Glypican 3 binds to GLUT1 and decreases glucose transport activity in hepatocellular carcinoma cells.
Glypican 3 (GPC3), a member of heparin sulfate proteoglycans, is attached to the cell surface by a glycosylphosphatidylinositol anchor and is reported to be overexpressed in liver cancers. In order to identify GPC3 binding proteins on the cell surface, we constructed a cDNA containing the C-terminal cell surface-attached form of GPC3 (GPC3c) in a baculoviral vector. The GPC3c bait protein was produced by expressing the construct in Sf21 insect cells and double purified using a His column and Flag immunoprecipitation. Purified GPC3c was used to uncover GPC3c-interacting proteins. Using an LC-MS/MS proteomics strategy, we identified glucose transporter 1 (GLUT1) as a novel GPC3 interacting protein from the HepG2 hepatoma cell lysates. The interaction was confirmed by immunoprecipitation (IP)-WB analysis and surface plasmon resonance (SPR). SPR result showed the interaction of GLUT1 to GPC3c with equilibrium dissociation constants (K(D) ) of 1.61 nM. Moreover, both incubation with GPC3c protein and transfection of Gpc3c cDNA into HepG2 cells resulted in reduced glucose uptake activity. Our results indicate that GPC3 plays a role in glucose transport by interacting with GLUT1.
Cho
Hye-Sim
HS
Department of Biochemistry, School of Dentistry, Kyungpook National University, Daegu, South Korea.
Ahn
Jung-Mo
JM
Han
Ho-Jae
HJ
Cho
Je-Yoel
JY
D000818:Animals; D001692:Biological Transport; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002460:Cell Line; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1 / Q000378:metabolism*; D053673:Glypicans / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D007313:Insecta; D008113:Liver Neoplasms / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods; D014162:Transfection
D051272:Glucose Transporter Type 1; D053673:Glypicans; C495677:SLC2A1 protein, human; D005947:Glucose