif present in the COOH-terminal tail of GLUT4 was found to be necessary, but not sufficient, for intracellular targeting. Contrary to previous studies, the NH2 terminus of GLUT4 did not affect the subcellular distribution of chimeras. Analysis of a chimera containing the COOH-terminal tail of GLUT4 by immunogold electron microscopy indicated that its subcellular distribution in basal cells was very similar to that of wild-type GLUT4 and that its content in the plasma membrane increased 6.8-10.5-fold in the presence of insulin. Furthermore, only the chimera containing the COOH terminus of GLUT4 enhanced insulin responsive 2-deoxyglucose uptake. GLUT1 and two other chimeras lacking the COOH terminus of GLUT4 were studied by immunogold electron microscopy and did not demonstrate insulin-mediated changes in subcellular distribution. The NH2-terminal cytoplasmic tail of GLUT4 did not confer intracellular sequestration and did not cause altered subcellular distribution in the presence of insulin. Intracellular targeting of one chimera to non-insulin- sensitive compartments was also observed. We conclude that the COOH terminus of GLUT4 is both necessary and sufficient to confer insulin- sensitive subcellular targeting of chimeric glucose transporters in L6 myoblasts.     

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

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.     

Insulin-regulated trafficking of dual-labeled glucose transporter 4 in primary rat adipose cells

In isolated rat adipose cells, physiologically relevant insulin target cells, glucose transporter 4 (GLUT4) subcellular trafficking can be assessed by transfection of exofacially HA-tagged GLUT4. To simultaneously visualize the transfected GLUT4, we fused GFP with HA-GLUT4. With the resulting chimeras, GFP-HA-GLUT4 and HA-GLUT4-GFP, we were able to visualize for the first time the cell-surface localization, total expression, and intracellular distribution of GLUT4 in a single cell. Confocal microscopy reveals that the intracellular proportions of both GFP-HA-GLUT4 and HA-GLUT4-GFP are properly targeted to the insulin-responsive aminopeptidase-positive vesicles. Dynamic studies demonstrate close similarities in the trafficking kinetics between the two constructs and with native GLUT4. However, while the basal subcellular distribution of HA-GLUT4-GFP and the response to insulin are indistinguishable from those of HA-GLUT4 and endogenous GLUT4, most of the GFP-HA-GLUT4 is targeted to the plasma membrane with little further insulin response. Thus, HA-GLUT4-GFP will be useful to study GLUT4 trafficking in vivo while GFP on the N-terminus interferes with intracellular retention.     

Identification of a key residue determining substrate affinity in the human glucose transporter GLUT1

Asn³³¹ in transmembrane segment 7 of the yeast Saccharomyces cerevisiae transporter Hxt2 has been identified as a single key residue for high-affinity glucose transport by comprehensive chimera approach. The glucose transporter GLUT1 of mammals belongs to the same major facilitator superfamily as Hxt2 and may therefore show a similar mechanism of substrate recognition. The functional role of Ile²⁸⁷ in human GLUT1, which corresponds to Asn³³¹ in Hxt2, was studied by its replacement with each of the other 19 amino acids. The mutant transporters were individually expressed in a recently developed yeast expression system for GLUT1. Replacement of Ile²⁸⁷ generated transporters with various affinities for glucose that correlated well with those of the corresponding mutants of the yeast transporter. Residues exhibiting high affinity for glucose were medium-sized, non-aromatic, uncharged and irrelevant to hydrogen-bond capability, suggesting an important role of van der Waals interaction. Sensitivity to phloretin, a specific inhibitor for the presumed exofacial glucose binding site, was decreased in two mutants, whereas that to cytochalasin B, a specific inhibitor for the presumed endofacial glucose binding site, was unchanged in the mutants. These results suggest that Ile²⁸⁷ is a key residue for maintaining high glucose affinity in GLUT1 as revealed in Hxt2 and is located at or near the exofacial glucose binding site.     

