Radioreceptor assay for epidermal growth factor.

An established cell line of human lung fibroblasts with a high number of surface receptorsfor mouse epidermal growth factor (mEGF) was used to develop a simple and highly sensitive radioreceptor assay for EGF. ¹²⁵I-Labeled mEGF competed mole for mole with unlabeled mEGF for specific receptors. Optimal range for discriminating EGF concentrations in body fluids and tissue extracts by a competitive binding assay was between 5 and 100 ng/ml. Interassay correlation of variation was 8.47% and the recovery of highly purified mEGF added to serum and urine samples was greater than 95%. Human serum and amniotic fluids contained about 24 and 4 ng/ml, respectively, of mEGF equivalents. Concentrations of mEGF in mouse urine and serum were highly variable and were 2- to 10-fold greater than that previously detected by radioimmune assay. Hypophysectomy nearly abolished submaxillary mEGF content in both male and female mice, but testosterone treatment of hypophysectomized animals restored normal concentrations of mEGF to the glands. mEGF added to culture medium disappeared with time as a function of the number of cellular EGF receptors indicating cellular degradation of the growth factor. The radioreceptor assay for EGF is based on the close biologic relationship between the cell receptor site and the native hormone and should prove to be a useful complementary tool to characterize the physiological role of EGF.     

Constitutive phosphorylation of the epidermal growth factor receptor blocks mitogenic signal transduction

The epidermal growth factor (EGF) receptor is phosphorylated by protein kinase C at Thr654. It has been proposed that the phosphorylation of this site is an important regulatory mechanism for the control of EGF receptor function. However, the physiological significance of the phosphorylation of EGF receptor Thr654 in intact cells is not understood. To address this question, the design of an experimental strategy is required that can be used to distinguish between the pleiotropic effects of kinase C activation and the specific effects of kinase C that are mediated by the phosphorylation of the EGF receptor at Thr654. The approach that we used was to examine the function of EGF receptors that are constitutively phosphorylated at residue 654. It was observed that the constitutive phosphorylation of the EGF receptor blocked mitogenic signal transduction by the receptor. These data are consistent with the hypothesis that the phosphorylation of the EGF receptor at residue 654 in intact cells inhibits EGF-stimulated cellular proliferation.     

An integrated model of epidermal growth factor receptor trafficking and signal transduction

Endocytic trafficking of many types of receptors can have profound effects on subsequent signaling events. Quantitative models of these processes, however, have usually considered trafficking and signaling independently. Here, we present an integrated model of both the trafficking and signaling pathway of the epidermal growth factor receptor (EGFR) using a probability weighted-dynamic Monte Carlo simulation. Our model consists of hundreds of distinct endocytic compartments and approximately 13,000 reactions/events that occur over a broad spatio-temporal range. By using a realistic multicompartment model, we can investigate the distribution of the receptors among cellular compartments as well as their potential signal transduction characteristics. Our new model also allows the incorporation of physiochemical aspects of ligand-receptor interactions, such as pH-dependent binding in different endosomal compartments. To determine the utility of this approach, we simulated the differential activation of the EGFR by two of its ligands, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). Our simulations predict that when EGFR is activated with TGF-alpha, receptor activation is biased toward the cell surface whereas EGF produces a signaling bias toward the endosomal compartment. Experiments confirm these predictions from our model and simulations. Our model accurately predicts the kinetics and extent of receptor downregulation induced by either EGF or TGF-alpha. Our results suggest that receptor trafficking controls the compartmental bias of signal transduction, rather than simply modulating signal magnitude. Our model provides a new approach to evaluating the complex effect of receptor trafficking on signal transduction. Importantly, the stochastic and compartmental nature of the simulation allows these models to be directly tested by high-throughput approaches, such as quantitative image analysis.     

A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor.

The ErbB family includes four homologous transmembrane tyrosine kinases. Whereas ErbB-1 binds to the epidermal growth factor (EGF), both ErbB-3 and ErbB-4 bind to the Neu differentiation factors (NDFs, or neuregulins), and ErbB-2, the most oncogenic family member, is an orphan receptor whose function is still unknown. Because previous lines of evidence indicated the existence of interreceptor interactions, we used ectopic expression of individual ErbB proteins and their combinations to analyze the details of receptor cross talks. We show that 8 of 10 possible homo-and heterodimeric complexes of ErbB proteins can be hierarchically induced by ligand binding. Although ErbB-2 binds neither ligand, even in a heterodimeric receptor complex, it is the preferred heterodimer partner of the three other members, and it favors interaction with ErbB-3. Selective receptor overexpression in human tumor cells appears to bias the hierarchical relationships. The ordered network is reflected in receptor transphosphorylation, ErbB-2-mediated enhancement of ligand affinities, and remarkable potentiation of mitogenesis by a coexpressed ErbB-2. The observed superior ability of ErbB-2 to form heterodimers, in conjunction with its uniquely high basal tyrosine kinase activity, may explain why ErbB-2 overexpression is associated with poor prognosis.     

