Signal transduction by epidermal growth factor occurs through the subclass of high affinity receptors

Many cell types display two classes of epidermal growth factor receptor (EGFR) as judged from EGF binding studies; i.e., a major class of low affinity EGFR and a minor class of high affinity EGFR. We have studied their respective contribution to the cascade of events elicited by EGF in human A431 carcinoma cells, using anti-EGFR mAb 2E9. This antibody specifically blocks EGF binding to low affinity EGFR, without activating receptors in intact cells, and thus enables us to study the effects of exclusive EGF binding to high affinity EGFR. We show that blocking of low affinity EGFR by mAb 2E9 has almost no effect on the activation of the receptor protein-tyrosine kinase by EGF, suggesting that EGFR kinase activation occurs exclusively through the subclass of high affinity EGFR (5-10%). In addition, we provide evidence that high affinity EGFR exists both in monomeric and dimeric forms, and that cross-phosphorylation of low affinity EGFR by high affinity EGFR may take place in dimers of both receptor types. We demonstrate that the following early cellular response to EGF are also unimpaired in the presence of mAb 2E9: (a) inositol phosphate production, (b) release of Ca2+ from intracellular stores, (c) rise in intracellular pH, (d) phosphorylation of EGF on threonine residue 654, (e) induction of c-fos gene expression, and (f) alteration in cell morphology. As possible nonspecific side effects, we observed that the EGF induced Ca2+ influx and fluid-phase pinocytosis were inhibited in A431 cells in the presence of mAb 2E9. We conclude, therefore, that the activation of the EGFR signal transduction cascade can occur completely through exclusive binding of EGF to the subclass of high affinity EGFR.     

Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells.

We report a highly specific fluorescence lifetime imaging microscopy (FLIM) method for monitoring epidermal growth factor receptor (EGFR) phosphorylation in cells based on fluorescence resonance energy transfer (FRET). EGFR phosphorylation was monitored using a green fluorescent protein (GFP)-tagged EGFR and Cy3-conjugated anti-phosphotyrosine antibodies. In this FRET-based imaging method, the information about phosphorylation is contained only in the (donor) GFP fluorescence lifetime and is independent of the antibody-derived (acceptor) fluorescence signal. A pixel-by-pixel reference lifetime of the donor GFP in the absence of FRET was acquired from the same cell after photobleaching of the acceptor. We show that this calibration, by acceptor photobleaching, works for the GFP-Cy3 donor-acceptor pair and allows the full quantitation of FRET efficiencies, and therefore the degree of exposed phosphotyrosines, at each pixel. The hallmark of EGFR stimulation is receptor dimerisation [1] [2] [3] [4] and concomitant activation of its intracellular tyrosine kinase domain [5] [6] [7]. Trans-autophosphorylation of the receptor [8] [9] on specific tyrosine residues couples the activated dimer to the intracellular signal transduction machinery as these phosphorylated residues serve as docking sites for adaptor and effector molecules containing Src homology 2 (SH2; reviewed in [10]) and phosphotyrosine-binding (PTB) [11] domains. The time-course and extent of EGFR phosphorylation are therefore important determinants of the underlying pathway and resulting cellular response. Our results strongly suggest that secondary proteins are recruited by activated receptors in endosomes, indicating that these are active compartments in signal transduction.     

Ligand-induced dimer-tetramer transition during the activation of the cell surface epidermal growth factor receptor-A multidimensional microscopy analysis

