ARF6 Promotes the Formation of Rac1 and WAVE-Dependent Ventral F-Actin Rosettes in Breast Cancer Cells in Response to Epidermal Growth Factor

Coordination between actin cytoskeleton assembly and localized polarization of intracellular trafficking routes is crucial for cancer cell migration. ARF6 has been implicated in the endocytic recycling of surface receptors and membrane components and in actin cytoskeleton remodeling. Here we show that overexpression of an ARF6 fast-cycling mutant in MDA-MB-231 breast cancer-derived cells to mimick ARF6 hyperactivation observed in invasive breast tumors induced a striking rearrangement of the actin cytoskeleton at the ventral cell surface. This phenotype consisted in the formation of dynamic actin-based podosome rosette-like structures expanding outward as wave positive for F-actin and actin cytoskeleton regulatory components including cortactin, Arp2/3 and SCAR/WAVE complexes and upstream Rac1 regulator. Ventral rosette-like structures were similarly induced in MDA-MB-231 cells in response to epidermal growth factor (EGF) stimulation and to Rac1 hyperactivation. In addition, interference with ARF6 expression attenuated activation and plasma membrane targeting of Rac1 in response to EGF treatment. Our data suggest a role for ARF6 in linking EGF-receptor signaling to Rac1 recruitment and activation at the plasma membrane to promote breast cancer cell directed migration.     

Sequential appearance of epidermal growth factor in plasma membrane-associated and intracellular vesicles during endocytosis

Receptor-mediated internalization of epidermal growth factor (EGF) occurs by a process involving initially clathrin-coated pits on the cell surface and the subsequent formation of ligand-containing endosomes. Using a modified acid wash technique, cell surface-bound EGF was removed. Utilizing sucrose density centrifugation, the residual cell-associated EGF was separated into plasma membrane-associated and intracellular vesicle-associated forms. Using these procedures we have identified a transient form of cell-associated EGF that is still attached to the plasma membrane but not accessible to the extracellular fluid. This form of EGF appears to be the precursor for endosomic EGF. We suggest that this intermediate form represents the receptor-ligand complex shown by electronmicroscopy to be located in narrow-necked plasma membrane invaginations (Willingham, M. C., and Pastan, I. (1980) Cell 21, 67-77).     

The F-BAR Protein PACSIN2 Regulates Epidermal Growth Factor Receptor Internalization

Signaling via growth factor receptors, including the epidermal growth factor (EGF) receptor, is key to various cellular processes, such as proliferation, cell survival, and cell migration. In a variety of human diseases such as cancer, aberrant expression and activation of growth factor receptors can lead to disturbed signaling. Intracellular trafficking is crucial for proper signaling of growth factor receptors. As a result, the level of cell surface expression of growth factor receptors is an important determinant for the outcome of downstream signaling. BAR domain-containing proteins represent an important family of proteins that regulate membrane dynamics. In this study, we identify a novel role for the F-BAR protein PACSIN2 in the regulation of EGF receptor signaling. We show that internalized EGF as well as the (activated) EGF receptor translocated to PACSIN2-positive endosomes. Furthermore, loss of PACSIN2 increased plasma membrane expression of the EGF receptor in resting cells and increased EGF-induced phosphorylation of the EGF receptor. As a consequence, EGF-induced activation of Erk and Akt as well as cell proliferation were enhanced in PACSIN2-depleted cells. In conclusion, this study identifies a novel role for the F-BAR-domain protein PACSIN2 in regulating EGF receptor surface levels and EGF-induced downstream signaling.     

Epidermal growth factor induces the accumulation of calpactin II on the cell surface during membrane ruffling

Confluent and proliferatively quiescent T51B rat liver epithelial cells provide a cellular model for the study of epidermal growth factor (EGF) effects in non-neoplastic cells. Immunoreactive calpactin II, a well-known substrate for EGF-receptor kinase, was found predominantly in the cytosol, although a second immunoreactive pool was found in a Triton X-100-extractable membrane fraction. Stimulation with EGF resulted in a rapid and transient (2-5 min) formation of ruffles at the cell surface and at the cell-cell contacts. Both calpactin II and filamentous actin were found co-localized at the membrane ruffles. Immunoprecipitations of membrane-bound calpactin II from 32P-labeled cells indicate a transient EGF-dependent phosphorylation of calpactin II correlating with membrane ruffling. These results suggest a temporal (2-5 min) function for calpactin II at the plasma membrane during the EGF-induced mitogenesis of T51B cells.     

