Epidermal Growth Factor Receptor Sites Research Articles

Systematic Analysis of the Epidermal Growth Factor Receptor by Mass Spectrometry Reveals Stimulation-dependent Multisite Phosphorylation

Multisite phosphorylation of proteins is a general mechanism for modulation of protein function and molecular interactions. Definition of phosphorylation sites and elucidation of the functional interplay between multiple phosphorylated residues in proteins are, however, a major analytical challenge in current molecular cell biology and proteomic research. In the present study, we used mass spectrometry to determine the major phosphorylated residues of the human epidermal growth factor (EGF) receptor at various well defined cellular conditions. Activation of EGF receptor was achieved by several types of stimulation, i.e. by sodium pervanadate, EGF, and integrin-dependent adhesion. The contribution of cell-matrix adhesion was also determined by activating the EGF receptor by EGF in cells kept in suspension. We developed an analytical strategy that combined miniaturized sample preparation techniques and MALDI tandem mass spectrometry and determined a total of nine phosphorylation sites in the EGF receptor. We discovered one novel phosphorylation site (Ser967) and revealed constitutive phosphorylation of Thr669, Ser967, Ser1002, and Tyr1045 and stimulation-dependent differential phosphorylation of Tyr1068, Tyr1086, Ser1142, Tyr1148, and Tyr1173. The EGF receptor was purified from HeLa cells or ECV304 cells by immunoprecipitation and SDS-PAGE and then digested with trypsin. Phosphopeptides in the range of 0.8-3.7 kDa were recovered by combinations of IMAC, perfusion chromatography, and graphite powder chromatography and subsequently detected and sequenced by MALDI quadrupole time-of-flight tandem mass spectrometry. Two phosphorylation sites were detected in the peptide 1137GSHQISLDNPDYQQDFFPK1155; however, only Tyr1148 was phosphorylated upon EGF treatment; in contrast Ser1142 was only phosphorylated by integrin-dependent adhesion in the absence of EGF treatment, suggesting differential phosphorylation of this region by distinct stimuli. This MALDI MS/MS-based analytical approach demonstrates the feasibility of systematic analysis of signaling molecules by mass spectrometry and provides new insights into the dynamics of receptor signaling processes.

An Enhancement of Uterine Epidermal Growth Factor Receptor Associated with Embryo Implantation in Rats.

The present study was designed to investigate the physiological significance of epidermal growth factor (EGF) receptors expressed on rat uterine epithelial cells during embryo implantation. The number of uterine EGF receptor sites, as determined with 125I-labelled EGFas a ligand, progressively increased from the 3rd to the 7th day of pregnancy. Analysis of the EGF receptor by Scatchard transformation of EGF-binding data and affinity labelling with 125I-labelled EGF indicated that EGF receptor proteins were noticeably expressed during postimplantation. Determination for EGF receptor sites was further carried out in the uterine tissues of rats, which were superovulating after transplantation of pituitary tissue into a capsule of kidney or subcutaneous injection of 50 IU pregnant mare serum gonadotropin (PMSG) and 50 IU human chorionic gonadotropin (hCG). The transplantation caused an increase in the number of implantation sites (24.5 ± 3.9, n=4) as compared to control rats (14.0 ± 0.6, n=7). Nevertheless, PMSG- and hCG-primed rats totally failed to initiate the implantation process, whereas the number of EGF receptor sites significantly increased during preimplantation (3rd day). In pituitary-transplanted rats on the 7th day, the number of EGF receptor sites was increased as observed in control rats (30.0 ± 0.5 fmol/mg proteins). In PMSG-and hCG-primed rats, however, the EGF receptor was noticeably reduced (7.3 ± 0.4 fmol/mg proteins). These results strongly suggest that functional EGF receptor proteins were noticeably expressed during postimplantation and that its drastic enhancement depends upon the interaction of blastocysts with uterine epithelial cells.

A transforming growth factor related to epidermal growth factor is expressed by fetal mouse salivary mesenchyme cells in culture

Fetal mouse salivary mesenchyme cells secrete a protein with an apparent MW of 15 Kd that is immunologically related to epidermal growth factor (EGF). Conditioned medium collected from these cells in culture stimulates the growth of primary mouse mammary epithelial cells cultured within collagen gels, competes for binding to EGF receptor sites on these mammary epithelial cells and stimulates the anchorage-independent growth of normal rat kidney fibroblast cells within soft agarose. Prior immunoprecipitation of salivary mesenchyme cell conditioned medium with anti-EGF antibodies effectively removes or attenuates all of these effects confirming that an EGF-like factor is involved in these responses.

