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. Epidermal growth factor (EGF), a polypeptide isolated from the submaxillary glands of male mice (1), stimulates the proliferation of various epidermal and epithelial tissues, both in vvo and in culture (2). It is a potent mitogen for human fibroblasts (3, 4), human glial cells (5), and mouse cell lines such as 3T3 (6, 7), stimulating cell proliferation at very low concentrations (1-10 ng/ml). EGF has also been isolated from urine of pregnant women (8). Both human and murine EGF are single-chain polypeptides of approximately 6000 molecular weight (8-10). The factors from both species have identical biological properties and compete for the membrane receptor site(s) in a wide variety of vertebrate cells (8). These observations suggest a vitally important function for this growth-regulatory system.Binding of EGF to responsive cells is highly specific (>90%), and at saturating EGF concentrations, 40,000 to 100,000 molecules of EGF are bound per cell (3,11). The presence of such a large number of high-affinity EGF receptors on the cell surface affords a system which should allow the isolation of receptor, e.g., from placenta (12), in quantities sufficient for biochemical characterization. The technique currently used for identification of hormone receptors involves solubilization and affinity purification (13-17). The major drawback of this procedure is that disruption of membrane structure by solubilization may lead to structural alterations in the receptor resulting in a loss of binding affinity or specificity. An alternative approach to this problem is affinity labeling of receptor using modified hormone (18-21). In this technique, the hormonereceptor complex is formed and covalently crosslinked before disruption or solubilization of membranes, thereby allowing the binding to take place under more physiological conditions. In this communication, we report the use of a novel photoactivable derivative of EGF in identification and specific radiolabeling of a membrane receptor for EGF on 3T3 cells.
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