Specific Radiolabeling of a Cell Surface Receptor for Epidermal Growth Factor

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 74; no. 7; pp. 2790 - 2794
• Main Authors: Das, Manjusri, Miyakawa, Tokichi, Fox, C. Fred, Pruss, Rebecca M., Aharonov, Aharon, Herschman, Harvey R.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 01-07-1977; National Acad Sciences
• Subjects: 3T3 cells; Affinity Labels; Biochemistry; Cell Line; Cell Membrane - metabolism; Crosslinking; Cultured cells; Electrophoresis; Epidermal Growth Factor - metabolism; Gels; Peptides - metabolism; Photochemistry; Radioactive decay; Receptors; Receptors, Drug - isolation & purification; Receptors, Drug - metabolism; Serum albumins; Sodium; Temperature; Time Factors
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.74.7.2790

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 with125I, 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.