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PubMed2006

Implication of phosphorylation of the myosin II regulatory light chain in insulin-stimulated GLUT4 translocation in 3T3-F442A adipocytes.

In adipocytes, insulin stimulates glucose transport primarily by promoting the translocation of GLUT4 to the plasma membrane. Requirements for Ca(2+)/calmodulin during insulin-stimulated GLUT4 translocation have been demonstrated; however, the mechanism of action of Ca(2+) in this process is unknown. Recently, myosin II, whose function in non-muscle cells is primarily regulated by phosphorylation of its regulatory light chain by the Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK), was implicated in insulin-stimulated GLUT4 translocation. The present studies in 3T3-F442A adipocytes demonstrate the novel finding that insulin significantly increases phosphorylation of the myosin II RLC in a Ca(2+)-dependent manner. In addition, ML-7, a selective inhibitor of MLCK, as well as inhibitors of myosin II, such as blebbistatin and 2,3-butanedione monoxime, block insulin-stimulated GLUT4 translocation and subsequent glucose transport. Our studies suggest that MLCK may be a regulatory target of Ca(2+)/calmodulin and may play an important role in insulin-stimulated glucose transport in adipocytes.

3T3 Cells↗
PubMed2001

Expression of a prenylation-deficient Rab4 inhibits the GLUT4 translocation induced by active phosphatidylinositol 3-kinase and protein kinase B.

The small GTPase Rab4 has been shown to participate in the subcellular distribution of GLUT4 under both basal and insulin-stimulated conditions in adipocytes. In the present work, we have characterized the effect of Rab4 DeltaCT, a prenylation-deficient and thus cytosolic form of Rab4, in this process. We show that the expression of Rab4 DeltaCT in freshly isolated adipocytes inhibits insulin-induced GLUT4 translocation, but only when this protein is in its GTP-bound active form. Further, it not only blocks the effect of insulin, but also that of a hyperosmotic shock, but does not interfere with the effect of zinc ions on GLUT4 translocation. Rab4 DeltaCT was then shown to prevent GLUT4 translocation induced by the expression of an active form of phosphatidylinositol 3-kinase or of protein kinase B, without altering the activities of the enzymes. Our results are consistent with a role of Rab4 DeltaCT acting as a dominant negative protein towards Rab4, possibly by binding to Rab4 effectors.

Adipocytes↗
PubMed2003

Insulin and dexamethasone induce GLUT4 gene expression in foetal brown adipocytes: synergistic effect through CCAAT/enhancer-binding protein alpha.

Treatment of foetal brown adipocytes in primary culture with either dexamethasone or insulin, at physiological concentrations, for 24 h up-regulates the expression of the GLUT4 gene, producing a synergistic effect on mRNA accumulation (20-fold increase), in the amount of protein in the total membrane fraction (8-fold increase) and in the transactivation of a full-promoter GLUT4 -chloramphenicol acetyltransferase gene ( CAT ) construct (7-fold increase). However, GLUT1 expression remains essentially unmodified regardless of the presence of the hormones. As a consequence, exposure of brown adipocytes to dexamethasone and insulin results in a dramatic increase of glucose uptake (12-fold). Dexamethasone induces the expression of CCAAT/enhancer-binding protein (C/EBP) alpha, insulin promotes myocyte enhancer factor-2 DNA-binding activity and both combined produces a significant increase in C/EBPalpha DNA-binding activity. Moreover, co-transfection with a wild-type C/EBPalpha construct transactivates a full-promoter GLUT4 - CAT fusion gene, whereas a dominant-negative C/EBPalpha expression vector impairs the hormonal effects. Our results show that the synergism between insulin and glucocorticoids on glucose uptake is a consequence of the activation of the GLUT4 promoter by the transcription factor C/EBPalpha.

Adipocytes↗
PubMed2005

AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain.