Ganglioside-mediated modulation of cell growth. Specific effects of GM3 on tyrosine phosphorylation of the epidermal growth factor receptor

Glycosphingolipids added exogenously to 3T3 cells in culture were shown to inhibit cell growth, alter the membrane affinity to platelet-derived growth factor binding, and reduce platelet-derived growth factor-stimulated membrane phosphorylation (Bremer, E., Hakomori, S., Bowen-Pope, D. F., Raines, E., and Ross, R. (1984) J. Biol. Chem. 259, 6818-6825). This approach has been extended to the epidermal growth factor (EGF) receptor of human epidermoid carcinoma cell lines KB and A431. GM3 and GM1 gangliosides inhibited both KB cell and A431 cell growth, although GM3 was a much stronger inhibitor of both KB and A431 cell growth. Neither GM3 nor GM1 had any affect on the binding of 125I-EGF to its cell surface receptor. However, GM3 and, to a much lower extent, GM1 were capable of inhibiting EGF-stimulated phosphorylation of the EGF receptor in membrane preparations of both KB and A431 cells. Further characterization of GM3-sensitive receptor phosphorylation was performed in A431 cells, which had a higher content of the EGF receptor. The following results were of particular interest. (i) EGF-dependent tyrosine phosphorylation of the EGF receptor and its inhibition by GM3 were also demonstrated on isolated EGF receptor after adsorption on the anti-receptor antibody-Sepharose complex, and the receptor phosphorylation was enhanced on addition of phosphatidylethanolamine. (ii) Phosphoamino acid analysis of the EGF receptor indicated that the reduction of phosphorylation induced by GM3 was entirely in the phosphotyrosine and not in the phosphoserine nor phosphothreonine content. (iii) The inhibitory effect of GM3 on EGF-dependent receptor phosphorylation could be reproduced in membranes isolated from A431 cells that had been cultured in medium containing 50 nmol/ml GM3 to effect cell growth inhibition. The membrane fraction isolated from such growth-arrested cells was found to be less responsive to EGF-stimulated receptor phosphorylation. These results suggest that membrane lipids, especially GM3, can modulate EGF receptor phosphorylation in vitro as well as in situ.     

Specificity in the synergism between retinoic acid and EGF on the growth of adult human skin fibroblasts

Vitamin A (retinol) and five retinoids were tested for their ability to enhance epidermal growth factor (EGF) stimulation of adult human skin fibroblast growth in vitro. The retinoids utilized in this study were RO-1-5488 (all-trans-retinoic acid), RO-4-3780 (13-cis-retinoic acid), RO-10-9359, RO-10-1670, and RO-21-6583. Retinol and each retinoid were capable of stimulating fibroblast growth alone (0-86%), while 13-cis and all-trans-retinoic acid were the most potent in potentiating the EGF promotion of fibroblast growth. Other growth factors tested in addition to EGF were nerve growth factor (NGF), fibroblast growth factor (FGF), and thrombin. While EGF and FGF stimulated fibroblast growth to the same degree (2.3-fold), only growth stimulated by EGF was potentiated by retinoic acid. Since retinoic acid might enhance the EGF stimulation of cell growth by increasing either EGF receptor number or binding affinity, the binding of 125I-labeled EGF was carried out in the presence of retinoic acid and the data were subjected to a Scatchard-type analysis. No change in EGF receptor number or affinity was seen in the presence of retinoic acid. The data indicate a specific interaction between retinoid acid and EGF which results in the potentiation of the EGF-stimulated cell growth. Furthermore, the mechanism of this interaction does not seem to involve the initial binding of EGF to its plasma membrane receptor or the available number of EGF receptors located on the cell surface.     

