Loss of alpha 2-macroglobulin and epidermal growth factor surface binding induced by phenothiazines and naphthalene sulfonamides

We have found that certain naphthalenesulfonamides [e.g., N-6(-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7)] and phenothiazines [e.g., trifluoperazine (TFP)] induce a loss of cell-surface receptors for alpha 2-macroglobulin, and epidermal growth factor (EGF) in fibroblasts. The loss of alpha 2-macroglobulin receptors is independent of receptor occupancy and is rapidly reversed upon removal of these agents from the culture medium. The extent of EGF receptor loss is less than for alpha 2-macroglobulin, and the EGF receptors do not reappear at the surface when W-7 is removed. Receptor loss was measured as a change in the capacity for binding iodinated ligands; no change in affinity of binding was observed. This receptor loss could reflect inactivation of receptors or internalization. W-7 did not induce a loss of cell surface beta 2-microglobulin, a membrane protein which is excluded from coated pits and which is not internalized, indicating that the effect of W-7 was specific for membrane receptors and not a result of bulk depletion of plasma membrane. The loss of alpha 2-macroglobulin and EGF receptors occurs at concentrations which do not cause an increase in the pH of endocytic vesicles or the cytoplasm, indicating that these agents act by a mechanism distinct from the effect of other weak bases. Since both TFP and W-7 are potent inhibitors of calmodulin, we investigated the possibility that inhibition of calmodulin was responsible for the loss of receptors. Three lines of evidence suggest that calmodulin inhibition is not responsible for the inhibition of binding and endocytosis: 1) Promethazine, a phenothiazine that is a poor inhibitor of calmodulin, is nearly as effective as TFP at inhibiting endocytosis; calmidazolium, a potent inhibitor of several calmodulin functions, did not cause a loss of binding; 2) the microinjection of calmodulin into cells did not reverse the effects of W-7; using pressure microinjection, we introduced up to a 100-fold excess of calmodulin over native levels into individual gerbil fibroma cells; using rhodamine-labeled alpha 2-macroglobulin, we saw that the W-7 induced inhibition of receptor-mediated endocytosis was the same in injected and uninjected cells; 3) we injected calcineurin, a calmodulin-binding protein, into cells (1-3 pg/cell) and observed no effect on the receptor-mediated endocytosis of rhodamine-labeled alpha 2-macroglobulin. These data indicated that cell surface receptor numbers can be regulated by a cellular component that is not cytoplasmic calmodulin but that shares some drug sensitivities with calmodulin.     

Characterization and Functional Analysis of the Calmodulin-Binding Domain of Rac1 GTPase

Rac1, a member of the Rho family of small GTPases, has been shown to promote formation of lamellipodia at the leading edge of motile cells and affect cell migration. We previously demonstrated that calmodulin can bind to a region in the C-terminal of Rac1 and that this interaction is important in the activation of platelet Rac1. Now, we have analyzed amino acid residue(s) in the Rac1-calmodulin binding domain that are essential for the interaction and assessed their functional contribution in Rac1 activation. The results demonstrated that region 151-164 in Rac1 is essential for calmodulin binding. Within the 151-164 region, positively-charged amino acids K153 and R163 were mutated to alanine to study impact on calmodulin binding. Mutant form of Rac1 (K153A) demonstrated significantly reduced binding to calmodulin while the double mutant K153A/R163A demonstrated complete lack of binding to calmodulin. Thrombin or EGF resulted in activation of Rac1 in CHRF-288-11 or HeLa cells respectively and W7 inhibited this activation. Immunoprecipitation studies demonstrated that higher amount of CaM was associated with Rac1 during EGF dependent activation. In cells expressing mutant forms of Rac1 (K153A or K153A/R163A), activation induced by EGF was significantly decreased in comparison to wild type or the R163A forms of Rac1. The lack of Rac1 activation in mutant forms was not due to an inability of GDP-GTP exchange or a change in subcelllular distribution. Moreover, Rac1 activation was decreased in cells where endogenous level of calmodulin was reduced using shRNA knockdown and increased in cells where calmodulin was overexpressed. Docking analysis and modeling demonstrated that K153 in Rac1 interacts with Q41 in calmodulin. These results suggest an important role for calmodulin in the activation of Rac1 and thus, in cytoskeleton reorganization and cell migration.     