The epidermal growth factor receptor (EGFR) is a member of the erbB tyrosine kinase family of receptors. For many years it has been believed that receptor activation occurs via a monomer-dimer transition that is associated with a conformational change to activate the kinase. However, little is known about the quaternary state of the receptor at normal levels of expression (<10(5) receptors/cell). We employed multidimensional microscopy techniques to gain insight into the state of association of the human EGFR, in the absence and presence of ligand, on the surface of intact BaF/3 cells (50,000 receptors/cell). Image correlation microscopy of an EGFR-enhanced green fluorescent protein chimera was used to establish an average degree of aggregation on the submicron scale of 2.2 receptors/cluster in the absence of ligand increasing to 3.7 receptors/cluster in the presence of ligand. Energy transfer measurements between mixtures of fluorescein isothiocyanate-EGF and Alexa 555-EGF were performed using fluorescence lifetime imaging microscopy as a function of the donor: acceptor labeling ratio to gain insight into the spatial disposition of EGFR ligand binding sites on the nanometer scale. In the context of a two-state F?rster resonance energy transfer (FRET)/non-FRET model, the data are consistent with a minimum transfer efficiency of 75% in the FRET population. The microscopy data are related to biophysical data on the EGFR in the A431 cell line and the three-dimensional structure of the ligated EGFR extracellular domain. In the context of a monomer-dimer-oligomer model, the biophysical data are consistent with a significant fraction of ligated EGFR tetramers comprising two dimers juxtaposed in a side-by-side (or slightly staggered) arrangement. Our data are consistent with a specific higher order association of the ligand-bound EGFR on the nanometer scale and indicate the existence of distinct signaling entities beyond the level of the EGFR dimer which could play an important role in receptor transactivation.     

Unligated epidermal growth factor receptor forms higher order oligomers within microclusters on A431 cells that are sensitive to tyrosine kinase inhibitor binding

Characterization of the association states of the unligated epidermal growth factor receptor (EGFR) is important in understanding the mechanism of EGFR tyrosine kinase activation in a tumor cell environment. We analyzed, in detail, the association states of unligated, immunotagged EGFR on the surface of intact epidermoid carcinoma A431 cells, using AlexaFluor488 and AlexaFluor546 anti-EGFR antibody, mAb528, as probes. Image correlation microscopy revealed the presence of unligated EGFR in submicron scale clusters containing an average of 10-30 receptors (mean cluster density = 32 +/- 9 clusters per square micron). Lifetime-based F?rster resonance energy transfer (FRET) techniques as a function of acceptor:donor labeling ratio disclosed a clustering of the unligated EGFR in clusters containing an average of four receptors on the nanometer (<10 nm) scale. The relationship between the nanoscale and submicron scale associations was determined using a new analysis that combines nanoscale information from lifetime-detected FRET imaging with submicron scale information obtained with image correlation microscopy. This analysis revealed the presence of monomers (or small oligomers) and larger clusters containing 15-30 receptors that were partially associated on the sub-10 nm scale. Pretreatment of the cells with the tyrosine kinase inhibitor AG1478 caused a partial dispersal of the submicron clusters (mean cluster density = 85 +/- 15 clusters per square micron; mean degree of association = 4-10 receptors per cluster) and reduced the level of FRET down to our limit of detection. These results are consistent with a higher order nanoscale receptor organization of the unligated receptor population that is partially controlled by the kinase domains. The ramifications of the results to mechanisms of EGFR activation in a tumor cell environment are discussed.     

Fluorescence resonance energy transfer-based stoichiometry in living cells

Imaging of fluorescence resonance energy transfer (FRET) between fluorescently labeled molecules can measure the timing and location of intermolecular interactions inside living cells. Present microscopic methods measure FRET in arbitrary units, and cannot discriminate FRET efficiency and the fractions of donor and acceptor in complex. Here we describe a stoichiometric method that uses three microscopic fluorescence images to measure FRET efficiency, the relative concentrations of donor and acceptor, and the fractions of donor and acceptor in complex in living cells. FRET stoichiometry derives from the concept that specific donor-acceptor complexes will give rise to a characteristic FRET efficiency, which, if measured, can allow stoichiometric discrimination of interacting components. A first equation determines FRET efficiency and the fraction of acceptor molecules in complex with donor. A second equation determines the fraction of donor molecules in complex by estimating the donor fluorescence lost due to energy transfer. This eliminates the need for acceptor photobleaching to determine total donor concentrations and allows for repeated measurements from the same cell. A third equation obtains the ratio of total acceptor to total donor molecules. The theory and method were confirmed by microscopic measurements of fluorescence from cyan fluorescent protein (CFP), citrine, and linked CFP-Citrine fusion protein, in solutions and inside cells. Together, the methods derived from these equations allow sensitive, rapid, and repeatable detection of donor-, acceptor-, and donor-acceptor complex stoichiometry at each pixel in an image. By accurately imaging molecular interactions, FRET stoichiometry opens new areas for quantitative study of intracellular molecular networks.     