The amino terminus of GLUT4 functions as an internalization motif but not an intracellular retention signal when substituted for the transferrin receptor cytoplasmic domain

Previous studies have demonstrated that the amino-terminal cytoplasmic domain of GLUT4 contains a phenylalanine-based targeting motif that determines its steady state distribution between the surface and the interior of cells (Piper, R. C., C. Tai, P. Kuleza, S. Pang, D. Warnock, J. Baenziger, J. W. Slot, H. J. Geuze, C. Puri, and D. E. James. 1993. J. Cell Biol. 121:1221). To directly measure the effect that the GLUT4 amino terminus has on internalization and subsequent recycling back to the cell surface, we constructed chimeras in which this sequence was substituted for the amino-terminal cytoplasmic domain of the human transferrin receptor. The chimeras were stably transfected into Chinese hamster ovary cells and their endocytic behavior characterized. The GLUT4-transferrin receptor chimera was recycled back to the cell surface with a rate similar to the transferrin receptor, indicating that the GLUT4 sequence was not promoting intracellular retention of the chimera. The GLUT4-transferrin receptor chimera was internalized at half the rate of the transferrin receptor. Substitution of an alanine for phenylalanine at position 5 slowed internalization of the chimera by twofold, to a level characteristic of bulk membrane internalization. However, substitution of a tyrosine increased the rate of internalization to the level of the transferrin receptor. Neither of these substitutions significantly altered the rate at which the chimeras were recycled back to the cell surface. These results demonstrate that the major function of the GLUT4 amino-terminal domain is to promote the effective internalization of the protein from the cell surface, via a functional phenylalanine-based internalization motif, rather than retention of the transporter within intracellular structures.     

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.     

Evidence that functional erythrocyte-type glucose transporters are oligomers

In this study we tested the hypothesis that functional erythrocyte-type glucose transporters (GLUT1) exist as oligomeric complexes by expressing chimeric transporter proteins in Chinese hamster ovary cells harboring endogenous GLUT1 transporters. The chimeric transporters were GLUT1-4c, in which the 29 C-terminal residues of human GLUT1 were replaced by the 30 C-terminal residues of rat skeletal muscle glucose transporter (GLUT4), and GLUT1n-4, containing the N-terminal 199 residues of GLUT1 and the 294 C-terminal residues of GLUT4. Endogenous GLUT1 was quantitatively co-immunoprecipitated by using an anti-GLUT4 C-terminal peptide antibody from detergent extracts of Chinese hamster ovary cells expressing either of the chimeric proteins, as detected by immunoblotting the precipitates with an anti-GLUT1 C-terminal peptide antiserum. No co-immunoprecipitation of native GLUT1 with native GLUT4 from extracts of 3T3-L1 adipocytes, which contain both these transporters, was observed with the same antibody. These data are consistent with the hypothesis that GLUT1 transporters exist as homodimers or higher order oligomers and that a major determinant of oligomerization is located within the first 199 residues of GLUT1.     

Activation of cell surface glucose transporters measured by photoaffinity labeling of insulin-sensitive 3T3-L1 adipocytes.

Several studies have demonstrated that the intrinsic catalytic activity of cell surface glucose transporters is highly regulated in 3T3-L1 adipocytes expressing GLUT1 (erythrocyte/brain) and GLUT4 (adipocyte/skeletal muscle) glucose transporter isoforms. For example, inhibition of protein synthesis in these cells by anisomycin or cycloheximide leads to marked increases in hexose transport without a change in the levels of cell surface glucose transporter proteins (Clancy, B. M., Harrison, S. A., Buxton, J. M., and Czech, M. P. (1991) J. Biol. Chem. 266, 10122-10130). In the present work the exofacial hexose binding sites on GLUT1 and GLUT4 in anisomycin-treated 3T3-L1 adipocytes were labeled with the cell-impermeant photoaffinity reagent [2-3H]2-N-[4-(1-azitrifluoroethyl)benzoyl]-1,3-bis- (D-mannos-4-yloxy)-2-propylamine [( 2-3H] ATB-BMPA) to determine which isoform is activated by protein synthetic blockade. As expected, a 15-fold increase in 2-deoxyglucose uptake in response to insulin was associated with 1.7- and 2.6-fold elevations in plasma membrane GLUT1 and GLUT4 protein levels, respectively. Anisomycin treatment of cultured adipocytes for 5 h produced an 8-fold stimulation of hexose transport but no increase in the content of glucose transporters in the plasma membrane fraction as measured by protein immunoblot analysis. Cell surface GLUT1 levels were also shown to be unaffected on 3T3-L1 adipocytes in response to anisomycin using an independent method, the binding of an antiexofacial GLUT1 antibody to intact cells. In contrast, anisomycin fully mimicked the action of insulin to stimulate (about 4-fold) the radiolabeling of GLUT1 transporters specifically immunoprecipitated from intact 3T3-L1 adipocytes irradiated after incubation with [2-3H] ATB-BMPA. Photolabeling of GLUT4 under these conditions was also significantly enhanced (1.8-fold) by anisomycin treatment, but this effect was only 15% of that caused by insulin. These results suggest that: 1) the photoaffinity reagent [2-3H]ATB-BMPA labels those cell surface glucose transporters present in a catalytically active state rather than total cell surface transporters as assumed previously and 2) inhibition of protein synthesis in 3T3-L1 adipocytes stimulates sugar transport primarily by enhancing the intrinsic catalytic activity of cell surface GLUT1, and to a lesser extent, GLUT4 proteins.     