Comparison of rapid changes in surface morphology and coated pit formation of PC12 cells in response to nerve growth factor, epidermal growth factor, and dibutyryl cyclic AMP

Scanning and transmission electron microscopic studies were carried out on the rapid cell surface response of PC12 pheochromocytoma cells to treatment with nerve growth factor (NGF), epidermal growth factor (EGF), and dibutyryl cyclic AMP. EGF induced a rapidly initiated series of surface changes identical to those previously observed with NGF. Ruffles appear over the dorsal surface of the cells by 30 s, are prominent at 3 min, and are absent by 7 min. Microvilli disappear as dorsal ruffles become prominent. Peripheral ruffles are seen by 3 min, are prominent on most of the cells by 7 min, and are virtually absent by 15 min. Large blebs are present on 50% of the cells by 2 h and are markedly decreased by 4 h. Within 30 s after NGF or EGF addition, an increase in the density of 60-130-nm coated pits per unit membrane is detectable. This reaches a maximum of two- to threefold in from 1 to 3 min and gradually decreases. Combined treatment with NGF and EGF increases surface ruffling and, after an early peak in coated pits which at 3 min is similar in magnitude to that observed for the separately administered factors, maintains a greater number of pits per unit area than either treatment alone. 3-d pretreatment with NGF greatly reduces the response of the cells to EGF both with respect to surface ruffling and coated pit formation while 4-h NGF pretreatment has no effect on the EGF response. Dibutyryl cyclic AMP induced none of the rapidly onsetting changes caused by NGF or EGF, and therefore it seems unlikely that cyclic AMP mediates these surface changes. Changes in cell surface architecture induced by NGF and EGF on PC12 cells and by NGF in normal sympathetic neurons (as previously described) indicates that such responses may be a widespread phenomenon associated with the interaction of at least some peptide growth factors/hormones with their receptors. These responses may represent or reflect primary events in the mechanism by which these factors act.     

Epidermal growth factor receptor distribution during chemotactic responses

To determine the distribution of the epidermal growth factor (EGF) receptor (EGFR) on the surface of cells responding to EGF as a chemoattractant, an EGFR-green fluorescent protein chimera was expressed in the MTLn3 mammary carcinoma cell line. The chimera was functional and easily visualized on the cell surface. In contrast to other studies indicating that the EGFR might be localized to certain regions of the plasma membrane, we found that the chimera is homogeneously distributed on the plasma membrane and becomes most concentrated in vesicles after endocytosis. In spatial gradients of EGF, endocytosed receptor accumulates on the upgradient side of the cell. Visualization of the binding of fluorescent EGF to cells reveals that the affinity properties of the receptor, together with its expression level on cells, can provide an initial amplification step in spatial gradient sensing.     

Bipartite Tetracysteine Display Reveals Allosteric Control of Ligand-Specific EGFR Activation

Aberrant activation of the epidermal growth factor receptor (EGFR), a prototypic receptor tyrosine kinase, is critical to the biology of many common cancers. The molecular events that define how EGFR transmits an extracellular ligand binding event through the membrane are not understood. Here we use a chemical tool, bipartite tetracysteine display, to report on ligand-specific conformational changes that link ligand binding and kinase activation for full-length EGFR on the mammalian cell surface. We discover that EGF binding is communicated to the cytosol through formation of an antiparallel coiled coil within the intracellular juxtamembrane (JM) domain. This conformational transition is functionally coupled to receptor activation by EGF. In contrast, TGFα binding is communicated to the cytosol through formation of a discrete, alternative helical interface. These findings suggest that the JM region can differentially decode extracellular signals and transmit them to the cell interior. Our results provide new insight into how EGFR communicates ligand-specific information across the membrane.     

Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components.