Epidermal growth factor receptors and EGF‐responsiveness of the human breast‐carcinoma cell line PMC42

PMC42 is an early-passage, well-differentiated, pleiomorphic human breast-cancer cell line. It expresses an average of 2.4 x 10(5) EGF receptor (EGFR) sites per cell when cultures are assayed using a radioreceptor assay. This is a relatively high figure for breast-carcinoma cell lines. Its responses to epidermal growth factor (EGF) were analyzed morphologically and by flow cytometry and immunocytochemistry. PMC42 responded in vitro to EGF with morphological changes, inhibition of "doming" and an increase in DNA synthesis in serum-containing medium in confluent or near-confluent cultures. When EGF receptor sites were localized immunocytochemically in PMC42, they were found to be variably expressed on the membrane of cells grown in the absence of EGF, with discrete areas of strong and weak cells of different morphological appearances; flow cytometry indicated a 10-fold range in EGFR levels between individual cells. Cultures maintained in the presence of EGF were less heterogeneous in their morphology and had a predominantly cytoplasmic, relatively uniform localization of EGFR with down-regulation of membrane EGFR levels. EGF-stimulated cultures were stained simultaneously for the presence of EGFR and the proliferation-related antigen Ki-67 and analyzed by both flow cytometry and immunocytochemistry. No correlation was observed between Ki-67 positivity and level of EGFR expression by individual cells. Electron microscopic localization of receptor sites demonstrated that the receptors were concentrated predominantly on the lateral and basal membranes. These results show that the PMC42 line reflects a number of the properties of heterogeneous breast tumours in situ. It may also continue to express growth-factor receptors in a manner that reflects their topographical distribution on normal luminal breast epithelial cells.

Visualization of Epidermal Growth Factor Receptors in Human Epidermis

The localization of epidermal growth factor (EGF) receptors in normal human epidermis was examined with two independent experimental methods. The distribution of EGF receptor sites was studied using light microscopic autoradiography with [125I]EGF and direct immunocytochemical techniques with EGF receptor antibodies and protein A-colloidal gold complexes. Direct visualization by autoradiography indicated that the concentration of EGF receptors was greatest in the lower epidermal layers. Ultrastructural morphometric analysis of protein A-gold complexes showed that EGF receptors were primarily associated with the plasma membranes although intranuclear and cytoplasmic localization was also evident. This postembedment immunolocalization method also confirmed the relative differences in the number of EGF receptors found in individual epidermal layers (basalis greater than spinosum greater than granulosum greater than corneum layers). This inverse relationship between numbers of EGF receptors and the degree of epidermal differentiation and/or keratinization may suggest a physiologic role for EGF in these processes in human epidermis.

Characteristics of specific binding of epidermal growth factor (EGF) on human tumor cell lines.

Using seventeen human tumor cell lines derived from a variety of tissues, specific binding sites for epidermal growth factor (EGF), a mouse submandibular gland-derived growth factor, has been characterized. A significant amount of membrane-bound EGF receptors, although considerably varied, was demonstrated in all the tumor cell lines studied. Epidermoid carcinoma appeared to have more EGF receptors than adenocarcinoma. One small cell carcinoma of the lung, one choriocarcinoma of the stomach and three bone tumors also possessed EGF receptors comparable to those of epidermoid carcinoma, while one adenoacanthoma of the stomach had less EGF receptors comparable to adenocarcinoma. Among a variety of phorbol esters tested, tetradecanoyl phorbol acetate, a potent tumor promotor, was shown to be the most effective compound in inhibiting 125I-labeled EGF binding to its receptors. Our results indicate that human tumor cells contain varying amounts of membrane-bound receptors for EGF and that phorbol esters interact with these EGF receptor sites. However, the relationship between EGF receptor sites on tumor cells and cellular proliferation and/or differentiation awaits further study.

Culture of A-431 human epidermoid carcinoma cells in serum-free medium: effect of culture conditions on the binding of [125I]-epidermal growth factor.

Serum-free culture conditions that permit the continuous growth of A-431 human epidermoid carcinoma cells were developed. In Dulbecco's modified Eagle's synthetic nutritional medium (DME) supplemented with fetuin, insulin, transferrin, biotin, and oleic acid-fatty acid-free bovine serum albumin complex A-431 cells grew at a rate comparable to that observed in the presence of calf or fetal calf serum. Of the factors tested, oleic acid had the most pronounced stimulatory effect on the growth and [3H]-thymidine incorporation of A-431 cells in serum-free medium. A-431 cells have a high number of receptors for epidermal growth factor (EGF); they bind and rapidly internalize EGF. Nevertheless, EGF did not stimulate either the growth or the [3H]-thymidine incorporation of these cells. Analyses of [125I]-EGF binding data indicated that A-431 cells grown in the presence of calf serum had about 3.2-3.9 X 10(6) specific, saturable EGF receptor sites on their surface. Linear Scatchard plots indicated a single class of noninteracting receptors with an apparent equilibrium dissociation constant of about 2.8 X 10(-9) M. The average number of receptors of A-431 cells maintained in DME supplemented with only fetuin, insulin, and transferrin for several months was significantly less, 1.54 X 10(6), than that of A-431 stock cells cultured in the same medium for 2 days only (2.68 X 10(6)). The apparent dissociation constants for the same cell populations were, however, similar, 4.5 X 10(-9) M and 4.1 X 10(-9) M, respectively. Stimulation of growth by oleic acid resulted in about 20% decrease in the average number of receptor sites, with an increase in the apparent equilibrium dissociation constant.