Membrane topology of human ASBT (SLC10A2) determined by dual label epitope insertion scanning mutagenesis. New evidence for seven transmembrane domains

The membrane topology of the human apical sodium-dependent bile acid transporter (hASBT) remains unresolved. Whereas N-glycosylation analysis favors a 7 transmembrane (TM) model, membrane insertion scanning supports a 9TM topology. In order to resolve this controversy, we used dual label epitope insertion to systematically examine the topological framework of hASBT. Two distinct epitopes, hemagglutinin (HA) and FLAG, were individually inserted by inverted PCR mutagenesis at strategic positions along the hASBT sequence. Cell surface biotinylation and immunoblotting with epitope-specific and anti-hASBT antibodies confirmed expression and trafficking of the mutants to the plasma membrane. Confocal microscopy confirmed membrane localization of epitope-tagged hASBT in saponin-treated (permeabilized) and nonpermeabilized transfected COS-1 and MDCK cells. Tags at positions 116, 120, 186, 270, and 284 were accessible to the epitope antibodies in nonpermeabilized cells, indicative of the extracellular localization of loops 1 (99-130), 2 (180-191), and 3 (253-287). The corresponding positions in the 9TM model were predicted to be intracellular or membrane bound. Epitope mutants at residues 56, 92, 156, and 221 were only detected after treatment with saponin, indicating the intracellular localizations of loops 1 (50-73), 2 (150-160), 3 (215-227) as predicted by a 7TM model. Our results also confirm the exofacial and cytosolic localization of N- and C-terminal tails, respectively. With the exception of constructs inserted at position 120, epitope mutants displayed active, sodium-dependent taurocholate uptake. Consequently, our study strongly supports a 7TM topology for hASBT and refutes the previously proposed 9TM model.     

Carboxy terminus of glucose transporter 3 contains an apical membrane targeting domain

We previously demonstrated that distinct facilitative glucose transporter isoforms display differential sorting in polarized epithelial cells. In Madin-Darby canine kidney (MDCK) cells, glucose transporter 1 and 2 (GLUT1 and GLUT2) are localized to the basolateral cell surface whereas GLUTs 3 and 5 are targeted to the apical membrane. To explore the molecular mechanisms underlying this asymmetric distribution, we analyzed the targeting of chimeric glucose transporter proteins in MDCK cells. Replacement of the carboxy-terminal cytosolic tail of GLUT1, GLUT2, or GLUT4 with that from GLUT3 resulted in apical targeting. Conversely, a GLUT3 chimera containing the cytosolic carboxy terminus of GLUT2 was sorted to the basolateral membrane. These findings are not attributable to the presence of a basolateral signal in the tails of GLUTs 1, 2, and 4 because the basolateral targeting of GLUT1 was retained in a GLUT1 chimera containing the carboxy terminus of GLUT5. In addition, we were unable to demonstrate the presence of an autonomous basolateral sorting signal in the GLUT1 tail using the low-density lipoprotein receptor as a reporter. By examining the targeting of a series of more defined GLUT1/3 chimeras, we found evidence of an apical targeting signal involving residues 473-484 (DRSGKDGVMEMN) in the carboxy tail. We conclude that the targeting of GLUT3 to the apical cell surface in MDCK cells is regulated by a unique cytosolic sorting motif.     