We have recently demonstrated the formation of interconnecting canalicular cell processes in bone cells upon contact with basement membrane components. Here we have determined whether growth factors in the reconstituted basement membrane (Matrigel) were active in influencing the cellular network formation. Various growth factors including transforming growth factor beta (TGF-beta), epidermal growth factor (EGF), insulin-like growth factor 1, bovine fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) were identified in Matrigel. Exogenous TGF-beta blocked the cellular network formation. Conversely, addition of TGF-beta 1 neutralizing antibodies to Matrigel stimulated the cellular network formation. bFGF, EGF, and PDGF all promoted cellular migration and organization on Matrigel. Addition of bFGF to MC3T3-E1 cells grown on Matrigel overcame the inhibitory effect of TGF-beta. Some TGF-beta remained bound to type IV collagen purified from the Engelbreth-Holm-Swarm tumor matrix. These data demonstrate that reconstituted basement membrane contains growth factors which influence cellular behavior, suggesting caution in the interpretation of experiments on cellular activity related to Matrigel, collagen type IV, and possibly other extracellular matrix components.     

Primary amines do not prevent the endocytosis of epidermal growth factor into 3T3 fibroblasts

Various amines block the degradation of endocytosed epidermal growth factor (EGF) without affecting the binding of the hormone to its surface receptors. However, studies based on fluorescence microscopy demonstrate that amines block the internalization of alpha 2-macroglobulin and EGF by preventing it from clustering in clathrin coated pits. In order to resolve this controversy we have studied in detail the effect of various amines on the localization and processing of fluorescent and radiolabelled EGF. We have explored the effect of amines on EGF binding and localization, receptor mobility, membrane fluidity, receptor down regulation, hormone degradation and release of degradative products as a function of time and temperature. Our conclusions are as follows. 1. Primary amines prevent the formation of visible patches of fluorescent EGF and alpha 2-macroglobulin on the cell surface at least for 15 min, thus increasing the diffusion coefficients and the mobile fraction of EGF-receptor complexes on the cell surface. 2. Amines do not block the endocytosis of EGF and alpha 2-macroglobulin. On most cells fluorescent EGF and alpha 2-macroglobulin are clustered and endocytosed within 30-45 min at 37 degrees C. 3. Amines do not effect the internalization of 125I-labelled-EGF and the down regulation of EGF receptors. 4. Amines block the degradation of the endocytosed EGF.     

Inhibition of epidermal growth factor/transforming growth factor-alpha-stimulated cell growth by a synthetic peptide

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.     

Regulation of EGF signaling by cell polarity in MDCK kidney epithelial cells.

Although the presence of a dominant basolateral sorting signal ensures that the majority of newly synthesized epidermal growth factor (EGF) receptors are delivered directly to the basolateral surface in polarized epithelial cells, a fraction of the receptors are also delivered to the apical surface. Similar to most basolateral membrane proteins, the EGF receptor has an additional signal(s) that selectively targets molecules lacking a dominant basolateral signal to the apical surface. Although the physiological relevance of signal hierarchy is not known, alternative targeting may occur in different epithelial cell types or during development. The goal of this study, therefore, was to determine the effect of membrane domain location on EGF receptor function, focusing on EGF-induced MAP kinase signaling and DNA synthesis. Whereas ligand responsiveness was restricted to the basolateral domain in Madin-Darby canine kidney (MDCK) cells expressing a normal complement of receptors, apical ligand was effective if apical receptor density was increased by overexpression of an exogenous wild-type human gene. Unexpectedly, cells expressing apically localized, cytoplasmically truncated receptors, which behave as dominant negative mutations in other cell types, were also responsive to apical EGF. The cytoplasmically truncated molecules appear to have at least two effects: first, to increase the local concentration of ligand at the apical cell surface; and second, to facilitate activation of the relatively few native EGF receptors normally located at the apical surface. These results indicate that cell context is a critical determinant of receptor mutant protein phenotype.     