Recently, we described a 160 kDa protein (designated AS160, for Akt substrate of 160 kDa) with a predicted Rab GAP (GTPase-activating protein) domain that is phosphorylated on multiple sites by the protein kinase Akt. Phosphorylation of AS160 in adipocytes is required for insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The aim of the present study was to determine whether AS160 is in fact a GAP for Rabs, and, if so, what its specificity is. We first identified a group of 16 Rabs in a preparation of intracellular vesicles containing GLUT4 by MS. We then prepared the recombinant GAP domain of AS160 and examined its activity against many of these Rabs, as well as several others. The GAP domain was active against Rabs 2A, 8A, 10 and 14. There was no significant activity against 14 other Rabs. GAP activity was further validated by the finding that the recombinant GAP domain with the predicted catalytic arginine residue replaced by lysine was inactive. Finally, it was found by immunoblotting that Rabs 2A, 8A and 14 are present in GLUT4 vesicles. These results indicate that AS160 is a Rab GAP, and suggest novel Rabs that may participate in GLUT4 translocation.

Adipocytes↗
PubMed1995

Glucose-transporter (GLUT4) protein content in oxidative and glycolytic skeletal muscles from calf and goat.

It is well accepted that skeletal muscle is a major glucose-utilizing tissue and that insulin is able to stimulate in vivo glucose utilization in ruminants as in monogastrics. In order to determine precisely how glucose uptake is controlled in various ruminant muscles, particularly by insulin, this study was designed to investigate in vitro glucose transport and insulin-regulatable glucose-transporter protein (GLUT4) in muscle from calf and goat. Our data demonstrate that glucose transport is the rate-limiting step for glucose uptake in bovine fibre strips, as in rat muscle. Insulin increases the rate of in vitro glucose transport in bovine muscle, but to a lower extent than in rat muscle. A GLUT4-like protein was detected by immunoblot assay in all insulin-responsive tissues from calf and goat (heart, skeletal muscle, adipose tissue) but not in liver, brain, erythrocytes and intestine. Unlike the rat, bovine and goat GLUT4 content is higher in glycolytic and oxido-glycolytic muscles than in oxidative muscles. In conclusion, using both a functional test (insulin stimulation of glucose transport) and an immunological approach, this study demonstrates that ruminant muscles express GLUT4 protein. Our data also suggest that, in ruminants, glucose is the main energy-yielding substrate for glycolytic but not for oxidative muscles, and that insulin responsiveness may be lower in oxidative than in other skeletal muscles.

Animals↗
PubMed1998

Regulation of GLUT4 protein and glycogen synthase during muscle glycogen synthesis after exercise.

The pattern of muscle glycogen synthesis following its depletion by exercise is biphasic. Initially, there is a rapid, insulin independent increase in the muscle glycogen stores. This is then followed by a slower insulin dependent rate of synthesis. Contributing to the rapid phase of glycogen synthesis is an increase in muscle cell membrane permeability to glucose, which serves to increase the intracellular concentration of glucose-6-phosphate (G6P) and activate glycogen synthase. Stimulation of glucose transport by muscle contraction as well as insulin is largely mediated by translocation of the glucose transporter isoform GLUT4 from intracellular sites to the plasma membrane. Thus, the increase in membrane permeability to glucose following exercise most likely reflects an increase in GLUT4 protein associated with the plasma membrane. This insulin-like effect on muscle glucose transport induced by muscle contraction, however, reverses rapidly after exercise is stopped. As this direct effect on transport is lost, it is replaced by a marked increase in the sensitivity of muscle glucose transport and glycogen synthesis to insulin. Thus, the second phase of glycogen synthesis appears to be related to an increased muscle insulin sensitivity. Although the cellular modifications responsible for the increase in insulin sensitivity are unknown, it apparently helps maintain an increased number of GLUT4 transporters associated with the plasma membrane once the contraction-stimulated effect on translocation has reversed. It is also possible that an increase in GLUT4 protein expression plays a role during the insulin dependent phase.

Animals↗
PubMed2006

Altered GLUT4 translocation in skeletal muscle of 12/15-lipoxygenase knockout mice.