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.     

A simple assay for detection of small-molecule redox activity

In addition to selecting molecules of pharmacological interest, high-throughput screening campaigns often generate hits of undesirable mechanism, which cannot be exploited for drug discovery as they lead to obvious problems of specificity and developability. Examples of undesirable mechanisms are target alkylation/acylation and compound aggregation. Both types of "promiscuous" mechanisms have been described in the literature, as have methods for their detection. In addition to these mechanisms, compounds can also inhibit by oxidizing susceptible enzyme targets, such as metalloenzymes and cysteine-using enzymes. However, this redox phenomenon has been documented infrequently, and an easy method for detecting this behavior is missing. In this article, the authors describe direct proof of small-molecule oxidation of a cysteine protease by liquid chromatography/tandem mass spectrometry, develop a simple assay to predict this oxidizing behavior by compounds, and show the utility of this assay by demonstrating its ability to distinguish nuisance redox compounds from well-behaved inhibitors in 3 historical GlaxoSmithKline drug discovery efforts.     

Endosomal signaling of epidermal growth factor receptor stimulates signal transduction pathways leading to cell survival

In spite of intensified efforts to understand cell signaling from endosomes, there is no direct evidence demonstrating that endosomal signaling is sufficient to activate signal transduction pathways and no evidence to demonstrate that endosomal signaling is able to produce a biological outcome. The lack of breakthrough is due in part to the lack of means to generate endosomal signals without plasma membrane signaling. In this paper, we report the establishment of a system to specifically activate epidermal growth factor (EGF) receptor (EGFR) when it endocytoses into endosomes. We treated cells with EGF in the presence of AG-1478, a specific EGFR tyrosine kinase inhibitor, and monensin, which blocks the recycling of EGFR. This treatment led to the internalization of nonactivated EGF-EGFR complexes into endosomes. The endosome-associated EGFR was then activated by removing AG-1478 and monensin. During this procedure we did not observe any surface EGFR phosphorylation. We also achieved specific activation of endosome-associated EGFR without using monensin. By using this system, we provided original evidence demonstrating that (i) the endosome can serve as a nucleation site for the formation of signaling complexes, (ii) endosomal EGFR signaling is sufficient to activate the major signaling pathways leading to cell proliferation and survival, and (iii) endosomal EGFR signaling is sufficient to suppress apoptosis induced by serum withdrawal.     

A simple and sensitive in vivo luciferase assay for tRNA-mediated nonsense suppression.

We present a rapid assay for tRNA suppression in living Escherichia coli. An amber, ochre, or opal nonsense mutation in a cloned luxB gene from the bacterium Vibrio harveyi was suppressed. Because luciferase (Lux) activity depends completely on the appearance of the full-length luxB gene product, the amount of light produced was proportional to tRNA-mediated nonsense suppression in the cell. This luminometric assay was notably quicker, easier, and more sensitive than a traditional colorimetric assay employing beta-galactosidase. Assays required only one addition to a growing culture and were complete within 1 min. Light output was directly proportional to the amount of bacterial luciferase in a sample over a range of greater than or equal to 40,000-fold. Fewer than 100 cells were required for detection of Lux with ordinary instrumentation; assays were 80-fold more sensitive than simultaneous beta-galactosidase measurements. Assayed cells survived and could be recovered as colony formers. The beta-galactosidase colorimetric assay and the luciferase assay were similarly reproducible. Light from colonies expressing Lux was visible to the dark-adapted eye and useful for screening. A rapid assay that does not depend on the formation of permanent transformants can be based on electroporation followed by luminometry.     

A sensitive and simple assay of starch synthase activity with pyruvate kinase and luciferase

A coupled recording assay of cyclic AMP phosphodiesterase activity at and below 1 μm is presented. The method is especially useful in preparative work where speed is more important than high accuracy, but results agree well with a more accurate radioactive assay. Attention is drawn to the fact that, because the product of the coupled reaction, ATP, is also an intermediate, its instantaneous rate of formation can exceed the rate of the first step under certain conditions.     

