Tyrosine kinase-deficient mutant human insulin receptors (Met1153-->Ile) overexpressed in transfected rat adipose cells fail to mediate translocation of epitope-tagged GLUT4.
Insulin regulates essential pathways for growth, differentiation, and metabolism in vivo. We report a physiologically relevant system for dissecting the molecular mechanisms of insulin signal transduction related to glucose transport. This is an extension of our recently reported method for transfection of DNA into rat adipose cells in primary culture. In the present work, cDNA coding for GLUT4 with an epitope tag (HA1) in the first exofacial loop is used as a reporter gene so that GLUT4 translocation can be studied exclusively in transfected cells. Insulin stimulates a 4.3-fold recruitment of transfected epitope-tagged GLUT4 to the cell surface. Cells cotransfected with the reporter gene and the human insulin receptor gene show an increase in cell surface GLUT4 in the basal state (no insulin) to levels comparable to those seen with maximal insulin stimulation of cells transfected with the reporter gene alone. In contrast, cells overexpressing a naturally occurring tyrosine kinase-deficient mutant insulin receptor (Met1153-->Ile) show no increase in the basal cell surface GLUT4 and no shift in the insulin dose-response curve relative to cells transfected with the reporter gene alone. These results demonstrate that insulin receptor tyrosine kinase activity is essential in insulin-stimulated glucose transport in adipose cells.
Quon
M J
MJ
Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Guerre-Millo
M
M
Zarnowski
M J
MJ
Butte
A J
AJ
Em
M
M
Cushman
S W
SW
Taylor
S I
SI
D000273:Adipose Tissue / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D005947:Glucose / Q000378:metabolism; D051275:Glucose Transporter Type 4; D006801:Humans; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D017931:DNA Primers; D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495692:SLC2A4 protein, human; C495694:Slc2a4 protein, rat; D011972:Receptor, Insulin; D005947:Glucose