Calmodulin inhibits the epidermal growth factor receptor tyrosine kinase.

We demonstrate in this report that the epidermal growth factor (EGF) receptor from rat liver can be isolated by calmodulin affinity chromatography by binding in the presence of Ca2+ and elution with a Ca(2+)-chelating agent. The bulk of the EGF receptor is not eluted by a NaCl gradient in the presence of Ca2+. We ascertained the identity of the isolated receptor by immunoblot and immunoprecipitation using a polyclonal antibody against an EGF receptor from human origin. The purified receptor is autophosphorylated in tyrosine residues in an EGF-stimulated manner, and EGF-dependent phosphorylation of serine residues was also detected. Both the EGF and the transforming growth factor-alpha stimulate the tyrosine-directed protein kinase activity of the isolated receptor with similar affinities. Furthermore, we demonstrate that calmodulin inhibits the EGF-dependent tyrosine-directed protein kinase activity associated to the receptor in a concentration-dependent manner. This inhibition is partially Ca2+ dependent and is not displaced by increasing the concentration of EGF up to an EGF/calmodulin ratio of 10 (mol/mol). In addition, calmodulin was phosphorylated in an EGF-stimulated manner in the presence of a basic protein (histone) as cofactor and in the absence, but not in the presence, of Ca2+.     

Calmodulin binds to K-Ras, but not to H- or N-Ras, and modulates its downstream signaling

Activation of Ras induces a variety of cellular responses depending on the specific effector activated and the intensity and amplitude of this activation. We have previously shown that calmodulin is an essential molecule in the down-regulation of the Ras/Raf/MEK/extracellularly regulated kinase (ERK) pathway in cultured fibroblasts and that this is due at least in part to an inhibitory effect of calmodulin on Ras activation. Here we show that inhibition of calmodulin synergizes with diverse stimuli (epidermal growth factor, platelet-derived growth factor, bombesin, or fetal bovine serum) to induce ERK activation. Moreover, even in the absence of any added stimuli, activation of Ras by calmodulin inhibition was observed. To identify the calmodulin-binding protein involved in this process, calmodulin affinity chromatography was performed. We show that Ras and Raf from cellular lysates were able to bind to calmodulin. Furthermore, Ras binding to calmodulin was favored in lysates with large amounts of GTP-bound Ras, and it was Raf independent. Interestingly, only one of the Ras isoforms, K-RasB, was able to bind to calmodulin. Furthermore, calmodulin inhibition preferentially activated K-Ras. Interaction between calmodulin and K-RasB is direct and is inhibited by the calmodulin kinase II calmodulin-binding domain. Thus, GTP-bound K-RasB is a calmodulin-binding protein, and we suggest that this binding may be a key element in the modulation of Ras signaling.     

Mechanisms of vascular angiotensin II surface receptor regulation by epidermal growth factor

We investigated mechanisms by which epidermal growth factor (EGF) reduces angiotensin II (AngII) surface receptor density and stimulated actions in vascular smooth muscle cells (VSMC). EGF downregulated specific AngII radioligand binding in intact cultured rat aortic smooth muscle cells but not in cell membranes and also inhibited AngII-stimulated contractions of aortic segments. Inhibitors of cAMP-dependent kinases, PI-3 kinase, MAP kinase, cyclooxygenase, and calmodulin did not prevent EGF-mediated downregulation of AngII receptor binding, whereas the EGF receptor kinase inhibitor AG1478 did. Total cell AngII AT1a receptor protein content of EGF-treated and untreated cells, measured by immunoblotting, did not differ. Actinomycin D or cytochalasin D, which interacts with the cytoskeleton, but not the protein synthesis inhibitor cycloheximide, prevented EGF from downregulating AngII receptor binding. Consistently, EGF inhibited AngII-stimulated formation of inositol phosphates in the presence of cycloheximide but not in the presence of actinomycin D or cytochalasin D. In conclusion, EGF needs an intact signal transduction pathway to downregulate AngII surface receptor binding, possibly by altering cellular location of the receptors.     

