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~z~I-labeled human epidermal growth factor (hEGF) binds in a specific and saturable manner to human fibroblasts. At 37~ the cell-bound ~25I-hEGF initially may be recovered in a native form by acid extraction; upon subsequent incubation, the cell-bound ~25I-hEGF is degraded very rapidly, with the appearance in the medium of 125I-monoiodotyrosine. At 0~ cell-bound nSI-hEGF is not degraded but slowly dissociates from the cell.The data are consistent with a mechanism in which ~zSI-hEGF initially is bound to the cell surface and subsequently is internalized before degradation. The degradation is blocked by inhibitors of metabolic energy production (azide, cyanide, dinitrophenol), some protease inhibitors (Tos-Lys-

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The binding and internalization of epidermal growth factor (EGF) in human epithelioid carcinoma cells (A-431), which have approximately 2.6 X 106 receptors per cell, has been followed with 125I-labeled EGF and by fluorescence microscopy. We have prepared a fluorescent derivative of EGF that is biologically active and retains substantial binding affinity for cell receptors. After binding of this derivative to cells at 60, the cellular borders were prominently stained and the fluorescence on the remainder of the membrane was uniform. Upon warming of these cells to 370 for 10 min, the surface fluorescence diminished and randomly distributed endocytotic vesicles appeared in the cytoplasm. After 20 min at 370 these fluorescent vesicles formed a perinuclear ring. The binding of EGF to the surface of these cells was also visualized by immunofluorescence using rabbit antibodies to EGF and rhodamine-labeled goat anti-rabbit antibodies. We did not detect large fluorescent clusters or cap formation in these experiments. These data provide direct confirmation of the previous biochemical data that suggested that cell membrane-bound EGF is rapidly internalized.Epidermal growth factor (EGF), a small polypeptide (Mr 6045) originally isolated from the submaxillary gland of the mouse, is a potent mitogen for epidermal cells in vivo and for a wide variety of cells in culture (1, 2). The primary sequence of EGF and its physicochemical properties have been reported (3, 4).Specific, saturable receptors for EGF have been demonstrated on cultured cells that are responsive to EGF (5, 6). As a first step in examining the biochemical mechanism by which EGF exerts its growth-promoting effects, we have investigated the metabolic fate of 1251-labeled EGF (1251-EGF) subsequent to its binding to the plasma membrane. Based upon the observations that (i) cell-bound 1251-EGF is rapidly degraded to mono[125I]iodotyrosine, (ii) the degradation is inhibited by drugs such as chloroquine that inhibit lysosomal function, and (111) exposure of fibroblasts to EGF results in an apparent loss of plasma membrane receptors for EGF, it was postulated that the 1251-EGF-receptor complex was internalized and the hormone subsequently was degraded in lysosomes (7).In this report we provide visual evidence supporting this hypothesis. The fate of cell-bound EGF was followed either directly, by using a biologically active fluorescent derivative of EGF, or indirectly, by using immunofluorescence methods. In these studies we have used human epithelioid carcinoma cells (A-431) because of their capacity to bind much larger quantities of EGF compared to human fibroblasts, thus rendering visualization possible.MATERLALS AND METHODS Purified mouse EGF was isolated as described (8). Rabbit antiserum (1) against EGF was purified by affinity chromatography (9). Rhodamine-conjugated affinity-purified goat antibodies against rabbit IgG (R-GARIgG) were prepared as described (10). 125I-EGF was prepared by published procedures (7).Fluorescein-conjugated EGF (Fl-EGF) was sy...

We have prepared a conjugate of epidermal growth factor (EGF) and ferritin that retains substantial binding affinity for cell receptors and is biologically active. Glutaraldehyde-activated EGF was covalently linked to ferritin to produce a conjugate that contained EGF and ferritin in a 1:1 molar ratio. The conjugate was separated from free ferritin by affinity chromatography using antibodies to EGF. Monolayers of human epithelioid carcinoma cells (A-431) were incubated with EGF:ferritin at 4~ and processed for transmission electron microscopy. Under these conditions, ~ 6 • 105 molecules of EGF:ferritin bound to the plasma membrane of each cell. In the presence of excess native EGF, the number of bound ferritin particles was reduced by 99%, indicating that EGF:ferritin binds specifically to cellular EGF receptors. At 37~ cell-bound EGF:ferritin rapidly redistributed in the plane of the plasma membrane to form small groups that were subsequently internalized into pinocytic vesicles. By 2.5 min at 37~ 32% of the cell-bound EGF:ferritin was localized in vesicles. After 2.5 min, there was a decrease in the proportion of conjugate in vesicles with a concomitant accumulation of EGF:ferritin in multivesicular bodies. By 30 min, 84% of the conjugate was located in structures morphologically identified as multivesicular bodies or lysosomes. These results are consistent with other morphological and biochemical studies utilizing I~I-EGF and fluorescein-conjugated EGF.KEY WORDS hormone receptors growth factor 9 endocytosis lysosomes Epidermal growth factor (EGF) ~ initiates a complex series of cellular events which ultimately 1 Abbreviations used in this paper: DMEM/BSA, Dulbecco's Modified Eagle Medium containing 0.1% bovine serum albumin; EGF, epidermal growth factor; I~I-EGF, l~ZI-labeled epidermal growth factor; MVB, multivesicular body; PBS, 9.5 mM sodium phosphate (pH 7.4) containing 4.14 mM KCI and 0.137 M NaCI.results in increased DNA synthesis and cell division (see references 6 and 7 for reviews). Cultured cells that are responsive to EGF contain specific, saturable plasma membrane receptors for this hormone (9, 19). As a first step in examining the biochemical mechanism by which EGF exerts its growth-promoting effects, we have investigated the metabolic fate of EGF using both ~I-labeled EGF and fluorescein-conjugated EGF. Our studies (4, 17) have indicated that cell-bound EGF rapidly is internalized into endocytic vesicles and 382J. CELL BIOLOGY 9 The Rockefeller University Press.