Intermolecular transphosphorylation between insulin receptors and EGF-insulin receptor chimerae.
The insulin receptor, a glycoprotein consisting of two extracellular alpha- and two transmembrane beta-subunits, is thought to mediate hormone action by means of its tyrosine-specific protein kinase activity. To explore the mechanism of insulin receptor phosphorylation we have used NIH3T3 cells transfected with two receptor constructs: one encoding a chimeric receptor composed of the extracellular domain of the human EGF receptor and the cytosolic domain of the human insulin receptor beta-subunit, and a second construct encoding a kinase-defiecient human insulin receptor. Stimulation of these cells with EGF induced tyrosine autophosphorylation of the EGF-insulin receptor chimera (150 kd) and tyrosine phosphorylation of the beta-subunit of the kinase-deficient insulin receptor (95 kd). The phosphopeptides of the autophosphorylated cytoplasmic domain of the EGF-insulin receptor chimera were comparable to those of the transphosphorylated beta-subunit of the kinase-deficient insulin receptor and of the wild-type human insulin receptor. When immunoaffinity purified EGF-insulin receptor hybrids and kinase-deficient insulin receptors were used in a cell lysate phosphorylation assay, it was found that addition of EGF produced 32P-labeling of both receptor species. We conclude that EGF acting directly through the EGF-insulin receptor chimera causes transphosphorylation of the kinase-deficient insulin receptor. These data support the notion that autophosphorylation of the insulin receptor may proceed by an intermolecular mechanism.
Ballotti
R
R
INSERM U 145, Faculté de Médecine, Nice, France.
Lammers
R
R
Scimeca
J C
JC
Dull
T
T
Schlessinger
J
J
Ullrich
A
A
Van Obberghen
E
E
D000818:Animals; D002460:Cell Line; D002678:Chimera; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D014443:Tyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin