Toxic ligand conjugates as tools in the study of receptor-ligand interactions

We have constructed hybrid proteins in which the toxic A chains of ricin or diptheria toxin have been linked to either asialofetuin, fetuin, or epidermal growth factor (EGF). Both ASF-RTA and ASF-DTA are potent toxins on cultured rat hepatocytes, cells that display the asialoglycoprotein receptor. Toxicity of these two compounds is restricted to hepatocytes and can be blocked by asialoglycoproteins but not the native glycoproteins or asialoagalactoglycoprotein derivatives, indicating that the toxicity of the conjugates is mediated by the hepatic asialoglycoprotein receptor. The EGF-RTA conjugate is an extremely potent toxin on cells that can bind the hormone, but is only poorly effective on cells that are unable to bind EGF. The EGF-DTA conjugate, in contrast, is unable to kill 3T3 cells and is at least two orders of magnitude less effective than EGF-RTA on A431 cells, a cell line with 1-2 X 10(6) EGF receptors per cell. However, when EGF-RTA and EGF-DTA were tested on primary liver hepatocyte cultures, which were susceptible to both ASF-RTA and ASF-DTA, both EGF conjugates were potent toxins. Sensitivity of the hepatocyte cultures to ricin toxicity increases slightly during a 52-hr culture period. In contrast, sensitivity to EGF-RTA and ASF-RTA decline dramatically during this period. Receptors for both ligands remain plentiful on the cell surface during this time.     

Pertussis toxin-sensitive Gi protein involvement in epidermal growth factor-induced activation of phospholipase C-gamma in rat hepatocytes.

Treatment of rat hepatocytes with epidermal growth factor (EGF) produced an enhanced tyrosine phosphorylation of the EGF receptor and phospholipase C-gamma (PLC-gamma) in conjunction with the mobilization of Ca2+. Approximately 30% of the total PLC-gamma was tyrosine-phosphorylated with a maximum being reached after 30 s of incubation with EGF. Pretreatment of the rats with pertussis toxin prior to isolation of the hepatocytes blocked EGF-induced tyrosine phosphorylation of PLC-gamma and Ca2+ mobilization but had no effect on autophosphorylation of the EGF receptor or Ca2+ responses elicited by angiotensin II or phenylephrine. Under these conditions Gi protein alpha subunits were fully ADP-ribosylated. A 41-kDa Gi protein alpha subunit was found to be present in the anti-PLC-gamma immune complex after EGF stimulation as shown by in vitro ADP-ribosylation using [32P]NAD+ and activated pertussis toxin. The kinetics of association between PLC-gamma with Gi alpha protein reached a maximum after 1 min of incubation with EGF. Antibodies specific for the EGF receptor also coimmunoprecipitated a Gi protein alpha subunit. Treatment of hepatocytes with EGF caused first an increase and then a decrease in the amount of Gi protein alpha subunit associated with the EGF receptor. In contrast, studies with cultured rat liver (WB) cells, a cell line in which EGF stimulation of phosphoinositide hydrolysis is not inhibited by pertussis toxin, showed that a stable complex of Gi alpha was not formed with either PLC-gamma or EGF receptor immunoprecipitates. These results indicate that a pertussis toxin-sensitive Gi protein is uniquely involved in the signal transduction pathway mediating EGF-induced activation of PLC-gamma and Ca2+ mobilization in hepatocytes.     

Cyclic AMP in relation to proliferation of the epidermal cell: a new view.

Four agents known to increase the level of cellular cAMP by different means (cholera toxin, dibutyryl cAMP, methyl isobutyl xanthine and isoproterenol) increase the growth of colonies of cultured human epidermal cells and of keratinocytes derived from other stratified squamous epithelia. This effect is due to an increase in the overall rate of cell proliferation in the colonies. When added to cultures under hitherto optimum conditions for epidermal cell growth [in the presence of supporting 3T3 cells and epidermal growth factor (EGF)], most of the agents exert an effect of considerable magnitude, the toxin being the most potent. Since the toxin exerts an effect in the absence of supporting 3T3 cells, it must be able to act directly on the keratinocytes. It can also act in the absence of ECF and of medium conditioned by 3T3 cells, although proliferation is greatest when supporting 3T3 cells and EGF are present. The increased proliferation in the presence of the toxin is associated with an increased proportion of small cells known to include the multiplying fraction. The use of toxin makes the cultivation of keratinocytes from epidermis and other stratified squamous epithelia much easier and prolong the culture life of the cells. Whether cell proliferation in the intact epidermis is regulated through agents affecting cAMP (in a direction opposite to that suggested by much of the earlier literature) remains to be elucidated, but the existence of such a mechanism in cultured cells suggests that it may function in the intact epithelium.     

The role of binding ligand in toxic hybrid proteins: a comparison of EGF-ricin, EGF-ricin A-chain, and ricin

To analyze the influence of ricin B-chain on the toxicity of hybrid-protein conjugates, the rate of cellular uptake of conjugates, and the rate at which ricin A-chain (RTA) is delivered to the cytoplasm, we have constructed toxic hybrid proteins consisting of epidermal growth factor (EGF) coupled in disulfide linkage either to ricin or to RTA. EGF-ricin is no more toxic on A431 cells than EGF-RTA. The two conjugates demonstrate similar kinetics of cellular uptake (defined as antibody irreversible toxicity). EGF-RTA and EGF-ricin, like ricin, required a 2-2 1/2 hour period at 37 degrees before the onset of protein synthesis inhibition occurred. Our results suggest that RTA determines the processes which carry it, either in conjugate or toxin, from the plasma membrane binding site to the cytoplasm following endocytosis, and the ricin B chain is not required for these processes.     