Intracellular trafficking pathways in the assembly of connexins into gap junctions

Trafficking pathways underlying the assembly of connexins into gap junctions were examined using living COS-7 cells expressing a range of connexin-aequorin (Cx-Aeq) chimeras. By measuring the chemiluminescence of the aequorin fusion partner, the translocation of oligomerized connexins from intracellular stores to the plasma membrane was shown to occur at different rates that depended on the connexin isoform. Treatment of COS-7 cells expressing Cx32-Aeq and Cx43-Aeq with brefeldin A inhibited the movement of these chimera to the plasma membrane by 84 +/- 4 and 88 +/- 4%, respectively. Nocodazole treatment of the cells expressing Cx32-Aeq and Cx43-Aeq produced 29 +/- 16 and 4 +/- 7% inhibition, respectively. In contrast, the transport of Cx26 to the plasma membrane, studied using a construct (Cx26/43T-Aeq) in which the short cytoplasmic carboxyl-terminal tail of Cx26 was replaced with the extended carboxyl terminus of Cx43, was inhibited 89 +/- 5% by nocodazole and was minimally affected by exposure of cells to brefeldin A (17 +/-11%). The transfer of Lucifer yellow across gap junctions between cells expressing wild-type Cx32, Cx43, and the corresponding Cx32-Aeq and Cx43-Aeq chimeras was reduced by nocodazole treatment and abolished by brefeldin A treatment. However, the extent of dye coupling between cells expressing wild-type Cx26 or the Cx26/43T-Aeq chimeras was not significantly affected by brefeldin A treatment, but after nocodazole treatment, transfer of dye to neighboring cells was greatly reduced. These contrasting effects of brefeldin A and nocodazole on the trafficking properties and intercellular dye transfer are interpreted to suggest that two pathways contribute to the routing of connexins to the gap junction.     

Structural basis of GLUT1 inhibition by cytoplasmic ATP

Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)-mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231-13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1-ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7-8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1-GLUT4 chimera in which loop 6-7 of GLUT1 is substituted with loop 6-7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-terminal half of GLUT1 cytoplasmic loop 6-7.     

The glucose transporter 4 FQQI motif is necessary for Akt substrate of 160-kilodalton-dependent plasma membrane translocation but not Golgi-localized (gamma)-ear-containing Arf-binding protein-dependent entry into the insulin-responsive storage compartment

Newly synthesized glucose transporter 4 (GLUT4) enters into the insulin-responsive storage compartment in a process that is Golgi-localized gamma-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.     

Exofacial photolabelling of the human erythrocyte glucose transporter with an azitrifluoroethylbenzoyl-substituted bismannose.

The synthesis of 2-N-[4-(1'-azitrifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-++ +yloxy)-2- propylamine (ATB-BMPA) is described. This compound was used as an exofacial probe for the human erythrocyte glucose-transport system. A new method is described for directly estimating the affinity for exofacial ligands which bind to the erythrocyte glucose transporter. By using this equilibrium-binding method, the Ki for ATB-BMPA was found to be 338 +/- 37 microM at 0 degrees C and 368 +/- 59 microM at 20 degrees C. This was similar to the concentration of ATB-BMPA required to half-maximally inhibit D-galactose uptake (Ki = 297 +/- 53 microM). The new photoaffinity reagent labelled the glucose transporter in intact cells but, because of its improved selectivity, was also used to label the glucose transporter in isolated erythrocyte membranes. The ATB-BMPA-labelled glucose transporter was 80% immunoprecipitated by anti-(GLUT1-C-terminal peptide) antibody, which shows that the GLUT1 glucose transporter is the major isoform present in erythrocytes. The labelling of the glucose transporter at its exofacial site, and the adoption of an outward-facing conformation, renders the transport system resistant to thermolysin and trypsin treatment. Trypsin treatment of the unlabelled glucose transporter in erythrocyte membranes produced an 18 kDa fragment which was subsequently labelled by ATB-BMPA, but had low affinity for this exofacial ligand. This suggests that the trypsin-treated transporter adopts an inward-facing conformation. The ability of D-glucose to displace ATB-BMPA from the native transporter and from the 18 kDa trypsin fragment have been compared. The D-glucose concentration which was required to obtain half-maximal inhibition of ATB-BMPA labelling was 6-fold lower for the 18 kDa tryptic fragment.     