Enzyme-linked immunosorbent assay for the epidermal growth factor receptor

An enzyme-linked immunosorbent assay (ELISA) for the epidermal growth factor (EGF) receptor was developed using three different antibody preparations, one of which is commercially available. Using one of the antisera (986), the assay could detect as few as 200 × 10⁶ receptors. This is equal to 0.332 fmol. This sensitivity means that a minimum of 100 A-431 cells (human carcinoma) or 5,000 normal cells are needed to quantitate the number of EGF receptors. Two of the antisera (986 and 451) recognized EGF receptors from placental tissue. EGF receptors from as little as 667 ng of placental membrane protein were detectable. The assay is highly species specific, with the sensitivity for the EGF receptor from different species dependent on the antiserum used. The commercial antibody, 29.1, had especially strong reactivity against pig and dog EGF receptors. An ELISA using this antibody had the capacity to detect the number of EGF receptors in 10 μg of liver membrane protein. The assay is sensitive to receptor conformation. The binding of antisera 986 and 451 to 1% sodium dodecyl sulfate (SDS)-denatured receptor was reduced. The binding of antibody 29.1 was impaired by the presence of 1% Triton X-100 but not the same levels of Tween-20 or SDS. In addition to being a sensitive technique for the quantitation of the EGF receptor, this assay is very rapid, taking a total of 4 h. The microtiter dish format also allows hundreds of samples to be assayed at once. By using the appropriate antiserum and standards, the EGF receptor can be quantitated in tissues from humans, dogs, pigs, and mice.     

Human insulin-like growth factor I receptor 950tyrosine is required for somatotroph growth factor signal transduction.

Insulin-like growth factor I (IGF-I), a growth hormone (GH)-dependent growth factor exerts feedback regulation of GH by inhibiting GH gene expression. IGF-I inhibition of GH secretion is enhanced 3-5-fold in GC rat pituitary cells overexpressing the wild type 950Tyr human IGF-I receptor which autophosphorylates appropriately. To determine the critical amino acid sequence responsible for IGF-I signaling, insertion, deletion, and site-directed mutants were constructed to substitute for 950Tyr in exon 16 of the human IGF-I receptor beta-subunit transmembrane domain. All mutant transfectants bound IGF-I with a similar Kd to untransfected cells but had markedly increased (7-34-fold) IGF-I-binding sites. GH responsiveness to IGF-I was tested in mutant transfectants. Overexpressed site-directed and insertion mutant IGF-I receptors exhibited a modest suppressive effect on GH in response to the IGF-I ligand, similar to that observed in untransfected cells. Deletion mutant (IG-FIR delta 22) (amino acid 944-965) did not transduce the IGF-I signal to the GH gene. Site-directed and insertion mutants therefore did not enhance the IGF-I response of the endogenous rat receptor, unlike the 950Tyr wild type transfectants which enhanced the IGF-I signal. All mutant transfectants, except the deletion mutant, internalized radioactive ligand similarly to 950Tyr wild type transfectants. 950Tyr of the human IGF-I receptor is therefore required for IGF-I signal transduction in the pituitary somatotroph, but not for IGF-I-mediated internalization.     

Transactivation of the epidermal growth factor receptor is involved in 12-O-tetradecanoylphorbol-13-acetate-induced signal transduction

The mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion is still not well understood even though it is thought to be related to the protein kinase C/mitogen-activated protein kinase/AP-1 pathway. Recently, TPA was also found to induce epidermal growth factor receptor (EGFR) activity. Here, we investigated whether the EGFR is a necessary component for TPA-induced signal transduction associated with tumor promotion. We demonstrated that potent inhibitors of the EGFR, PD153035 and AG1478, blocked TPA-induced phosphorylation of extracellular signal-regulated kinases (ERKs), AP-1 activity, and cell transformation. Egfr gene deficiency blocked TPA-induced ERK activity and AP-1 binding activity. The blocking of the ectodomain of the EGFR by a monoclonal antibody depressed TPA-induced ERK activity and AP-1 DNA binding activity. The use of a neutralizing antibody for heparin-binding EGF, one of the ligands of EGFR, blocked TPA-induced phosphorylation of ERKs. BB-94, a potent inhibitor of matrix metalloproteinases, which are activators of ectodomain shedding of EGFR ligands, also blocked TPA-induced ERK activity, AP-1 DNA binding, and cell transformation but had no effect on EGF-induced signal transduction. Anti-EGFR, anti-heparin-binding EGF, and BB-94 each blocked TPA-induced EGFR phosphorylation, but only anti-EGFR could block EGF-induced EGFR phosphorylation. Based on these results, we conclude that the EGFR is required for mediating TPA-induced signal transduction. EGFR transactivation induced by TPA is a mechanism by which the EGFR mediates TPA-induced tumor promotion-related signal transduction.     

A biological assay for epidermal growth factor/urogastrone and related polypeptides

A bioassay for epidermal growth factor (EFG) which is rapid, specific, and extremely sensitive is described. As the bioassay detects as little as 25 pg of EGF, this assay is more sensitive than commonly used radioreceptor assays and nearly as sensitive as radioimmunoassays. This bioassay involves only the measurement of the proliferation of cultures of an EGF-requiring cell line and can be carried out in a quantitative manner over a 40-fold range of EGF concentrations.     

