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. 2008 May 20:9:25.
doi: 10.1186/1471-2121-9-25.

An intracellular motif of GLUT4 regulates fusion of GLUT4-containing vesicles

Catherine A Heyward  1 , Trevor R Pettitt, Sophie E Leney, Gavin I Welsh, Jeremy M Tavaré, Michael J O Wakelam
Affiliations

An intracellular motif of GLUT4 regulates fusion of GLUT4-containing vesicles

Catherine A Heyward et al. BMC Cell Biol. .

Abstract

Background: Insulin stimulates glucose uptake by adipocytes through increasing translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. Fusion of GLUT4-containing vesicles at the cell surface is thought to involve phospholipase D activity, generating the signalling lipid phosphatidic acid, although the mechanism of action is not yet clear.

Results: Here we report the identification of a putative phosphatidic acid-binding motif in a GLUT4 intracellular loop. Mutation of this motif causes a decrease in the insulin-induced exposure of GLUT4 at the cell surface of 3T3-L1 adipocytes via an effect on vesicle fusion.

Conclusion: The potential phosphatidic acid-binding motif identified in this study is unique to GLUT4 among the sugar transporters, therefore this motif may provide a unique mechanism for regulating insulin-induced translocation by phospholipase D signalling.

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Figures

Figure 1
Figure 1
ClustalX alignment of glucose transporter family sequences. Amino acid sequences of the known glucose transporter family members were aligned using ClustalX. The potential PtdOH binding motif SQWLxRML (shown by boxed region) is only found in GLUT4, located in the cytoplasmic loop between helices two and three. Lower panel shows the alignment of the GLUT4 region with the phage display peptide, with homologous residues in bold type.
Figure 2
Figure 2
Dual-tagged wild type and mutant GLUT4 translocates to the plasma membrane in response to insulin stimulation in 3T3-L1 adipocytes. 3T3-L1 adipocytes were transiently transfected with (a) wild type or (b) 4A mutant GLUT4HA-GFP and serum starved for 4 hr, stimulated with 83 nM insulin for 10 min then fixed for confocal microscopy as described in Materials and Methods. Green staining shows total GLUT4HA-GFP, red denotes surface exposed HA epitope.
Figure 3
Figure 3
Mutant GLUT4 shows less response to insulin than wild type GLUT4 in 3T3-L1 adipocytes. 3T3-L1 adipocytes were transfected with wild type (WT, black bars) or 4A mutant (MUT, grey bars) pGLUT4HA-GFP and serum-starved for 4 hr, then stimulated with 83 nM insulin for 30 min. GLUT4 translocation was measured by quantitative confocal microscopy as described in Materials and Methods. Data are means of 3 independent experiments ± SEM. A ratio of 1.0 was set for insulin-stimulated wild type cells to normalise the data between the experiments. * indicates values are significantly different (ANOVA P < 0.0005).
Figure 4
Figure 4
4A mutation causes defect in fusion of GLUT4-containing vesicles at the plasma membrane. 3T3-L1 adipocytes transfected with wild type (WT, black bars) or 4A mutant (MUT, grey bars) pGLUT4HA-GFP. Cells were serum-starved for 4 hr prior to stimulation with 83 nM insulin for 30 min. The ratio of Alexa633 to GFP signals at the plasma membrane was quantified as described in Materials and Methods. Histogram (a) shows distribution of ratios for wild type and mutant GLUT4-expressing cells expressed as a % of the total in each condition, and is representative of 3 independent experiments. Graph (b) shows means of 3 independent experiments ± SEM. The difference between wild type and mutant mean values is statistically significant (t-test P < 0.05, ANOVA P < 0.0005).
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