GLUT7: a new intestinal facilitated hexose transporter

The very last member of the SLC2A gene family of facilitated hexose transporters to be cloned was SLC2A7 (hGLUT7). It has been assigned to the class II of the GLUT family on the basis of sequence similarity, and its closest family member is GLUT5, an intestinal fructose transporter. GLUT7 is primarily expressed in the small intestine and colon, although mRNA has been detected in the testis and prostate as well. The protein is expressed in the apical membrane of the small intestine and colon, and it has a high affinity (<0.5 mM) for glucose and fructose. The abundance of the protein in the small intestine does change in parallel with the dietary carbohydrate. However, the distribution of GLUT7 along the small intestine does not entirely match with the availability of glucose and fructose, suggesting that the physiological substrate for this transporter has yet to be identified. Unlike GLUT13, the proton-coupled myoinositol transporter (HMIT), there is no evidence for the coupling of protons to the hexose movement via GLUT7. One area of study in which GLUT7 has provided a useful comparison with GLUT1 has been in the development of the hypothesis that the facilitated hexose transporters may have a selectivity filter at the exofacial opening of the translocation pore, which helps to determine which hexoses can be transported. If substantiated, the elucidation of this mechanism may prove useful in the design of hexose analogs for use in cancer imaging and therapeutics.     

Cysteine-scanning mutagenesis of transmembrane segment 7 of the GLUT1 glucose transporter

The human erythrocyte facilitative glucose transporter (Glut1) is predicted to contain 12 transmembrane spanning alpha-helices based upon hydropathy plot analysis of the primary sequence. Five of these helices (3, 5, 7, 8, and 11) are capable of forming amphipathic structures. A model of GLUT1 tertiary structure has therefore been proposed in which the hydrophilic faces of several amphipathic helices are arranged to form a central aqueous channel through which glucose traverses the hydrophobic lipid bilayer. In order to test this model, we individually mutated each of the amino acid residues in transmembrane segment 7 to cysteine in an engineered GLUT1 molecule devoid of all native cysteines (C-less). Measurement of 2-deoxyglucose uptake in a Xenopus oocyte expression system revealed that nearly all of these mutants retain measurable transport activity. Over one-half of the cysteine mutants had significantly reduced specific activity relative to the C-less protein. The solvent accessibility and relative orientation of the residues within the helix was investigated by determining the sensitivity of the mutant transporters to inhibition by the sulfhydryl directed reagent p-chloromercuribenzene sulfonate (pCMBS). Cysteine replacement at six positions (Gln(282), Gln(283), Ile(287), Ala(289), Val(290), and Phe(291)), all near the exofacial side of the cell membrane, produced transporters that were inhibited by incubation with extracellular pCMBS. Residues predicted to be near the cytoplasmic side of the cell membrane were minimally affected by pCMBS. These data demonstrate that the exofacial portion of transmembrane segment 7 is accessible to the external solvent and provide evidence for the positioning of this alpha-helix within the glucose permeation pathway.     

Ligand-regulated chimeric receptor approach reveals distinctive subcellular localization and signaling properties of the Toll-like receptors