Regulation of the epidermal growth factor receptor phosphorylation state by sphingosine in A431 human epidermoid carcinoma cells

The regulation of protein phosphorylation by sphingosine in A431 human epidermoid carcinoma cells was examined. Sphingosine is a competitive inhibitor of phorbol ester binding to protein kinase C (Ca2+/phospholipid-dependent enzyme) and potently inhibits phosphotransferase activity in vitro. Addition of sphingosine to intact A431 cells caused an inhibition of the phorbol ester-stimulated phosphorylation of two protein kinase C substrates, epidermal growth factor (EGF) receptor threonine 654 and transferrin receptor serine 24. We conclude that sphingosine inhibits the activity of protein kinase C in intact A431 cells. However, further experiments demonstrated that sphingosine-treatment of A431 cells resulted in the regulation of the EGF receptor by a mechanism that was independent of protein kinase C. First, sphingosine caused an increase in the threonine phosphorylation of the EGF receptor on a unique tryptic peptide. Second, sphingosine caused an increase in the affinity of the EGF receptor in A431 and in Chinese hamster ovary cells expressing wild-type (Thr654) and mutated (Ala654) EGF receptors. Sphingosine was also observed to cause an increase in the number of EGF-binding sites expressed at the surface of A431 cells. Examination of the time course of sphingosine action demonstrated that the effects on EGF binding were rapid (maximal at 2 mins) and were observed prior to the stimulation of receptor phosphorylation (maximal at 20 mins). We conclude that sphingosine is a potently bioactive molecule that modulates cellular functions by: 1) inhibiting protein kinase C; 2) stimulating a protein kinase C-independent pathway of protein phosphorylation; and 3) increasing the affinity and number of cell surface EGF receptors.     

pp60cSrc Is a Caspase-3 Substrate and Is Essential for the Transformed Phenotype of A431 Cells

The protein tyrosine kinase c-Src is a major signal transduction element in many growth factor receptor signals for proliferation and transformation. We showed recently that c-Src is a mediator of antiapoptotic signals through regulation of the antiapoptotic gene Bcl-X_L. A431 cells overexpress the EGF receptor (EGFR) and possess high Src activity. In A431 cells, Src is activated by the EGFR, and inhibition of the EGF receptor results in c-Src inhibition. In this study we show that (i) inhibition of the EGFR kinase or Src kinase by specific inhibitors results in growth inhibition and inhibition of colony formation in soft agar. The relative efficacies of the EGFR kinase inhibitor and of the Src kinase inhibitor are similar suggesting the major role src plays in the oncogenic signaling of EGFR in A431 cells. (ii) The Src kinase inhibitor PP1 sensitizes A431 cells to CDDP-induced apoptosis. (iii) CDDP induces caspase-3-dependent cleavage of the c-Src C-terminal portion and a concomitant reduction in Bcl-X_L levels. We conclude that c-Src is an important antiapoptotic signaling molecule downstream of the EGF receptor that contributes to the transformed phenotype of A431 cells.     

