Affinity labeling of a cell surface receptor for epidermal growth factor.
The membrane receptor for epidermal growth factor (EGF) on 3T3 cells has been identified and specifically labeled radiochemically using a photoreactive derivative of EGF. Photoreactive EGF, labeled with 125I, was incubated with 3T3 cells and then photolyzed in situ to generate a nitrene capable of reacting with a wide variety of chemical bonds. Analysis of the system by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed, besides the band of EGF, only 1 other major radioactive band which migrated at approximately 190,000 daltons. This band was absent when a nonresponsive and nonbinding variant of 3T3 was used. A direct proportionality between binding activity and crosslinked complex formation was demonstrated using a variety of binding conditions. The crosslinked complex in intact cells is accessible to the action of a macromolecule like trypsin at 4 degrees, suggesting a cell surface location for this complex. Upon incubation of cells at 37 degrees, radioactivity from previously formed EGF-receptor crosslinked complex is converted by cellular action to 3 forms of mol wt less than or equal to 58,000 daltons. These are not accessible to trypsin action upon intact cells.
D000345:Affinity Labels; D001386:Azides; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000378:metabolism*; D007097:Imines; D007202:Indicators and Reagents; D010455:Peptides / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000032:analysis*; D013696:Temperature
D000345:Affinity Labels; D001386:Azides; D007097:Imines; D007202:Indicators and Reagents; D010455:Peptides; D011956:Receptors, Cell Surface; D004815:Epidermal Growth Factor