Possible involvement of GTP-binding proteins in growth regulation of human epidermoid carcinoma cell line A431

A431 cells, a human epidermoid carcinoma cell line, express an unusually large number of cell surface receptors for the epidermal growth factor (EGF). The growth rate of A431 cells was estimated by measuring [3H]thymidine incorporation at the logarithmic growth phase. The growth of the cells in protein-free medium was partially inhibited by exposure of the cells to pertussis toxin, islet-activating protein (IAP). The growth in both serum-containing and protein-free medium was inhibited by high concentrations of EGF, and these inhibitions were partially reversed by treatment of the cells with IAP. The effects of IAP were well correlated with the degree of ADP-ribosylation of a membrane 40-kDa protein. Thus, IAP sensitive G-proteins appear to be involved in the signal transduction of both positive and negative regulation of A431 cell growth. The possibility is also discussed that phosphatidylinositol turnover may participate in growth regulation.     

Developments in the mechanism of growth factor action: activation of protein kinase by epidermal growth factor

The interaction between epidermal growth factor (EGF) and its target cells has been used as a model for studying the regulation of cell proliferation. Many of the details of binding and subsequent internalization and degradation of this growth factor have been elucidated by following the fate of [125I]EGF in the presence of responsive cells. To investigate the membrane-localized biochemical consequences of EGF-receptor complex formation, a subcellular membrane system has been developed. In this system, EGF enhances phosphorylation of its receptor as well as other endogenous proteins. This EGF-stimulable protein kinase activity is not separated from the EGF receptor activity either by detergent solubilization or by affinity purification of the solubilized membranes. The data suggest that the EGF-binding activity and EGF-sensitive protein kinase activity reside in a single membrane protein.     

Rapid phosphorylation and reorganization of ezrin and spectrin accompany morphological changes induced in A-431 cells by epidermal growth factor

Addition of EGF to human carcinoma A-431 cells is known to induce membrane ruffling after approximately 2 min (Chinkers, M., J. A. McKanna, and S. Cohen. 1979. J. Cell Biol. 83:260-265) and the phosphorylation of a protein referred to as p81, a known substrate for various protein-tyrosine kinases (Cooper, J. A., D. F. Bowen-Pope, E. Raines, R. Ross, and T. Hunter. 1982. Cell. 31:263-273). Ezrin, a Mr approximately 80,000 cytoskeletal protein of the isolated chicken microvillar core, is present in actin-containing cell surface structures of a wide variety of cells (Bretscher, A. 1983. J. Cell Biol. 97:425-432). Ezrin was then found to be homologous to p81 and to be phosphorylated on tyrosine in response to EGF (Gould, K. L., J. A. Cooper, A. Bretscher, and T. Hunter. 1986. J. Cell Biol. 102:660-669). Here, the purification of ezrin from human placenta is described. Antibodies to human ezrin, together with antibodies to other microfilament-associated proteins, were used to follow the distribution and phosphorylation of these proteins in A-431 cells after EGF treatment. EGF induces the formation of microvillar-like surface structures on these cells within 30 s and these give way to membrane ruffles at approximately 2-5 min after EGF addition; the cells then round up after approximately 10-20 min. Ezrin is recruited into the microvillar-like structures and the membrane ruffles, and is phosphorylated on tyrosine and serine in a time course that parallels the formation and disappearance of these surface structures. Spectrin is recruited into the membrane ruffles and shows a similar rapid kinetics of phosphorylation, but only on serine residues, and remains phosphorylated through the rounding up of the cells. The microvillar- like structures and membrane ruffles are also enriched in fimbrin and alpha-actinin. Myosin becomes rapidly reorganized into a striated pattern that is consistent with it playing a role in cell rounding. These results show that two cortical proteins, ezrin and spectrin, become phosphorylated in a time course coincident with remodeling of the cell surface. The results are consistent with the notion that ezrin phosphorylation may play a role in the formation of cell surface projections whereas spectrin phosphorylation may be involved in remodelling of more planar areas of the cell surface.     