Comparison of epidermal growth factor (EGF) receptor-receptor interactions in intact A431 cells and isolated plasma membranes. Large scale receptor micro-aggregation is not detected during EGF-stimulated early events

We have used resonance energy transfer to monitor epidermal growth factor (EGF) receptor micro-aggregation at the surface of intact human epidermoid carcinoma (A431) cells. EGF molecules labeled with fluorescein isothiocyanate and eosin isothiocyanate were demonstrated to bind tightly to cellsurface receptors, to elicit immediate changes in cytosolic free [Ca2+], and to undergo endocytosis. Under conditions which maintain the integrity of the cell, we observed no energy transfer between the donor fluorescein isothiocyanate-labeled EGF molecules and the acceptor eosin isothiocyanate-labeled growth factors bound to receptors. However, after disruption of cells by Dounce homogenization, a significant degree of energy transfer was observed (approximately 10-20%) with membranes, indicative of receptor aggregation. These results suggest that EGF does not cause micro-aggregation of the majority of its receptors on the surface of intact A431 cells within the time period of the early events associated with growth factor action. Moreover, it appears that the A431 cells contain some component which imparts a constraint on the ability of EGF receptors to aggregate, and that some of this component is lost upon the disruption of cells.     

Modulation of type alpha transforming growth factor receptors by a phorbol ester tumor promoter

Epidermal growth factor (EGF) and an EGF-like transforming growth factor (eTGF) from retrovirally transformed cells bind to a common receptor type in A431 cells. We have investigated the effects of the tumor promoter phorbol myristate acetate [PMA] on EGF/eTGF receptors in intact A431 cells. Treatment with PMA at 37 degrees C induces a complete loss of high-affinity (Kd = 35-50 pM) binding sites for eTGF and EGF on the cell surface of A431 cells. This effect is half-maximal at 0.1 nM PMA, exhibits rapid kinetics, and persists for at least 4 hr in the presence of PMA. eTGF and PMA added to intact A431 cells induce the phosphorylation of immunoprecipitable 170kd EGF/eTGF receptors. The EGF/eTGF receptor isolated from control cells was found to contain phosphoserine and phosphothreonine. PMA and eTGF caused a marked increase in the level of these two phosphoamino acids. In addition, eTGF but not PMA caused the appearance of phosphotyrosine in the EGF/eTGF receptor in vivo. We conclude that the tumor-promoting phorbol diester regulates both the affinity and phosphorylation state of the A431 cell receptor for the type alpha transforming growth factors, eTGF and EGF.     

Rotational mobility of high-affinity epidermal growth factor receptors on the surface of living A431 cells.

The rotational diffusion of epidermal growth factor (EGF) bound to its specific receptor on the surface of human carcinoma A431 cells was studied by means of time-resolved phosphorescence anisotropy measurements. The rotational mobility was measured on the total population of EGF receptors by using a saturating concentration of EGF conjugated with a phosphorescent label, erythrosin, or on the subpopulation of high-affinity EGF receptors by using a low concentration of labeled EGF. At 4 degrees C, the rotational correlation times for both the high-affinity and total (mostly low affinity) receptor populations were in the range of 60-100 microns. Elevation of the temperature to 37 degrees C resulted in a lengthening of the rotational correlation time of the total receptor population to 200-300 microns, confirming a previous study of receptor microaggregation. The high-affinity EGF receptors were completely immobilized at 37 degrees C (rotational correlation time greater than 500 microns). The data are consistent with a model involving association of the cytoskeleton with the high-affinity receptors at 37 degrees C, but not at 4 degrees C.     