Teleocidin,a possible naturally occurring tumor promoter from streptomyces,modulates actions of epidermal growth factor on rat hepatoma cells

Epidermal growth factor (EGF) stimulated the proliferation of rat AH66 hepatoma cells in a low-serum culture. Teleocidin, a toxic substance isolated from Streptomyces, inhibited the binding of [¹²⁵I] EGF to cellular receptors and antagonized the mitogenic action of EGF. Insulin binding to AH66 cells was not affected by teleocidin, suggesting that the effect of teleocidin might be selective to the action of EGF. However, the inhibition of EGF binding by teleocidin was a transient phenomenon, and AH66 cells escaped from and became refractory to the teleocidin-inhibition of EGF binding after a prolonged treatment with teleocidin. In addition, teleocidin seemed to have no remarkable effect on the internalization of EGF-receptor complex. These results appeared to indicate that the effect of teleocidin on the mitogenic action of EGF might be related with a pathway of EGF action following the interaction of EGF with its receptors.     

IQGAP1 integrates Ca2+/calmodulin and B-Raf signaling

Ca(2+) and calmodulin modulate numerous cellular functions, ranging from muscle contraction to the cell cycle. Accumulating evidence indicates that Ca(2+) and calmodulin regulate the MAPK signaling pathway at multiple positions in the cascade, but the molecular mechanism underlying these observations is poorly defined. We previously documented that IQGAP1 is a scaffold in the MAPK cascade. IQGAP1 binds to and regulates the activities of ERK, MEK, and B-Raf. Here we demonstrate that IQGAP1 integrates Ca(2+) and calmodulin with B-Raf signaling. In vitro analysis reveals that Ca(2+) promotes the direct binding of IQGAP1 to B-Raf. This interaction is inhibited by calmodulin in a Ca(2+)-regulated manner. Epidermal growth factor (EGF) is unable to stimulate B-Raf activity in fibroblasts treated with the Ca(2+) ionophore A23187. In contrast, chelation of intracellular free Ca(2+) concentrations ([Ca(2+)](i)) significantly enhances EGF-stimulated B-Raf activity, an effect that is dependent on IQGAP1. Incubation of cells with EGF augments the association of B-Raf with IQGAP1. Moreover, Ca(2+) regulates the association of B-Raf with IQGAP1 in cells. Increasing [Ca(2+)](i) with Ca(2+) ionophores significantly reduces co-immunoprecipitation of B-Raf and IQGAP1, whereas chelation of Ca(2+) enhances the interaction. Consistent with these findings, increasing and decreasing [Ca(2+)](i) increase and decrease, respectively, co-immunoprecipitation of calmodulin with IQGAP1. Collectively, our data identify a previously unrecognized mechanism in which the scaffold protein IQGAP1 couples Ca(2+) and calmodulin signaling to B-Raf function.     

Binding of the Neuronal Protein B-50, but Not the Metabolite B-60, to Calmodulin

Protein kinase C phosphorylates the neurone-specific protein B-50 at a single Ser41 residue, which is also the point for a major proteolytic cleavage in vitro, and probably in vivo, that produces a B-50 phosphorylation-inhibiting N-terminal fragment and a large C-terminal metabolite B-60 (B-50(41-226]. The intact purified protein will bind to calmodulin in the absence of calcium, but the interaction has an absolute requirement for dephospho-B-50. In an attempt to unify two aspects of B-50 biochemistry, we have examined the interaction of B-50 binding to calmodulin and B-50 proteolysis. HPLC- and affinity-purified B-50 bound to calmodulin, but purified B-60 did not. To ensure that this effect was not due to the phosphorylation state of pure, isolated B-60, the metabolite was generated in vitro using a Triton extract of synaptosomal plasma membranes, which contains the as yet uncharacterized B-50 protease. B-60 derived from dephospho-B-50 also failed to bind calmodulin. The results demonstrate a direct connection between B-50 binding to calmodulin and B-50 proteolysis. The position of the proposed calmodulin-binding domain within intact B-50 is discussed in light of the failure of calmodulin to bind B-60.     

Thioridazine enhances lysosomal accumulation of epidermal growth factor and toxicity of conjugates of epidermal growth factor with Pseudomonas exotoxin

Thioridazine, a phenothiazine calmodulin inhibitor, aggravated the cytotoxic effect of a conjugate (EGF-PE) of epidermal growth factor (EGF) coupled with Pseudomonas exotoxin against cultured HeLa cells. Other phenothiazine calmodulin inhibitors, trifluoperazine and chlorpromazine, also intensified the cytotoxic effect of EGF-PE, whereas N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7) had no such effect. By using iodinated epidermal growth factor ( [125I]EGF), the effect of thioridazine on intracellular transport of EGF was examined. The release of radioactivity associated with [125I]EGF into medium was slow in the presence of thioridazine. The Percoll gradient centrifugation pattern showed that thioridazine delayed both the appearance of [125I]EGF in lysosomes and the disappearance of [125I]EGF from the lysosomes. The pH value in lysosomes was 5.28 in thioridazine-treated HeLa cells, while that in untreated cells was 5.15. Thioridazine was found to inhibit lysosomal enzyme activities of cathepsin B and acid phosphatase, but not beta-hexosaminidase when cell extracts were treated with the drug. Electron microscopy showed an increased number of electron-dense bodies, possibly autophagosomes/lysosomes in HeLa cells grown for 48 h with 3 micrograms/ml thioridazine. The potentiating action of EGF-PE by thioridazine is discussed in relation to the altered lysosomal function in treated cells.     

Roles of epidermal growth factor and Na+/H+ exchanger-1 in esophageal epithelial defense against acid-induced injury

Epidermal growth factor (EGF) is predominantly secreted by salivary glands and activates Na(+)/H(+) exchanger-1 (NHE-1), which regulates intracellular pH (pH(i)). We investigated the roles of EGF and NHE-1 in esophageal epithelial defense against acid using human esophageal epithelial cell lines and a rat chronic esophagitis model. Esophageal epithelial cells were incubated with acidified medium in the absence or presence of EGF. Cell viability and changes in pH(i) were measured. Chronic acid reflux esophagitis was induced in rats with and without sialoadenectomy. Esophageal lesion index, epithelial proliferation, and expression of EGF receptors and NHE-1 were examined. EGF protected esophageal epithelial cells against acid in a dose-dependent manner, and the cytoprotective effect of EGF was completely blocked by treatment with NHE-1 inhibitors. Tyrosine kinase, calmodulin, and PKC inhibitors significantly inhibited cytoprotection by EGF, whereas MEK, phosphatidylinositol 3-kinase, and PKA inhibitors had no effect. EGF significantly increased pH(i) recovery after NH(4)Cl pulse acidification, and this increase in pH(i) recovery was significantly blocked by inhibitors of calmodulin and PKC. Sialoadenectomy led to an increase in the severity of chronic esophagitis but affected neither epithelial proliferation nor expression of EGF receptors. Expression of NHE-1 mRNA was increased in esophagitis and upregulated in rats with sialoadenectomy. The increasing severity of esophagitis in rats with sialoadenectomy was prevented by exogenous administration of EGF. In conclusion, EGF protects esophageal epithelial cells against acid through NHE activation via Ca(2+)/calmodulin and the PKC pathway. Deficiency in endogenous EGF is associated with increased severity of esophagitis. EGF and NHE-1 play crucial roles in esophageal epithelial defense against acid.     

