Dynamics of insulin-stimulated translocation of GLUT4 in single living cells visualised using green fluorescent protein.
Insulin increases glucose uptake by promoting the translocation of the GLUT4 isoform of glucose transporters to the plasma membrane. We have studied this process in living single cells by fusing green fluorescent protein (GFP) to the N-terminus (GFP-GLUT4) or C-terminus (GLUT4-GFP), of GLUT4. Both chimeras were expressed in a perinuclear compartment of CHO cells, and in a vesicular distribution through the cytosol. Insulin promoted an increase in plasma membrane fluorescence as a result of net translocation of the chimeras to the cell surface. GLUT4-GFP, but not GFP-GLUT4, was re-internalised upon the removal of insulin suggesting that a critical internalisation signal sequence exists in the N-terminus of GLUT4. The use of GFP thus allows an analysis of GLUT4 trafficking in single living cells.
Dobson
S P
SP
Department of Biochemistry, School of Medical Sciences, University of Bristol, UK.
Livingstone
C
C
Gould
G W
GW
Tavaré
J M
JM
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers; D051275:Glucose Transporter Type 4; D049452:Green Fluorescent Proteins; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000378:metabolism*; D009124:Muscle Proteins; D016133:Polymerase Chain Reaction; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D017931:DNA Primers; D051275:Glucose Transporter Type 4; D007328:Insulin; D008164:Luminescent Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; C495692:SLC2A4 protein, human; D049452:Green Fluorescent Proteins; D011972:Receptor, Insulin