Consumption of EGF by A431 cells: evidence for receptor recycling

We examined the extent of EGF consumption by EGFR in A431 cells. When 125I-EGF was added to A431 cell cultures at low or high density, at concentrations which corresponded to 10-fold excess of ligand over receptor on the cell surface, most of the 125I-EGF was consumed within 2 h. The amounts of 125I-EGF consumed were much greater than available EGFR on the A431 cells, by a factor of 6.5 in low-density cultures and 5.8 in high-density cultures. When the concentration of 125I-EGF was increased in low density cultures, further consumption of 125I-EGF by the A431 cells was greatly reduced, partially due to a rapid down regulation of EGFR. However, when higher concentrations of 125I-EGF were added to high density cultures, with reduced receptor down regulation, the cells continued to consume a large fraction of the EGF in the culture medium. The consumption of 125I-EGF by these cultures was in excellent agreement with the measured amount of ligand internalized into the cell. EGF consumption was far in excess of the number of EGFR down regulated or degraded. Only a minor portion of the EGFR could have been replaced during the assay period by synthesis of new EGFR or from the intracellular pool of EGFR, and the fluid-phase uptake of EGF is only temporarily increased by exposure to EGF. Our results demonstrate that EGFR in high density A431 cell cultures recycled many times. The apparent level of recycling was dependent upon the concentration of EGF and followed Michaelis-Menton kinetics for ligand concentrations as high as 215 nM. At this EGF concentration, high-density cultures consumed 45 EGF molecules per receptor over a period of 6 h.     

Gefitinib induces epidermal growth factor receptor dimers which alters the interaction characteristics with ¹²⁵I-EGF

The tyrosine kinase inhibitor gefitinib inhibits growth in some tumor types by targeting the epidermal growth factor receptor (EGFR). Previous studies show that the affinity of the EGF-EGFR interaction varies between hosting cell line, and that gefitinib increases the affinity for some cell lines. In this paper, we investigate possible mechanisms behind these observations. Real-time interaction analysis in LigandTracer® Grey revealed that the HER2 dimerization preventing antibody pertuzumab clearly modified the binding of ¹²⁵I-EGF to EGFR on HER2 overexpressing SKOV3 cells in the presence of gefitinib. Pertuzumab did not affect the binding on A431 cells, which express low levels of HER2. Cross-linking measurements showed that gefitinib increased the amount of EGFR dimers 3.0–3.8 times in A431 cells in the absence of EGF. In EGF stimulated SKOV3 cells the amount of EGFR dimers increased 1.8–2.2 times by gefitinib, but this effect was cancelled by pertuzumab. Gefitinib treatment did not alter the number of EGFR or HER2 expressed in tumor cell lines A431, U343, SKOV3 and SKBR3. Real-time binding traces were further analyzed in a novel tool, Interaction Map, which deciphered the different components of the measured interaction and supports EGF binding to multiple binding sites. EGFR and HER2 expression affect the levels of EGFR monomers, homodimers and heterodimers and EGF binds to the various monomeric/dimeric forms of EGFR with unique binding properties. Taken together, we conclude that dimerization explains the varying affinity of EGF – EGFR in different cells, and we propose that gefitinib induces EGFR dimmers, which alters the interaction characteristics with ¹²⁵I-EGF.     

Biochemical and biophysical characterization of serotonin 5-HT2C receptor homodimers on the plasma membrane of living cells

While many studies have provided evidence of homodimerization and heterodimerization of G-protein-coupled receptors (GPCRs), few studies have used fluorescence resonance energy transfer (FRET) combined with confocal microscopy to visualize receptor dimerization on the plasma membrane, and there have been no reports demonstrating the expression of serotonin receptor dimers/oligomers on the plasma membrane of living cells. In the study presented here, biochemical and biophysical techniques were used to determine if 5-HT(2C) receptors exist as homodimers on the plasma membrane of living cells. Immunoprecipitation followed by Western blotting revealed the presence of immunoreactive bands the predicted size of 5-HT(2C) receptor monomers and homodimers that were detergent and cross-linker sensitive. Bioluminescence resonance energy transfer (BRET) was assessed in HEK293 cells expressing 5-HT(2C) receptors labeled with Renilla luciferase and yellow fluorescent protein. BRET levels were not altered by pretreatment with serotonin. Confocal microscopy provided direct visualization of FRET on the plasma membrane of live cells expressing 5-HT(2C) receptors labeled with cyan (donor) and yellow (acceptor) fluorescent proteins. FRET, assessed by acceptor photobleaching, was dependent on the donor/acceptor ratio and independent of acceptor expression levels, indicating that FRET resulted from receptor clustering and not from overexpression of randomly distributed receptors, providing evidence for GPCR dimers/oligomers in a clustered distribution on the plasma membrane. The results of this study suggest that 5-HT(2C) receptors exist as constitutive homodimers on the plasma membrane of living cells. In addition, a confocal-based FRET method for monitoring receptor dimerization directly on the plasma membrane of living cells is described.     