Calcium/calmodulin inhibits direct binding of spectrin to synaptosomal membranes

Brain spectrin, through its beta subunit, binds with high affinity to protein-binding sites on brain membranes quantitatively depleted of ankyrin (Steiner, J., and Bennett, V. (1988) J. Biol. Chem. 263, 14417-14425). In this study, calmodulin is demonstrated to inhibit binding of brain spectrin to synaptosomal membranes. Submicromolar concentrations of calcium are required for inhibition of binding, with half-maximal effects at pCa = 6.5. Calmodulin competitively inhibits binding of spectrin to protein(s) in stripped synaptosomal membranes, with Ki = 1.3 microM in the presence of 10 microM calcium. A reversible receptor-mediated process, and not proteolysis, is responsible for inhibition since the effect of calcium/calmodulin is reversed by the calmodulin antagonist trifluoperazine and by chelation of calcium with sodium [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The target of calmodulin is most likely the spectrin attachment protein(s) rather than spectrin itself since: (a) membrane binding of the brain spectrin beta subunit, which does not associate with calmodulin, is inhibited by calcium/calmodulin, and (b) red cell spectrin which binds calmodulin very weakly, is inhibited from interacting with membrane receptors in the presence of calcium/calmodulin. Ca2+/calmodulin inhibited association of erythrocyte spectrin with synaptosomal membranes but had no effect on binding of erythrocyte or brain spectrin to ankyrin in erythrocyte membranes. These experiments demonstrate the potential for differential regulation of spectrin-membrane protein interactions, with the consequence that Ca2+/calmodulin can dissociate direct spectrin-membrane interactions locally or regionally without disassembly of the areas of the membrane skeleton stabilized by linkage of spectrin to ankyrin. A membrane protein of Mr = 88,000 has been identified that is dissociated from spectrin affinity columns by calcium/calmodulin and is a candidate for the calmodulin-sensitive spectrin-binding site in brain.     

Drug-protein interactions: binding of chlorpromazine to calmodulin, calmodulin fragments, and related calcium binding proteins

The quantitative binding of a phenothiazine drug to calmodulin, calmodulin fragments, and structurally related calcium binding proteins was measured under conditions of thermodynamic equilibrium by using a gel filtration method. Plant and animal calmodulins, troponin C, S100 alpha, and S100 beta bind chlorpromazine in a calcium-dependent manner with different stoichiometries and affinities for the drug. The interaction between calmodulin and chlorpromazine appears to be a complex, calcium-dependent phenomenon. Bovine brain calmodulin bound approximately 5 mol of drug per mol of protein with apparent half-maximal binding at 17 microM drug. Large fragments of calmodulin had limited ability to bind chlorpromazine. The largest fragment, containing residues 1-90, retained only 5% of the drug binding activity of the intact protein. A reinvestigation of the chlorpromazine inhibition of calmodulin stimulation of cyclic nucleotide phosphodiesterase further indicated a complex, multiple equilibrium among the reaction components and demonstrated that the order of addition of components to the reaction altered the drug concentration required for half-maximal inhibition of the activity over a 10-fold range. These results confirm previous observations using immobilized phenothiazines [Marshak, D.R., Watterson, D.M., & Van Eldik, L.J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6793-6797] that indicated a subclass of calcium-modulated proteins bound phenothiazines in a calcium-dependent manner, demonstrate that the interaction between phenothiazines and calmodulin is more complex than previously assumed, and suggest that extended regions of the calmodulin molecule capable of forming the appropriate conformation are required for specific, high-affinity, calcium-dependent drug binding activity.     

Calmodulin binding to Drosophila NinaC required for termination of phototransduction.

The ninaC locus encodes two unconventional myosins, p132 and p174, consisting of fused protein kinase and myosin head domains expressed in Drosophila photoreceptor cells. NinaC are the major calmodulin-binding proteins in the retina and the NinaC-calmodulin interaction is required for the normal subcellular localization of calmodulin as well as for normal photo-transduction. In the current report, we present evidence for two calmodulin-binding sites in NinaC, C1 and C2, which have different in vitro binding properties. C1 was found to be common to both p132 and p174 while C2 was unique to p174. To address the requirements for calmodulin binding at each site in vivo, we generated transgenic flies expressing ninaC genes deleted for either C1 or C2. We found that the spatial localization of calmodulin depended on binding to both C1 and C2. Furthermore, mutation of either site resulted in a defective photoresponse. A prolonged depolarization afterpotential (PDA) was elicited at lower light intensities than necessary to produce a PDA in wild-type flies. These results suggest that calmodulin binding to both C1 and C2 is required in vivo for termination of phototransduction.     