Biophysical integration of effects of epidermal growth factor and fibronectin on fibroblast migration

Cell migration is regulated simultaneously by growth factors and extracellular matrix molecules. Although information is continually increasing regarding the relevant signaling pathways, there exists little understanding concerning how these pathways integrate to produce the biophysical processes that govern locomotion. Herein, we report the effects of epidermal growth factor (EGF) and fibronectin (Fn) on multiple facets of fibroblast motility: locomotion speed, membrane extension and retraction activity, and adhesion. A surprising finding is that EGF can either decrease or increase locomotion speed depending on the surface Fn concentration, despite EGF diminishing global cell adhesion at all Fn concentrations. At the same time, the effect of EGF on membrane activity varies from negative to positive to no-effect as Fn concentration and adhesion range from low to high. Taking these effects together, we find that EGF and Fn regulate fibroblast migration speed through integration of the processes of membrane extension, attachment, and detachment, with each of these processes being rate-limiting for locomotion in sequential regimes of increasing adhesivity. Thus, distinct biophysical processes are shown to integrate for overall cell migration responses to growth factor and extracellular matrix stimuli.     

Cell surface changes associated with EGF and insulin-induced cell adhesion

The ability of insulin and epidermal growth factor (EGF) to restore cell surface function in cells damaged by serum deprivation has been examined. Both insulin (10^?7 M) and EGF (10^?8 M), when added for 2 h, resulted in reattachment of cells to the culture dish; with insulin, attachment was associated with increased amino acid uptake. Both hormones caused an increase in sialic acid and in free sulfhydryl groups associated with the cell membrane. The mobility of concanavalin A (Con A) on the upper cell surface was increased by EGF, and to a lesser extent by insulin. The effects of insulin and EGF on adhesion appear to be regulated by specific receptors for these hormones.     

Epidermal growth factor induces an increase in cell adhesion and an arrangement of actin skeleton in stress fibres in pituitary cultured cells from infantile rats but not adult rats

The rat anterior pituitary gland undergoes changes in its cyto-architecture during the second and third weeks of postnatal life. However, little is known about the factors that regulate these tissue conformational changes. The epidermal growth factor (EGF) is one of the growth factors that are synthesized by the pituitary gland, and almost all of the pituitary cells have EGF receptors (EGFR). In addition to the effects of the EGF on mitosis and differentiation, this growth factor can modulate cell adhesion, cell migration, and cytoskeletal organization. In this study we focussed our attention in examining the effects of EGF on the adhesion of cells to the extracellular matrix and on the actin cytoskeletal arrangement of pituitary cells from infantile and adult rats. Our results show that in infantile cells the EGF induces cell adhesion with increase in cell surface area. The arrangement of actin-F in infantile EGF-treated cells was in stress fibers and vinculin acquired a striped shape at the membrane border, suggesting the assembly of focal adhesion contacts. In contrast, in adult pituitary cells EGF does not induce any change in cell adhesion, and the cells maintain a rounded shape with an arrangement of actin-F in thin cortical bands even though, immuno-localization of the EGFR was observed in adult cells cultured in defined medium. We also looked for the EGFR in membrane preparations from infantile and adult pituitaries, and a marked difference in membrane EGFR was observed between them, the infantile pituitaries showing a significantly higher amount. Our results suggest that in infantile cells EGF induces the assembly of focal adhesion contacts, and that in adult cells the receptor of this growth factor is uncoupled of the signaling pathway by which a rearrangement of actin cytoskeleton occurs.     

Single-molecule analysis of epidermal growth factor binding on the surface of living cells

Global cellular responses induced by epidermal growth factor (EGF) receptor (EGFR) occur immediately with a less than 1% occupancy among tens of thousands of EGFR molecules on single cell surface. Activation of EGFR requires the formation of a signaling dimer of EGFR bound with a single ligand to each molecule. How sufficient numbers of signaling dimers are formed at such low occupancy rate is still not known. Here, we have analyzed the kinetics of EGF binding and the formation of the signaling dimer using single-molecule imaging and mathematical modeling. A small number of EGFR on the cell surface formed dimeric binding sites, which bound EGF two orders of magnitude faster than the monomeric binding sites. There was a positive cooperative binding of EGF to the dimeric binding sites through a newly discovered kinetic intermediate. These two mechanisms facilitate the formation of signaling dimers of EGF/EGFR complexes.