We have recently shown that 12(S)-hydroxyeicosatetraenoic acid plays a role in the organization of actin microfilaments in rat cardiomyocytes, and that inhibition of 12-lipoxygenase abrogates insulin-stimulated GLUT4 translocation in these cells. In the present study, we used mice that were null for the leukocyte 12/15-lipoxygenase to explore the implications of this enzyme for insulin action under IN VIVO conditions. Insulin induced a profound reduction in blood glucose in both control and knockout mice. However, significantly higher serum insulin levels were observed in these animals. GLUT4 expression in heart and skeletal muscle was unaffected in KO mice. Insulin-regulated serine phosphorylation of Akt and GSK3alpha and GSK3beta was unaltered in heart and skeletal muscle of knockout mice, suggesting unaltered insulin signaling. Fractionation of hind limb muscles showed that insulin had induced a prominent translocation of GLUT4 to skeletal muscle plasma membranes in control mice. However, this response was largely reduced in knockout animals. Our data show that the lack of leukocyte 12/15-lipoxygenase does not lead to the development of an insulin-resistant phenotype. However, perturbation of GLUT4 translocation in skeletal muscle of knockout mice may indicate latent insulin resistance, and supports our hypothesis that eicosanoids are involved in insulin-mediated regulation of muscle glucose transport.

Animals↗
PubMed1995

High fat diet-induced hyperglycemia: prevention by low level expression of a glucose transporter (GLUT4) minigene in transgenic mice.

High-fat intake leading to obesity contributes to the development of non-insulin-dependent diabetes mellitus (NIDDM, type 2). Similarly, mice fed a high-fat (safflower oil) diet develop defective glycemic control, hyperglycemia, and obesity. To assess the effect of a modest increase in the expression of GLUT4 (the insulin-responsive glucose transporter) on impaired glycemic control caused by fat feeding, transgenic mice harboring a GLUT4 minigene were fed a high-fat diet. Low-level tissue-specific (heart, skeletal muscle, and adipose tissue) expression of the GLUT4 minigene in transgenic mice prevented the impairment of glycemic control and accompanying hyperglycemia, but not obesity, caused by fat feeding. Thus, a small increase (< or = 2-fold) in the tissue level of GLUT4 prevents a primary symptom of the diabetic state in a mouse model, suggesting a possible target for intervention in the treatment of NIDDM.

Animals↗
PubMed1996

Activated phosphatidylinositol 3-kinase is sufficient to mediate actin rearrangement and GLUT4 translocation in 3T3-L1 adipocytes.

Insulin stimulation of 3T3-L1 adipocytes causes rapid translocation of actin and the GLUT4 glucose transporter to the plasma membrane. Both processes depend on the activity of phosphatidylinositol 3-kinase. Using single cell microinjection, we have transiently expressed a constitutively activated mutant of phosphatidylinositol 3-kinase, p110*, in 3T3-L1 adipocytes. Fluorescent detection of GLUT4 protein and actin within these cells demonstrates that expression of p110* is sufficient to cause translocation of GLUT4 to the plasma membrane and the formation of actin membrane ruffles. These effects are inhibited by wortmannin in the p110*-expressing cells, indicating that the phosphatidylinositol 3-kinase activity of the protein is required. Overexpression of an identical protein containing a point mutation in the kinase domain, p110*Deltakin, was incapable of mediating either action, confirming that neither the microinjection process nor a nonspecific effect of the protein was responsible for the observed effects. These data suggest that although insulin is capable of inducing numerous signaling pathways, the isolated activation of phosphatidylinositol 3-kinase can initiate the signaling cascade leading to both actin rearrangement and GLUT4 translocation in the absence of insulin stimulation.

3T3 Cells↗
PubMed1996

The carboxyl terminus of GLUT4 contains a serine-leucine-leucine sequence that functions as a potent internalization motif in Chinese hamster ovary cells.

To characterize the trafficking motifs contained in the carboxyl terminus of GLUT4, a chimera (GTCTR) was constructed in which the carboxyl-terminal 30 amino acids of GLUT4 were substituted for the amino-terminal cytoplasmic domain of the transferrin receptor (TR). The endocytic behavior of this chimera was characterized in Chinese hamster ovary cells. The GTCTR chimera had a more predominant intracellular distribution compared to the TR. Only 20% of the GTCTR chimera is on the surface at steady-state compared to 35% of the TR. The GTCTR chimera is internalized 50% more rapidly and recycled 20% more slowly than the TR. Acidification of the cytosol inhibited internalization of the GTCTR chimera, indicating that the chimera is internalized through clathrin-coated pits. Mutations of GTCTR were constructed in which a di-leucine sequence of the carboxyl domain of GLUT4 was mutated to a di-alanine sequence (GTCTR-AA) and serine residue 488, immediately preceding the di-leucine sequence, was mutated to either an alanine or aspartate residue. In each case, albeit to varying degrees, the substitutions shifted the distribution of the mutated GTCTR constructs toward the surface. The shift in the distribution of GTCTR-AA resulted from a 10-fold reduction in internalization, and the shift of serine 488 mutants resulted from a 3-fold reduction in the internalization rate compared to GTCTR. None of these mutations affected the recycling rate. These results demonstrate that the carboxyl terminus of GLUT4 contains a serine-leucine-leucine-based motif that, when expressed in non-insulin responsive cells, functions as a potent internalization motif which promotes more rapid internalization than does the native TR internalization motif.

