Subcellular distribution and activity of glucose transporter isoforms GLUT1 and GLUT4 transiently expressed in COS-7 cells.
In adipose and muscle cells, the glucose transporter isoform GLUT4 is mainly located in an intracellular, vesicular compartment from which it is translocated to the plasma membrane in response to insulin. In order to test the hypothesis that this preferential targeting of a glucose transporter to an intracellular storage site is conferred only by its primary sequence, we compared the subcellular distribution of the fat/muscle glucose transporter GLUT4 with that of the erythrocyte/brain-type glucose transporter GLUT1 after transient expression in COS-7 cells. Full-length cDNA was ligated into the expression vector pCMV that is driven by the cytomegalovirus promoter, and introduced into COS cells by the DEAE-dextran method. Cells were homogenized and fractionated by differential centrifugation to yield plasma membranes and a Golgi-enriched fraction of intracellular membranes (low-density microsomes). In these membrane fractions, the abundance of glucose transporters was assessed by immunoblotting with specific antibodies against GLUT1 and GLUT4, and their transport activity was assayed after solubilization and reconstitution into lecithin liposomes. Uptake rates of 2-deoxyglucose assayed in parallel samples were higher in cells expressing GLUT1 or GLUT4 as compared with control cells (transfection of pCMV without transporter cDNA). Reconstituted glucose transport activity in plasma membranes was about 5-fold higher after expression of GLUT1 and GLUT4 as compared with control cells. The relative amount of GLUT4 in the low-density microsomes as detected by reconstitution and immunoblotting exceeded that of the GLUT1, but was much lower than that observed in typical insulin-sensitive cells, e.g., rat fat cells or 3T3-L1 adipocytes. These data indicate that COS-7 cells transfected with glucose transporter cDNA express the active transport proteins and can be used for functional studies.
Schürmann
A
A
Institut für Pharmakologie und Toxikologie RWTH, Aachen, Germany.
Monden
I
I
Joost
H G
HG
Keller
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D005947:Glucose / Q000378:metabolism; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D015151:Immunoblotting; D007328:Insulin / Q000494:pharmacology; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins; D010957:Plasmids / Q000235:genetics; D014162:Transfection / Q000235:genetics
D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D005947:Glucose