Binding of calcium to anticoagulant protein S: role of the fourth EGF module

Protein S is an anticoagulant protein containing a Gla (enclosing gamma-carboxyglutamic acids) module, a TSR (thrombin sensitive region) module, four EGF (epidermal growth factor)-like modules, and a SHBG (sex hormone binding globulin)-like region. Protein S is a cofactor to activated protein C (APC) in the degradation of coagulation factors Va and VIIIa but also has APC-independent activities. The function of the fourth EGF module (EGF4) in protein S has so far not been clear. We have now investigated this module through studies of recombinant wild-type protein S and a naturally occurring mutant (Asn217Ser). The mutant has essentially normal APC anticoagulant activity and a previously reported secretion defect. In the wild-type protein, Asn217 is normally beta-hydroxylated. The binding of calcium to wild-type protein S is characterized by four high-affinity binding sites with K(D) values ranging from 10(-)(7) to 10(-)(9) M. Three of these binding sites are located in EGF modules. Using surface plasmon resonance, competition with a calcium chelator, and antibody-based methods, we found that one high-affinity binding site for calcium was lost in protein S Asn217Ser but that the mutation also affected the calcium-dependent conformation of EGF1. We conclude that binding of calcium to EGF4 of protein S, involving Asn217, is important for the maintenance of the structure of protein S. Also, the abolition of binding of calcium to EGF4, related to Asn217, impairs both the structure and function of EGF1.     

Evidence for a role of calmodulin in the regulation of prolactin gene expression

Epidermal growth factor (EGF) stimulates prolactin (PRL) gene expression in GH3 cells in a Ca2+-dependent manner (White, B. A., and Bancroft, F. C. (1983) J. Biol. Chem. 258, 4618-4622). The present report shows that the phenothiazine, calmidazolium (compound R 24571), blocks the ability of EGF plus Ca2+ to increase levels of PRL mRNA. Calmidazolium inhibition of this response is dose dependent in the range of 0.05-1.00 microM. Total inhibition of the response was consistently obtained at a level of calmidazolium (0.5 microM) that had no effect on total cytoplasmic RNA synthesis, total cytoplasmic protein synthesis, cell viability, or extent of EGF plus Ca2+-induced cell aggregation. The drug inhibited the increase in PRL mRNA when given immediately before or 48 h after treatment with EGF plus Ca2+. Another calmodulin inhibitor, W13, similarly blocked the ability of EGF plus Ca2+ to stimulate PRL mRNA, whereas the less active analog, W12, had little effect. These results implicate Ca2+-binding proteins such as calmodulin in the mechanism of action of EGF in GH3 cells, and, therefore, provide further evidence for a role of intracellular Ca2+ in the regulation of the expression of a specific eukaryotic gene, the PRL gene.     

Trifluoperazine binding to mutant calmodulins

Trifluoperazine (TFP) binding by 14 calmodulins, including 12 produced by site-directed mutagenesis, was determined. While vertebrate calmodulin binds 4.2 +/- 0.2 equiv of TFP, Escherichia coli expressed but unmutated calmodulins bind about 5.0 +/- 0.5 equiv of TFP. The cause for this difference is not known. The E. coli expressed proteins consist of two different series expressed from different calmodulin genes, CaMI and SYNCAM. The wild-type genes code for proteins that differ by nine conservative amino acid substitutions. Both these calmodulins bind 5 equiv of TFP with similar affinities, thus none of these conservative substitutions has any additional effect on TFP binding. Some altered calmodulins (deletion of EE83-84 or SEEE81-84, changing DEE118-120----KKK, M124----I,E120----K, or E82----K) have no appreciable effect on TFP binding. Other mutations affect either the binding of one TFP (deletion of E84) or about two TFP (changing E84----K, EEE82-84----KKK, E67----A, DEQ6-8----KKK, or E11----K). The mutations that affect TFP binding are localized to three regions of calmodulin: The amino-terminal alpha-helix, the central helix between the two globular ends of calmodulin, and a calcium-binding site in the second calcium-binding domain. The results are consistent with each of these regions either directly participating in drug binding or involved structurally in maintaining or inducing the correct conformation for TFP binding in the amino-terminal half of calmodulin.     

Epithelioid and fibroblastic rat kidney cell clones: epidermal growth factor (EGF) receptors and the effect of mouse sarcoma virus transformation.