Regulation by ceramide of epidermal growth factor signal transduction and mitogenesis in cell lines overexpressing the growth factor receptor.

Ceramide has emerged as a pleiotropic signal mediator of cellular responses including differentiation, proliferation, cell cycle arrest and apoptosis. In the present study we evaluated the effect of cell permeant ceramide analogues on ligand-induced tyrosine phosphorylation of the EGF receptor (EGFR), phospholipase Cy (PLCgamma) activity and cell proliferation. Treatment with N-acetylsphingosine (C2-cer) and N-hexanoylceramide (C6-cer) prevented EGF-induced tyrosine trans-phosphorylation of the receptor in two different cell lines overexpressing the human EGFR (A431 and EGF-T17 cells). In contrast, treatment of A431 and EGFR-T17 cells with C2-cer or C6-cer did not affect the ligand binding capacity of the receptor, an effect that was however observed after TPA-induced activation of PKC. In addition EGF-stimulated PLCgamma activity was transiently decreased in A431 cells treated with C6-cer and only a modest, albeit significant reduction on ligand-induced 3H-InsP3 generation was observed in EGFR-T17 cells pretreated with ceramide. We also examined the effect of C2-cer on serum (A431)- or EGF (EGFR-T 17)-induced cell proliferation. Treatment of EGFR-TI7 cells with C2-cer (0.1-10 microM) did not affect cell viability, but prevented EGF-induced 3H-thymidine incorporation in a dose-dependent manner. In contrast, 3H-thymidine incorporation in serum-stimulated A431 cells decreased only at the higher doses of C2-cer used (1-10 microM), being this effect accompanied by a slight, albeit significant (20-25%), reduction in cell viability.     

Confocal microscopic dual-laser dual-polarization FRET (2polFRET) at the acceptor side for correlating rotations at different distances on the cell surface

Relationship of donor and acceptor fluorescence anisotropies as well as efficiency of fluorescence resonance energy transfer (FRET) has been investigated in a confocal microscope in the context of FRET systems comprised of donor and acceptor-labeled MHCI and MHCII receptors on the surface of Kit-225 K6 human T-cells. The measurements have been carried out in a 2-laser, 5-signal platform where the total donor fluorescence intensity and 2 acceptor fluorescence intensities with their anisotropies – one at the donor's excitation wavelength, the other at the acceptor's excitation wavelength – have been detected. This configuration enabled the determination of FRET efficiency and correlating it with the two acceptor fluorescence anisotropies as a kind of calibration. Estimations for the FRET-enhanced donor fluorescence anisotropy, the directly excited acceptor fluorescence anisotropy, and the fluorescence anisotropy of sensitized emission have been obtained. Procedures for determining FRET by measuring only the total donor intensity and the acceptor intensity and its anisotropy, or two acceptor intensities and their anisotropies have been elaborated, the errors of which have been estimated based on the fluorescence anisotropy values obtained in the calibration with the method of flow cytometric energy transfer (FCET).The combined detection of the donor and acceptor fluorescence anisotropies enabled also the determination of the lower and upper limits of the orientation factor for FRET (κ²). An increase in range for κ² with increasing FRET efficiency has been observed, with average κ² values different from the dynamic random average of 2/3. These observations call for the need of κ² determination in proximity measurements, where the donor and acceptor orientations are not predictable.An increasing range of κ² with increasing intermolecular proximity of the MHCI and MHCII receptors has been observed. This indicates that molecular flexibility in the clusters of the MHCI and MHCII receptors reduces with increasing cluster density, i.e. a “fluidity gradient” exists in the clusters. More specifically, the local density dependent flexibility can also be taken as a direct proof for that the association of these receptors is non-random, but mediated by some type of physical interaction, a finding as a benefit of FRET detection by polarization spectroscopy.Two new quantities – the quenched donor fluorescence anisotropy and a fluorescence anisotropy analogue, the “dissymmetry index” of the polarized FRET efficiency components – have also been introduced for the characterization of the orientational dynamics of the excited state during FRET.     

