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A photoreactive derivative of epidermal growth factor (EGF) has been used to identify and specifically label a membrane receptor for EGF on mouse 3T3 cells. Photoactivable 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 one other major radioactive band, at a position indicating an apparent molecular weight of 190,000. 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. "Down regulated" cells, in which EGF binding activity was greatly reduced by prolonged incubation with an appropriate concentration of EGF, also had a decrease in covalent complex formation proportional to the decrease in EGF binding activity.
Das et al. (Fri,) studied this question.