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Functional cloning of TUG as a regulator of GLUT4 glucose transporter trafficking

Abstract

Insulin stimulates glucose uptake in fat and muscle by mobilizing the GLUT4 glucose transporter. GLUT4 is sequestered intracellularly in the absence of insulin, and is redistributed to the plasma membrane within minutes of insulin stimulation1,2. But the trafficking mechanisms that control GLUT4 sequestration have remained elusive. Here we describe a functional screen to identify proteins that modulate GLUT4 distribution, and identify TUG as a putative tether, containing a UBX domain, for GLUT4. In truncated form, TUG acts in a dominant-negative manner to inhibit insulin-stimulated GLUT4 redistribution in Chinese hamster ovary cells and 3T3-L1 adipocytes. Full-length TUG forms a complex specifically with GLUT4; in 3T3-L1 adipocytes, this complex is present in unstimulated cells and is largely disassembled by insulin. Endogenous TUG is localized with the insulin-mobilizable pool of GLUT4 in unstimulated 3T3-L1 adipocytes, and is not mobilized to the plasma membrane by insulin. Distinct regions of TUG are required to bind GLUT4 and to retain GLUT4 intracellularly in transfected, non-adipose cells. Our data suggest that TUG traps endocytosed GLUT4 and tethers it intracellularly, and that insulin mobilizes this pool of retained GLUT4 by releasing this tether.

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Figure 1: Expression cloning of TUG.
Figure 2: Interaction of TUG with GLUT4.
Figure 3: Subcellular localization of TUG.
Figure 4: Mapping of TUG domains in non-adipose cells.

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Acknowledgements

We thank P. Bickel for help with cDNA library construction, G. Paradis for assistance with flow cytometry, J. Cresswell for technical assistance, and N.-W. Chi, J. Avruch, J. Dziura and K. Calia for advice and encouragement. We thank I. Verma for the pCL-Eco plasmid, G. Nolan for retroviral packaging cell lines, M. Charron for GLUT4 antisera, M. Krieger for CHO cells expressing the ecotropic murine retrovirus receptor, and N. Watson and S. Zarnegar for help with microscopy. This work used the W.M. Keck Foundation Biological Imaging Facility at the Whitehead Institute, and the Center for Cell Imaging at Yale University. This work was supported by a New Investigator Award from the Chestnut Hill Charitable Foundation to J.S.B., by a Career Development Award from the American Diabetes Association to J.S.B., by a Pilot and Feasibility Award from the NIH-sponsored Boston Area Diabetes Endocrinology Research Center to J.S.B., by a postdoctoral fellowship from the Ares-Serono Foundation to T.-S.T., and by NIH grants to H.F.L. and J.S.B.

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Correspondence to Jonathan S. Bogan.

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Bogan, J., Hendon, N., McKee, A. et al. Functional cloning of TUG as a regulator of GLUT4 glucose transporter trafficking. Nature 425, 727–733 (2003). https://doi.org/10.1038/nature01989

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