Toll-like receptors (TLRs) are sensors for the detection of invading infectious agents and can initiate innate immune responses. Because the innate immune system induces an appropriate defense against different pathogens, different TLR signaling domains may have unique properties that are responsible for eliciting distinctive responses to different types of pathogens. To test this hypothesis, we created ligand-regulated TLR chimeric receptors composed of the extracellular region of TLR4 and the transmembrane and cytoplasmic regions of other TLRs and expressed these chimeras in macrophages lacking endogenous TLR4. Interestingly, the chimeras between TLR4 and either TLR3, TLR7, or TLR9 were localized completely intracellularly whereas other chimeras were expressed on the cell surface. Lipopolysaccharide (LPS), a ligand for these chimeras, induced the activation of nuclear factor kappa B and mitogen-activated protein kinases and the subsequent production of pro-inflammatory cytokines in macrophages expressing TLR4, TLR4/TLR5, or TLR4/TLR8 chimeras but not in macrophages expressing TLR4/TLR1, TLR4/TLR2, or TLR4/TLR6 chimeras. Co-expression of unresponsive chimeras in some combinations (chimeras with TLR1+TLR2 or TLR2+TLR6 but not TLR1+TLR6) resulted in LPS responsiveness, indicating functional complementarity. Furthermore, the pair of TLR2+TLR6 chimera required approximately 10-fold less LPS to induce the same responses compared with the TLR1+TLR2 pair. Finally, LPS induced effective interferon-beta production and subsequent Stat1 phosphorylation in macrophages expressing full-length TLR4 but not other cell surface TLR chimeras. These results suggest that the functions of TLRs are diversified not only in their extracellular regions for ligand recognition but also in their transmembrane and cytoplasmic regions for subcellular localization and signaling properties.     

Light inactivation of water transport and protein-protein interactions of aquaporin-Killer Red chimeras

Aquaporins (AQPs) have a broad range of cellular and organ functions; however, nontoxic inhibitors of AQP water transport are not available. Here, we applied chromophore-assisted light inactivation (CALI) to inhibit the water permeability of AQP1, and of two AQP4 isoforms (M1 and M23), one of which (M23) forms aggregates at the cell plasma membrane. Chimeras containing Killer Red (KR) and AQPs were generated with linkers of different lengths. Osmotic water permeability of cells expressing KR/AQP chimeras was measured from osmotic swelling-induced dilution of cytoplasmic chloride, which was detected using a genetically encoded chloride-sensing fluorescent protein. KR-AQP1 red fluorescence was bleached rapidly (~10% per second) by wide-field epifluorescence microscopy. After KR bleaching, KR-AQP1 water permeability was reduced by up to 80% for the chimera with the shortest linker. Remarkably, CALI-induced reduction in AQP4-KR water permeability was approximately twice as efficient for the aggregate-forming M23 isoform; this suggests intermolecular CALI, which was confirmed by native gel electrophoresis on cells coexpressing M23-AQP4-KR and myc-tagged M23-AQP4. CALI also disrupted the interaction of AQP4 with a neuromyelitis optica autoantibody directed against an extracellular epitope on AQP4. CALI thus permits rapid, spatially targeted and irreversible reduction in AQP water permeability and interactions in live cells. Our data also support the utility of CALI to study protein-protein interactions as well as other membrane transporters and receptors.     

Human IL-Rbeta chains form IL-2 binding homodimers

Two types of functional interleukin-2 receptor (IL-2Ralpha/IL-2Rbeta/gammac and IL-2Rbeta/gammac) have already been characterized in humans. Here we describe a new form consisting of IL-2Rbeta/beta homodimers that assemble spontaneously in the absence of gammac. Co-transfection of COS-7 cells with constructs expressing IL-2Rbeta chains tagged with either HA or MYC sequences results in the formation of IL-2Rbeta:HA/IL-2Rbeta:MYC complexes detectable by coimmunoprecipitation. The formation of these IL-2Rbeta:HA/IL-2Rbeta:MYC dimers is also observed in the absence of IL-2. Moreover, in COS cells expressing chimeras of IL-2Rbeta fused to fluorescence reporters such as IL-2Rbeta:ECFP and IL-2Rbeta:EYFP, we also observed specific FRET at the surface of living cells, as expected for dimer formation. Transiently transfected COS-7 cells expressing IL-2Rbeta bind 125I-labeled IL-2 (homodimers, Kd = 1nM) as cells expressing both IL-2Rbeta and gammac chains (heterodimers, Kd = 1 nM). IL-2Rbeta/IL-2Rbeta could represent either a decoy receptor or a new form of IL-2R involved in signaling when gammac expression is low.