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The metabolism of the receptor for epidermal growth factor (EGF) has been measured by labeling the receptor in vivo with radioactive amino acid precursors and then determining, by immunoprecipitation with specific anti-EGF receptor antisera, the rate of degradation of the receptor when the cells are placed in a nonradioactive medium. In human fibroblasts the rate of EGF receptor degradation (t1i2 = 10.1 h) was faster than the rate of degradation of total cell protein . When EGF was added to the nonradioactive medium, the half-life of prelabeled receptor was decreased to 1 .2 h in human fibroblasts . These data demonstrate by direct analysis of receptor protein that during "down regulation" the EGF receptor is rapidly degraded . Enhanced receptor degradation was observed 5-10 min after the addition of EGF . The EGF-induced degradation of the receptor was blocked by methylamine, chloroquine, iodoacetate, or incubation at 25°C . We have also shown that EGF-induced down regulation in human fibroblasts results in a decrease in the total amount of EGF receptor protein present .The amount of EGF receptor protein has been quantitated by radiolabeling cellular protein and immunoprecipitation of the receptor . The EGF receptor constitutes^-0.0035% of the cellular protein in human fibroblasts .Epidermal growth factor (EGF)' is a low molecular weight polypeptide that elicits mitogenic and nonmitogenic responses in a variety of cell types (reviewed in references 1 and 2). A specific membrane receptor for EGF has been identified and purified (3-5). The receptor is a 170,000-dalton glycoprotein having a single polypeptide chain . In addition to a binding site for EGF, the receptor has intrinsic protein kinase activity (5, 6) that is tyrosine specific (7) and activated by the binding of EGF (8) . The protein kinase characteristics of the EGF receptor are similar to properties of protein kinase activities possessed by (a) receptors for other hormones that modulate cell growth, such as platelet-derived growth factor (9) and insulin (10), and (b) certain viral transforming proteins that are also membrane localized (11) .The binding of EGF to its receptors on the surface of cells produces, with continued incubation at 37°C, a decrease in the binding capacity of the cells for '25I-EGF (12). This 'Abbreviation used in this paper: EGF, epidermal growth factor. 1048 phenomenon has been observed for several polypeptide hormone-receptor systems and is referred to as "down regulation." Studies of EGF-induced down regulation have employed chemical (12) or morphological (13-17) techniques to determine the fate of cell-bound EGF labeled with ' 251-, fluorescein, or ferritin . These studies have demonstrated that immediately after the formation of EGF-receptor complexes on the cell surface, the labeled ligand is internalized with the formation of endocytotic vesicles containing the labeled ligand in the vesicle lumen. The ultimate fate of internalized EGF is degradation within lysosomes. The fate ofthe occupied receptor during these ...

To localize epidermal growth factor (EGF) receptors in normal human epidermis and other skin structures, two different light microscopic methods were used. EGF binding [( 125I]EGF/R) to the extracellular portion of the EGF receptor was studied by incubating intact skin samples with [125I]EGF, sectioning the tissues, and performing autoradiography. Immunoreactive EGF receptor molecules (IR-EGF/R) were localized with a mono-specific anti-EGF receptor antibody using a 2-step indirect immunocytochemical method (horseradish peroxidase) and detergent permeabilized tissues. This latter method measured the total pool of EGF receptors: occupied and/or internalized forms, precursor forms, and partially degraded forms of the EGF receptor that retain immunoreactivity. Both the [125I]EGF/R and IR-EGF/R localization studies indicated that EGF receptors were present in basal epidermal keratinocytes, sebocytes, outer root sheath cells in hair follicles, smooth muscle cells of arrector pili muscles, and dermal arteries. The highest levels of [125I]EGF/R and IR-EGF/R were found in the dermal ducts of eccrine sweat glands. The distribution of both [125I]EGF/R and IR-EGF/R was not consistent with the concept that EGF exclusively is involved in cellular division and proliferation in normal human epidermis and its appendages, i.e., EGF receptors were also found in tissues that do not undergo rapid proliferation. The present study indicates that EGF may have a more complex regulatory role in the skin than was previously thought.

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