A direct radioimmunoassay for human epidermal growth factor receptor using 32P-autophosphorylated receptor

A simple and reproducible radioimmunoassay of the epidermal growth factor (EGF) receptor which uses 32P-labeled EGF receptor and anti-receptor monoclonal antibodies is reported. In vitro phosphorylation of A431 cell membranes with [gamma-32P]ATP in the presence of 20% dimethyl sulfoxide (which stimulates autophosphorylation of the EGF receptor) and 10 microM Na3VO4 (a potent inhibitor of phosphotyrosyl protein phosphatase) provides radiolabeled EGF receptor for radioimmunoassay without further purification. The most selective phosphorylation of the EGF receptor is achieved at ATP concentrations of 0.1-0.2 microM, which corresponds to the reported Km value for the autophosphorylation reaction of the EGF receptor (W. Weber, P.J. Bertics, and G.N. Gill, 1984, J. Biol. Chem. 259, 14631-14939). The incorporation of 32P into EGF receptors increases in proportion to the increase of ATP concentration up to 6 mol of labeled phosphate at 2.0 microM ATP. The label is entirely on tyrosine residues. The cell membranes can be stored at -70 degrees C for 3 months without loss of immunoreactivity and autophosphorylating activity. Standard curves for the radioimmunoassay were constructed employing either A431 cell membranes or whole cell homogenates containing a known amount of EGF receptor. The assay can detect 7 X 10(10) EGF receptor molecules or 20 ng of the receptor protein, and can quantitatively distinguish the difference in EGF receptor numbers between A431 cells and 29E2 and KB cells with 10-fold and 15-fold fewer receptors than A431 cells, respectively. 29E2 cells and KB cells express twofold more immunoreactive EGF receptors than EGF-binding sites. In contrast, A431 cells possess the same number of immunoreactive sites and receptor sites for EGF binding. To assess total EGF receptor expression, it is necessary to use a method which detects EGF receptors regardless of their intrinsic kinase activity, or capacity to bind EGF. This radioimmunoassay detects immunoreactive receptor molecules, even those which do not bind EGF.     

Epidermal growth factor receptor of A431 cells. Characterization of a monoclonal anti-receptor antibody noncompetitive agonist of epidermal growth factor action

A monoclonal antibody to the epidermal growth factor (EGF) receptor of A431 cells, denoted 2D1-IgM, was generated after fusion of immunized BALB/c mouse spleen cells with SP2/0-Ag14 myeloma cells. Specific binding of 2D1-IgM to the A431 cell-surface receptor for EGF was demonstrated by indirect immunofluorescence, immunoprecipitation, and immunoblot analysis. Scatchard analysis of 125I-EGF binding to A431 cells demonstrated that 2D1-IgM treatment did not change the number of EGF receptors, but caused an increase in the affinity of EGF receptors from a population of low affinity to a uniform population of high affinity. Like EGF, 2D1-IgM induced phosphorylation of EGF receptors and EGF receptor clustering. As in the case of EGF, a biphasic growth response with stimulation of DNA synthesis at low and inhibition at high concentrations of 2D1-IgM was evident in A431 cells. The intrinsic "EGF-like" bioactivity of 2D1-IgM was enhanced by the presence of EGF. These results suggest that the binding of 2D1-IgM to the EGF receptor at a different site from that to which EGF binds can initiate an effective EGF-like biological response; and the EGF-like biological effects of 2D1-IgM may be mediated by a population of high affinity EGF receptors which may be involved in the control of cellular growth.     