Retinoids specifically enhance the number of epidermal growth factor receptors.

Retinoids elicit many biological and biochemical responses from cells in vitro. One widely used criterion for the responsiveness of cells to retinoids is inhibition of growth; retinoids reduce the saturation density and/or growth rate of many normal and tumorigenic cell lines. Propagation of eukaryotic cells has been demonstrated to be dependent on the presence of macromolecular growth factors such as epidermal growth factor (EGF), which can stimulate proliferation of epithelial and fibroblastic cell lines. We now describe the effect of retinoids on the binding of EGF to its receptor. Retinoic acid enhances binding of 125I-labelled EGF to various fibroblastic and epidermal cell lines. It has no marked effect on the affinity of this growth factor for its receptor, but increases the number of EGF receptor sites. Retinoic acid has little effect on the binding of concanavalin A (Con A) and insulin, indicating the specific nature of the action of retinoids on cell-surface glycoproteins. Treatment of cells with the phorbol ester 12-o-tetradecanoyl phorbol-13-acetate (TPA) and retinoic acid shows poor antagonism between these compounds on EGF binding. It has been previously shown that retinoids induce or stimulate differentiation of embryonal carcinoma cells. EGF binding can be used as a marker to monitor differentiation of these cells.

Cell cycle regulation by growth factors and nutrients in normal and transformed cells

SV3T3 cells, originally responsive to epidermal growth factor (EGF) and displaying density-dependent inhibition of growth, lose responsiveness to the growth factor after several passages and then proliferate without restriction, but continue to display EGF receptor sites at the cell surface. Proliferation of primary fetal rat hepatocytes is not stimulated by EGF, but cells bind it to an extent comparable to that of responsive 3T3 cells. Therefore presence of EGF receptors does not imply that cells are responsive to the growth factor. The relevance of some growth-factor-induced events for DNA synthesis initiation is dicussed. In various primary and secondary cell cultures, Ca++-levels appear to be involved in controlling cell proliferation. In contrast, in 3T3-4a cells, levels of Ca++ ions are not tightly coupled to DNA synthesis initiation; effects of growth factors are not mediated by extracellular Ca++ ions, but cells have a Ca++-sensitive restriction, point in G1. In various cell types in primary or secondary culture or in 3T3-4a cells, polyamine, levels are not tightly coupled to induction of proliferation. Therefore growth-factor-induced ornithine decarboxylase is not an event essential for DNA synthesis initiation. Normal but not transformed cells have a spermidine/spermine-sensitive restriction point in G1. Although rRNA synthesis appears to be necessary for induction of proliferation, preliminary data obtained by double-beam flow microfluorometry suggest that cellular RNA levels might not affect rate of entry into S phase and, furthermore, that 3T3-4a cells can enter S without accumulating RNA above levels present in quiescent cells. It appears that none of the events induced during the prereplicative phase that have been studied in 3T3 cells are essential for DNA synthesis initiation under normal culture conditions.

Density-dependent regulation of growth of BSC-1 cells in cell culture: control of growth by serum factors.

BSC-1 cells grow slowly, to high cell density, in medium with 0.1% calf serum. An increase in the serum concentration increases both the growth rate of the cells and the final cell density. The serum can be replaced to some extent by epidermal growth factor (EGF). Initiation of DNA synthesis in BSC-1 cells that have spread into a "wound" in a crowded cell layer requires the addition of a trace of serum or EGF, if the cells have previously been deprived of serum. The binding of 125I-labeled EGF to low-density and high-density BSC-1 cells has been studied. Binding is faster to low-density cells. Cells at low cell density also bind much more EGF per cell than cells at high cell density. The fraction of bound 125I-labeled EGF that is present on the cell surface as intact EGF is larger at low than at high cell density. The results indicate that the number of available EGF receptors per cell decreases drastically as the cell density increases. It is suggested that a decrease in the number of available EGF receptor sites per cell, and the accompanying decrease in sensitivity of the cells to EGF, contributes to density-dependent regulation of growth of these cells.