Fibroblastic and epithelioid clones have been isolated from the normal rat kidney line, NRK. These clones were studied for their ability to bind epidermal growth factor (EGF), susceptibility to transformation by mouse sarcoma virus (MSV), and alteration in EGF binding upon sarcoma virus transformation. The epithelioid clones bound much more EGF than the fibroblastic clones; Scatchard plots on two of these clones, one epithelioid and one fibroblastic, showed that the higher EGF binding (1.3 x 10(5) molecules per cell for the epithelioid clone and 1.3 x 10(4) molecules per cell for the fibroblastic clone) was due to a greater number or receptors on the epithelioid cells rather than to a difference in the apparent affinity constant. When the clones were transformed by Moloney murine sarcoma virus the EGF binding decreased, the effect being greater with the fibroblastic clones. In 20 out of 20 independently isolated sarcoma virus transformed fibroblastic clones, the level of EGF binding was either greatly reduced or completely eliminated. In contrast to EGF, another growth factor, multiplication stimulating activity (MSA), bound to a greater extent to the fibroblastic clones than the epithelioid clones, and its binding was not decreased by sarcoma virus transformation. The results show that loss of EGF binding ability correlates with expression of the murine sarcoma virus transformation.     

Keloid fibroblasts in culture: abnormal growth behaviour and altered response to the epidermal growth factor

Keloid fibroblasts were propagated in culture and their proliferative behaviour and response to the Epidermal Growth Factor (EGF) were studied. Keloid fibroblasts grew at a rate which was approximately one-half that of normal age, sex and race matched control fibroblasts. Keloid fibroblasts were stimulated to grow in the presence of EGF (10 ng/ml), but to a lesser degree than the normal control fibroblasts. Scatchard analysis of the binding data obtained using 125I-labeled EGF revealed no difference in binding affinity or receptor numbers between keloid and normal fibroblasts.     

Calcium effects on epidermal growth factor receptor-mediated endocytosis in normal and SV40-transformed human fibroblasts

Lowering of extracellular Ca²⁺ levels will reversibly arrest the growth of human fibroblasts (WI38). Simian virus₄₀(SV₄₀)-transformed WI38 cells do not exhibit this Ca²⁺-dependent arrest. One possibility for this difference in Ca²⁺ requirement is that extracellular or surface membrane-bound Ca²⁺ may be required for growth factor receptor-mediated endocytosis and this Ca²⁺ requirement may differ in normal versus transformed cells. In this study we have evaluated the role of Ca²⁺ in the binding, internalization, and degradation of epidermal growth factor (EGF) in the WI38 and SV₄₀ WI38 cell. The binding of [¹²⁵I]EGF to the cell surface is not significantly altered by lowering of Ca²⁺ to 10^?5-M levels in either the normal or transformed cell. At this Ca²⁺ level, growth of the normal cell is inhibited. The subsequent internalization of EGF is reduced nearly threefold in the normal cell but not in the transformed cell following Ca²⁺ deprivation. Degradation of the EGF-receptor complex is also sensitive to Ca²⁺. A twofold reduction in the rate of release of acid-soluble ¹²⁵I occurs in the normal but not the transformed cell under conditions of lowered medium Ca²⁺. In contrast, 2-chloro-10-3-aminopropyl phenothiazine (CP), an inhibitor of the Ca²⁺-dependent regulator protein calmodulin, causes an inhibition of [¹²⁵I]EGF internalization and degradation in both the normal and transformed WI38 cell, and a marked inhibition of [¹²⁵I]EGF binding to the cell surface receptor of the transformed cell but not the normal cell.     

Association of EGF and LDL receptors with the cytoskeleton of cultured keratinocytes

In this paper we demonstrate that isolated cytoskeletons of normal keratinocytes cultured under differentiation inducing conditions exhibit a high level of epidermal growth factor (EGF) binding. This binding is approximately 300% higher than the binding of intact cells. In contrast, various squamous carcinoma cell lines or normal keratinocytes cultured under differentiation retarding conditions exhibit EGF binding to isolated cytoskeletons which is around 10-20% of the binding to intact cells. Incubation of normal keratinocytes in the presence of arotinoid ethyl sulfone resulted in a marked decrease of the ability of the cells to differentiate, and a decrease of EGF binding to isolated cytoskeletons. These results suggest a close relationship between the differentiation capacity of the cells and the presence of cytoskeleton-associated EGF receptors. Similar results were obtained for low density lipoprotein (LDL) binding.