Effect of Ca2+-Mg2+ exchange on the flexibility and/or conformation of the small domain in monomeric actin.

A fluorescence resonance energy transfer (FRET) parameter, f' (defined as the average transfer efficiency, (E), normalized by the actual fluorescence intensity of the donor in the presence of acceptor, F(DA)), was previously shown to be capable of monitoring both changes in local flexibility of the protein matrix and major conformational transitions. The temperature profile of this parameter was used to detect the change of the protein flexibility in the small domain of the actin monomer (G-actin) upon the replacement of Ca2+ by Mg2+. The Cys-374 residue of the actin monomer was labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) to introduce a fluorescence donor and the Lys-61 residue with fluorescein-5-isothiocyanate (FITC) to serve as an acceptor. The f' increases with increasing temperature over the whole temperature range for Mg-G-actin. This parameter increases similarly in the case of Ca-G-actin up to 26 degrees C, whereas an opposite tendency appears above this temperature. These data indicate that there is a conformational change in Ca-G-actin above 26 degrees C that was not detected in the case of Mg-G-actin. In the temperature range between 6 degrees C and 26 degrees C the slope of the temperature profile of f' is the same for Ca-G-actin and Mg-G-actin, suggesting that the flexibility of the protein matrix between the two labels is identical in the two forms of actin.     

Modulation of oncogene expression by epidermal growth factor and gamma-interferon in A431 squamous carcinoma cells

Epidermal growth factor (EGF) acts, in a dose dependent manner, as both a mitogen and an inhibitor of growth of the A431 squamous carcinoma cell line. gamma-interferon (IFN) also inhibits A431 cell growth. The dual effects of EGF on A431 growth and expression of the oncogenes, EGF receptor (EGFR) and Ha-ras, were evaluated with or without gamma-IFN. A mitogenic level (10pM) of EGF had no effect on expression of EGFR 10 kb mRNA or protein. gamma-IFN combined with 10pM EGF caused an initial drop in EGFR mRNA not reflected at the protein level; at 72 hours, the level of EGFR 10kb mRNA rose and inhibition of cell growth was observed. Treatment with a cytostatic amount (10nM) of EGF resulted in decreased expression of EGFR 10kb mRNA and protein within 24 hours; combined treatment with gamma-IFN caused rapid cell death. Expression of Ha-ras mRNA paralleled that of EGFR mRNA upon treatment with 10pM EGF and/or gamma-IFN, but differed with 10nM EGF.     

Treatment of A431 cells with epidermal growth factor (EGF) induces desensitization of EGF-stimulated phosphatidylinositol turnover

Epidermal growth factor (EGF) stimulates the turnover of phosphoinositides in A431 cells. In cells that were pretreated with EGF for 30 min at 37 degrees C and then washed to remove surface-bound hormone, a 70-100% decrease in the EGF-stimulated production of inositol monophosphate, inositol bisphosphate, and inositol triphosphate was noted when the cells were exposed to the agonist a second time. Since only a 15% decrease in receptor number was observed in these pretreated cells, the loss of responsiveness to EGF for the production of inositol phosphates could not be attributed to a down-regulation of the EGF receptors. These data suggest that pretreatment of A431 cells with high concentrations of EGF leads to a desensitization of the EGF receptor. This desensitization of the receptor by EGF is apparent within 10-15 min of the addition of EGF and is maximal by 30 min. The desensitization appears to be homologous in nature since pretreatment of cells with EGF did not diminish their responsiveness to bradykinin; and conversely, pretreatment with bradykinin did not diminish the subsequent responsiveness of the cells to EGF. Desensitization to EGF was observed in cells in which protein kinase C had been down-regulated by prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate, implying that EGF receptor desensitization is independent of protein kinase C. The desensitizing effects of EGF on growth factor-induced phosphatidylinositol turnover could be prevented by pretreatment of the cells with the calmodulin antagonist trifluoperazine, suggesting that calmodulin may be involved in the regulation of EGF receptor sensitivity.