Visualization by fluorescence of the binding and internalization of epidermal growth factor in human carcinoma cells A-431.

Abstract

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...

Similarities and differences between the effects of epidermal growth factor and Rous sarcoma virus.

“…We found 7 x 106 saturable EGF binding sites per A431 cell, in general agreement with published values (27), whereas there were 14 x 106 sites per SRA431 (clone 6) cell . We suspect that this difference is not because the SRA431 cells were infected but rather because the A431 cells used to derive the infected lines, and for control experiments, had not been cloned .…”

“…EGF binding was determined essentially as described (26,27). EGF additions to A431 and SRA431 cells were performed with subconfluent cultures, because we had found earlier that A431 cells only showed phosphorylation changes in response to EGF when cultured at low density (15) .…”

Epidermal growth factor activation of NF-κB is mediated through IκBα degradation and intracellular free calcium

“…In this report, the capacity of EGF to induce NF-kB activation in cell lines commonly used for signal transduction studies is documented and elements of the mechanism are described. activity was measured following EGF treatment of human carcinoma A-431 cells, which overexpress EGF receptors (2.0610 6 receptors per cell) (Haigler et al, 1978) and mouse embryo ®broblasts (MEF), which express a more normal level of receptors (5.0610 4 receptors per cell) (J Baulida and G Carpenter, unpublished results). As demonstrated by electrophoretic mobility shift (EMS) assays in Figure 1, EGF addition to A-431 cells induced two shifted complexes with an oligonucleotide probe containing the NF-kB binding site.…”

Effect of lipid mimetics of GM3 and lyso-GM3 dimer on EGF receptor tyrosine kinase and EGF-induced signal transduction

“…Cell culture-Human epidermoid carcinoma A431 cells [10,11] were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT) as described previously [1], but with addition of 1 mM sodium pyruvate, 100 IU/ml penicillin, and 10 µg/ml streptomycin (Mediatech, Inc, Herndon, VA) at 37°C, 5% CO 2 .…”