Amino Acid Sequence↗
PubMed1996

Differential effects of GLUT1 or GLUT4 overexpression on hexosamine biosynthesis by muscles of transgenic mice.

Transgenic mice that overexpress GLUT1 or GLUT4 in skeletal muscle were studied; the former but not the latter develop insulin resistance. Because increased glucose flux via the hexosamine biosynthesis pathway has been implicated in glucose-induced insulin resistance, we measured the activity of glutamine:fructose-6-phosphate amidotransferase (GFAT; rate-limiting enzyme) and the concentrations of UDP-N-acetyl hexosamines (major products of the pathway) as well as UDP-hexoses and GDP-mannose in hind limb muscles and liver in both transgenic models and controls. GFAT activity was increased 60-70% in muscles of GLUT1 but not in GLUT4 transgenics. GFAT mRNA abundance was unchanged. The concentrations of all nucleotide-linked sugars were increased 2-3-fold in GLUT1 and were unchanged in GLUT4-overexpressing muscles. Similar results were obtained in fed and fasted mice. GFAT and nucleotide sugars were unchanged in liver, where the transgene is not expressed. We concluded that 1) glucose transport appears to be rate limiting for synthesis of nucleotide sugars; 2) chronically increased glucose flux increases muscle GFAT activity posttranscriptionally; 3) increased UDP-glucose likely accounts for the marked glycogen accumulation in muscles of GLUT1-overexpressing mice; and 4) glucose flux via the hexosamine biosynthetic pathway is increased in muscles of GLUT1-overexpressing but not GLUT4-overexpressing mice; products of the pathway may contribute to insulin resistance in GLUT1 transgenics.

Animals↗
PubMed1998

A sequence element in the GLUT4 gene that mediates repression by insulin.

Prolonged treatment of 3T3-L1 adipocytes decreases expression of GLUT4, the insulin-responsive glucose transporter. Expression of promoter-reporter gene constructs that contained 2900 or 785 base pairs of 5'-flanking region of the murine GLUT4 gene was down-regulated by insulin (p < 0.0005), whereas expression of constructs that contained 641, 469, or 78 base pairs of 5'-flanking region was not. Nuclear extract from 3T3-L1 adipocytes protected the region from -707 to -681 in the GLUT4 5'-flanking region from DNase I digestion. Using an oligonucleotide probe that corresponded to this footprinted region, two major protein-DNA complexes were identified by a gel mobility shift assay. Southwestern analysis identified four protein bands with molecular masses from 38 to 46 kDa that bound to the insulin-responsive region probe. A reporter gene construct in which bases -706 to -676 were deleted was not repressed by insulin treatment, confirming that this sequence is necessary for the repression of the GLUT4 promoter by insulin in 3T3-L1 adipocytes. This sequence does not show homology to previously described insulin response elements and thus represents a distinct mechanism of gene regulation by insulin.

Adipocytes↗
PubMed1998

Syndet, an adipocyte target SNARE involved in the insulin-induced translocation of GLUT4 to the cell surface.