Is epidermal growth factor present in human blood? Interference with the radioreceptor assay for epidermal growth factor

Human whole blood serum reduces the binding of [125I]labeled mouse epidermal growth factor to cultured human fibroblasts as well as does 70-100 ng/ml mouse epidermal growth factor. However, at most 1-2% of the binding inhibitor detected in human whole blood serum is related to epidermal growth factor--the remaining consists of other factors released during preparation of serum, predominantly the platelet-derived growth factor, which are capable of altering the binding properties of the epidermal growth factor receptor. This accounts for much of the differences between values reported for epidermal growth factor concentration in blood by investigators using different assay procedures.     

A screen for genes that influence fibroblast growth factor signal transduction in Drosophila

The misexpression of an activated form of the FGF receptor (FGFR) Breathless in conjunction with downstream-of-FGF-receptor (Dof), an essential signaling molecule of the FGF pathway, in the Drosophila eye imaginal discs impairs eye development and results in a rough eye phenotype. We used this phenotype in a gain-of-function screen to search for modifiers of FGF signaling. We identified 50 EP stocks with insertions defining at least 35 genes that affect the rough eye phenotype. Among these genes, 4 appear to be specific for FGFR signaling, but most of the genes also influence other signaling pathways, as assessed by their effects on rough eyes induced by other activated receptor tyrosine kinases (RTKs). Analysis of loss-of-function alleles of a number of these genes in embryos indicates that in many cases the products are provided maternally and are involved in germ cell development. At least two of the genes, sar1 and robo2, show a genetic interaction with a hypomorphic dof allele, suggesting that they participate in FGF-mediated morphogenetic events during embryogenesis.     

Nerve growth factor mediates signal transduction through trk homodimer receptors.

We have investigated the molecular nature of the high affinity nerve growth factor (NGF) receptors by using cell lines expressing gp75LNGFR and gp140trk. Our results suggest that gp75LNGFR and gp140trk interact with NGF independently and that only gp140trk mediates NGF signaling. NGF binds to gp140trk with picomolar affinity and induces its phosphorylation on tyrosine residues regardless of the presence of gp75LNGFR. NGF-gp140trk complexes display the slow dissociation rate and rapid internalization characteristics of high affinity NGF receptors. Cross-linking studies reveal the existence of gp75LNGFR and gp140trk homodimers. However, we were unable to detect gp75LNGFR-gp140trk heterodimers. Coexpression in COS cells of wild-type and kinase deficient mutants reveals that gp140trk receptors can undergo intermolecular phosphorylation, indicating the formation of functional homodimers. Moreover, these kinase deficient mutants inhibit NGF-induced signaling through wild-type gp140trk receptors. These results indicate that the functional high affinity NGF receptors consist of gp140trk homodimeric (or oligomeric) complexes.     

Comparative effects of hepatocyte growth factor and epidermal growth factor on motility,morphology, mitogenesis,and signal transduction of primary rat hepatocytes

Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) are major hepatacyte mitogens, but HGF, also known as scatter factor (SF), has also been shown as a potent motogen for epithelial and endothelial cells. The mechanisms by which HGF is a stronger motogen compared to other mitogens are not understood. Here we report a comparative study of the effect of the two growth factors on cultured primary rat hepatocytes regarding their differential effects on morphology, mitogenicity, and motility as well as the phosphorylation of cytoskeletal-associated proteins. Using three different motility assays, both HGF and EGF increased the motility of hepatocytes, but HGF consistently elicited a significantly greater motility response than EGF. Additionally, HGF induced a more flattened, highly spread morphology compared to EGF. To examine if HGF and EGF phosphorylated different cytoskeletal elements as signal transduction targets in view of the observed variation in morphology and motility, primary cultures of ³²P-loaded rat hepatocytes were stimulated by either HGF or EGF for up to 60 min. Both mitogens rapidly stimulated four isoforms of MAP kinase with similar kinetics and also rapidly facilitated the phosphorylation of cytoskeletal-associated F-actin. Two cytoskeletal-associated proteins, however, were observed to undergo rapid phosphorylation by HGF and not EGF during the time points described. One protein of 28 kDa was observed to become phosphorylated fivefold over controls, while the EGF-stimulated cells showed only a slight increase in the phosphorylation of this protein. Another protein with an apparent mwt of 42 kDa was phosphorylated 20-fold at 1 min and remained phosphorylated over 50-fold over control up to the 60 min time point. This protein was observed to become phosphorylated by EGF only after 10 min, and to a lesser extent (20-fold). Taken together, the data suggest that HGF and EGF stimulate divergent as well as redundant signal transduction pathways in the hepatocyte cytoskeleton, and this may result in unique HGF- or EGF-specific motility, morphology, and mitogenicity in hepatocytes. © 1994 Wiley-Liss, Inc.