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...

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Collagen and/or procollagen was demonstrated on the surface of monolayers of fibroblasts from normal rat kidney by indirect immunofluorescence with affinity-purified antibodies to collagen.

By the use of a rat kidney cell line infected with a temperature-sensitive Rous sarcoma virus, we have shown that, at permissive temperatures where the cells are transformed, concanavalin A induces a clustering of its cell membrane receptors into patches, and the intracellular smooth muscle myosin-like protein is in a disordered state. By contrast, with infected cells grown at nonpermissive temperatures, the addition of concanavalin A does not alter the uniform distribution of its receptors, and the smooth muscle myosin-like protein is arranged in an ordered filamentous structure. These results are consistent with the hypothesis that the myosin protein is part of an intracellular aggregating-disaggregating complex. In the normal cell it is in its aggregated state and inhibits the lateral mobility of the concanavalin A receptors in the membrane; in the transformed cell the complex is relatively disaggregated and permits the concanava in A receptors to be mobile.The addition of protein synthesis inhibitors to infected cells grown at the permissive temperature causes the cells to change from the transformed phenotype to the normal. Removal of the reversible inhibitors causes the cells to revert to the transformed phenotype. These results show that (i) protein synthesis, presumably of an unstable product of the transforming gene of the temperature-sensitive virus, is required to maintain the transformed state in these infected cells at the permissive temperature; and (ii) protein synthesis is not required for the intracellular myosin-containing complex to revert from its disordered transformed state to its ordered normal state. This suggests that the product of the transforming gene directly or indirectly causes the disaggregation of the myosin-containing complex in the process of transformation.A number of different receptor molecules in the cell membranes of cultured fibroblasts show a greater lateral mobility in transformed than in normal cells (1-4). It has been suggested (5, 6) that these differences in receptor mobility are directly connected to the loss of control of normal cell growth which occurs upon transformation. That is, if cell growth requires the mobility of receptors in the cell membrane (to allow membrane expansion and cytokinesis to occur, for example), then normal cell growth can be inhibited by restricting receptor mobility by means of mechanisms that the transformed cell is no longer able to use (5). Several molecular mechanisms have-been proposed to account for these mobility differences, no one of which has yet been generally accepted. We have a working hypothesis (5,7, 8) to explain these mobility differences. (a) There is an intracellular smooth muscle actomyosin-like protein complex present in nonmuscle cells (9) that forms at least part of the microfilament assemblies that have been observed inside these cells (10-12). (b) These actomyosin-like proteins are, at least in part, peripheral proteins (13) bound to the cytoplasmic surfaces of these cell membranes (8), attached noncoval...