In adipocytes, insulin stimulates the translocation of the glucose transporter, GLUT4, from an intracellular storage compartment to the cell surface. Substantial evidence exists to suggest that in the basal state GLUT4 resides in discrete storage vesicles. A direct interaction of GLUT4 storage vesicles with the plasma membrane has been implicated because the v-SNARE, vesicle-associated membrane protein-2 (VAMP2), appears to be a specific component of these vesicles. In the present study we sought to identify the cognate target SNAREs for VAMP2 in mouse 3T3-L1 adipocytes. Membrane fractions were isolated from adipocytes and probed by far Western blotting with the cytosolic portion of VAMP2 fused to glutathione S-transferase. Two plasma membrane-enriched proteins, p25 and p35, were specifically labeled with this probe. By using a combination of immunoblotting, detergent extraction, and anion exchange chromatography, we identified p35 as Syntaxin-4 and p25 as the recently identified murine SNAP-25 homologue, Syndet (mSNAP-23). By using surface plasmon resonance we show that VAMP2, Syntaxin-4, and Syndet form a ternary SDS-resistant SNARE complex. Microinjection of anti-Syndet antibodies into 3T3-L1 adipocytes, or incubation of permeabilized adipocytes with a synthetic peptide comprising the C-terminal 24 amino acids of Syndet, inhibited insulin-stimulated GLUT4 translocation to the cell surface by approximately 40%. GLUT1 trafficking remained unaffected by the presence of the peptide. Our data suggest that Syntaxin-4 and Syndet are important cell-surface target SNAREs within adipocytes that regulate docking and fusion of GLUT-4-containing vesicles with the plasma membrane in response to insulin.

3T3 Cells↗
PubMed1998

Inhibition of insulin-induced GLUT4 translocation by Munc18c through interaction with syntaxin4 in 3T3-L1 adipocytes.

Insulin induces the translocation of vesicles containing the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane in adipocytes. SNARE proteins have been implicated in the docking and fusion of these vesicles with the cell membrane. The role of Munc18c, previously identified as an n-Sec1/Munc18 homolog in 3T3-L1 adipocytes, in insulin-regulated GLUT4 trafficking has now been investigated in 3T3-L1 adipocytes. In these cells, Munc18c was predominantly associated with syntaxin4, although it bound both syntaxin2 and syntaxin4 to similar extents in vitro. In addition, SNAP-23, an adipocyte homolog of SNAP-25, associated with both syntaxins 2 and 4 in 3T3-L1 adipocytes. Overexpression of Munc18c in 3T3-L1 adipocytes by adenovirus-mediated gene transfer resulted in inhibition of insulin-stimulated glucose transport in a virus dose-dependent manner (maximal effect, approximately 50%) as well as in inhibition of sorbitol-induced glucose transport (by approximately 35%), which is mediated by a pathway different from that used by insulin. In contrast, Munc18b, which is also expressed in adipocytes but which did not bind to syntaxin4, had no effect on glucose transport. Furthermore, overexpression of Munc18c resulted in inhibition of insulin-induced translocation of GLUT4, but not of that of GLUT1, to the plasma membrane. These results suggest that Munc18c is involved in the insulin-dependent trafficking of GLUT4 from the intracellular storage compartment to the plasma membrane in 3T3-L1 adipocytes by modulating the formation of a SNARE complex that includes syntaxin4.

3T3 Cells↗
PubMed1999

Okadaic acid activates atypical protein kinase C (zeta/lambda) in rat and 3T3/L1 adipocytes. An apparent requirement for activation of Glut4 translocation and glucose transport.

Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, is known to provoke insulin-like effects on GLUT4 translocation and glucose transport, but the underlying mechanism is obscure. Presently, we found in both rat adipocytes and 3T3/L1 adipocytes that okadaic acid provoked partial insulin-like increases in glucose transport, which were inhibited by phosphatidylinositol (PI) 3-kinase inhibitors, wortmannin and LY294002, and inhibitors of atypical protein kinase C (PKC) isoforms, zeta and lambda. Moreover, in both cell types, okadaic acid provoked increases in the activity of immunoprecipitable PKC-zeta/lambda by a PI 3-kinase-dependent mechanism. In keeping with apparent PI 3-kinase dependence of stimulatory effects of okadaic acid on glucose transport and PKC-zeta/lambda activity, okadaic acid provoked insulin-like increases in membrane PI 3-kinase activity in rat adipocytes; the mechanism for PI 3-kinase activation was uncertain, however, because it was not apparent in phosphotyrosine immunoprecipitates. Of further note, okadaic acid provoked partial insulin-like increases in the translocation of hemagglutinin antigen-tagged GLUT4 to the plasma membrane in transiently transfected rat adipocytes, and these stimulatory effects on hemagglutinin antigen-tagged GLUT4 translocation were inhibited by co-expression of kinase-inactive forms of PKC-zeta and PKC-lambda but not by a double mutant (T308A, S473A), activation-resistant form of protein kinase B. Our findings suggest that, as with insulin, PI 3-kinase-dependent atypical PKCs, zeta and lambda, are required for okadaic acid-induced increases in GLUT4 translocation and glucose transport in rat adipocytes and 3T3/L1 adipocytes.

3T3 Cells↗
PubMed1999

Opposite translational control of GLUT1 and GLUT4 glucose transporter mRNAs in response to insulin. Role of mammalian target of rapamycin, protein kinase b, and phosphatidylinositol 3-kinase in GLUT1 mRNA translation.

Prolonged exposure of 3T3-L1 adipocytes to insulin increases GLUT1 protein content while diminishing GLUT4. These changes arise in part from changes in mRNA transcription. Here we examined whether there are also specific effects of insulin on GLUT1 and GLUT4 mRNA translation. Insulin enhanced association of GLUT1 mRNA with polyribosomes and decreased association with monosomes, suggesting increased translation. Conversely, insulin arrested the majority of GLUT4 transcripts in monosomes. Insulin inactivates the translational suppressor eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) through the mammalian target of rapamycin (mTOR). Hence, we examined the effect of rapamycin on GLUT1 mRNA translation and protein expression. Rapamycin abrogated the insulin-mediated increase in GLUT1 protein synthesis through partial inhibition of GLUT1 mRNA translation and partial inhibition of the rise in GLUT1 mRNA. 4E-BP1 inhibited GLUT1 mRNA translation in vitro. Because phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB), in concert with mTOR, inactivate 4E-BP1, we explored their role in GLUT1 protein expression. Cotransfection of cytomegalovirus promoter-driven, hemagglutinin epitope-tagged GLUT1 with dominant inhibitory mutants of PI3K or PKB inhibited the insulin-elicited increase in hemagglutinin-tagged GLUT1 protein. These results unravel the opposite effects of insulin on GLUT1 and GLUT4 mRNA translation. Increased GLUT1 mRNA translation appears to occur via the PI3K/PKB/mTOR/4E-BP1 cascade.

3T3 Cells↗
PubMed2004

Neomycin prevents the wortmannin inhibition of insulin-stimulated Glut4 translocation and glucose transport in 3T3-L1 adipocytes.

Insulin stimulates the movement of the facilitative glucose transporter glucose transporter-4 (Glut4) from an intracellular compartment to the plasma membrane in adipocytes and muscle cells, resulting in an increased rate of glucose uptake. Insulin-stimulated Glut4 translocation and glucose transport are abolished by wortmannin, a specific inhibitor of phosphatidylinositol 3'-kinase (PI3K). Here, we demonstrate that neomycin, a drug that masks the cellular substrate of PI3K, phosphatidylinositol 4,5-bisphosphate (PIP), prevents wortmannin inhibition of insulin-stimulated (2)Glut4 translocation and glucose transport without activating protein kinase B, a downstream effector of PI3K. These results suggest that PIP(2) may have an important regulatory function in insulin-stimulated Glut4 translocation and glucose transport.

3T3 Cells↗
PubMed2000

Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice.

We have previously demonstrated that the important cis-acting elements regulating transcription of the human GLUT4 gene reside within 895 base pairs (bp) upstream of the transcription initiation site (Thai, M. V., Guruswamy, S., Cao, K. T., Pessin, J. E., and Olson, A. L. (1998) J. Biol. Chem. 273, 14285-14292). Our studies demonstrated that an MEF2 binding site within this region was necessary, but not sufficient, for GLUT4 promoter function in transgenic mice. We have identified a second regulatory element (Domain I) that functions cooperatively with the MEF2 domain in regulating GLUT4 transcription. Using a yeast-one hybrid screen, we obtained a partial cDNA and generated an antibody directed against a protein binding specifically to Domain I. Sequence analysis of the partial cDNA indicates that the protein binding to Domain I is a novel protein. The antibody specifically labels two proteins of approximately 70 and 50 kDa in Western blot analysis. These molecular masses correspond to Domain I binding proteins identified by UV-cross-linking nuclear extracts to a Domain I probe. The antibody raised against the Domain I binding protein inhibited formation of a Domain I-protein complex in electrophoretic mobility shift assays. We conclude that we have identified an authentic, novel, Domain I binding protein required for transcriptional regulation of the human GLUT4 promoter.

Amino Acid Sequence↗