Synthesis of a cytotoxic insulin cross-linked to diphtheria toxin fragment A capable of recognizing insulin receptors

Insulin has been cross-linked via a disulfide bond to the diphtheria toxin fragment A which is catalytically active in ADP-ribosylating elongation factor-2 but does not retain binding sites for toxin receptors. The purified conjugate proved to be cytotoxic to mouse Swiss/3T3 cells which are toxin resistant but express insulin receptors. This cytotoxicity coincided with a decrease in protein synthesis and with drastic morphology changes. In contrast, IN-2 cells, which are insulin-nonresponsive variants derived from mouse $\text{BALBc}\text{3T3}$ cells, were resistant to the conjugate. Thus, the conjugate (a chimeric insulin) appears to mediate entry of the toxic fragment A into 3T3 cells through insulin receptors.     

An epidermal growth factor-ricin A chain (EGF-RTA)-resistant mutant and an epidermal growth factor-Pseudomonas endotoxin (EGF-PE)-resistant mutant have distinct phenotypes

H2Oe12 is a mutant HeLa cell line selected for resistance to the toxicity of a chimeric protein conjugate composed of epidermal growth factor (EGF) and the toxic A chain of ricin (RTA). ET-28 is a mutant KB cell line selected for resistance to the toxicity of a chimeric protein conjugate composed of EGF and Pseudomonas exotoxin (PE). In this report we describe the presence or absence, in these mutants, of cross-resistance to the two toxic conjugates and the effects of ammonium chloride, leupeptin, and adenovirus cotreatments on toxin efficacies. ET-28 cells, the EGF-PE-resistant cells, are resistant to both EGF-PE and EGF-RTA. In contrast, H2Oe12 cells, the EGF-RTA-resistant cells, are as sensitive to EGF-PE toxicity as are their parent HeLa cells. Ammonium chloride cotreatment substantially reduces the resistance of H2Oe12 cells to EGF-RTA but has little or no effect on the resistance of ET-28 cells to either EGF-RTA or EGF-PE. Leupeptin has no effect on the toxicity of either chimeric conjugate on any of the four cell lines, effect on the toxicity of either chimeric conjugate on any of the four cell lines, despite its demonstrated ability to inhibit cellular degradation of EGF. In contrast, adenovirus cotreatment enhances the toxicity of EGF-RTA and EGF-PE on all cells tested, and completely nullifies the relative resistance of H2Oe12 and ET-28 cells to these toxic conjugates. H2Oe12 and ET-28 cells appear to be altered in distinct, possibly endosomal, functions.     

Association of ganglioside-protein conjugates into cell and Sendai virus. Requirement for the HN subunit in viral fusion

We have prepared several electron and light microscopic labels of epidermal growth factor (EGF) to analyse the morphologic features of its binding and internalization by cultured cells. These include a ferritin conjugate of EGF, a covalent conjugate of EGF and horseradish peroxidase (EGF-HRP), a colloidal gold marker system using EGF-HRP as a primary antigen, and a covalent complex of EGF with rhodamine-labelled lactalbumin. All of the light and electron microscopic labels showed similar patterns of binding. EGF initially bound to diffusely distributed cell surface receptors at 4 degrees C. The EGF-receptor complexes clustered into clathrin-coated pits on the cell surface only when the temperature was raised to 37 degrees C. In KB and Swiss 3T3 cells, this was followed by rapid internationalization into receptosomes, compartmentalization into the Golgi system, clustering in the clathrin-coated regions of the Golgi, and finally delivery into lysosomes from the Golgi. This general pathway was seen in Swiss 3T3 cells which have a low number of EGF receptors, KB cells which have a moderate number of receptors and A431 cells that have a high number of receptors. However, the ruffling activity induced in A431 cells by EGF produced some internalization through macropinosomes, making the pathway of entry more difficult to evaluate. Double label experiments showed that EGF is internalized together with alpha 2-macroglobulin and adenovirus particles. These data clarify the route of entry of EGF in different cell types using multiple labels, and shows that it enters cells through the same coated pit entry pathway as most other ligands previously examined.     

Epidermal growth factor labeled beta-amanitin-poly-L-ornithine: preparation and evidence for specific cytotoxicity

Poly-L-ornithine with an average molecular weight of 32K was reacted with beta-amanitin hydroxysuccinimide ester to form an amide-linked toxin conjugate. Loading of the polymeric chain with amanitin was high, corresponding to up to 35% of the total weight. To this amatoxin vehicle we attached a targeting molecule, human recombinant leucine-21 epidermal growth factor (hrEGFL), via a disulfide-containing linker moiety. A typical average stoichiometry of the hrEGFL labeled toxin conjugate was (L-Orn)164(beta-amanitin)19(COC2H4SSC2H4CO-hrEGFL)2. The affinity for EGF receptors of hrEGFL bound in this conjugate was tested by using A 431 cells. The affinity was eight times lower than that of unsubstituted hrEGFL but regarded as high enough for studying specific toxicity effects with cells bearing EGF receptors. We found that beta-amanitin in the labeled conjugate was able to inhibit the growth of A 431 cells at a concentration of 28 nM, 80 times lower than for native beta-amanitin and 20 times lower than for poly-L-ornithine-bound beta-amanitin without the hrEGFL label. The approximately 20-fold enhancement of cytotoxicity suggests a specific internalization of the toxin conjugate mediated by the hormone label. This idea is supported by the fact that also in another transformed fibroblast cell line, with an increased though smaller number of EGF receptors than A 431 cells, the corresponding enhancement of cytotoxicity was demonstrable but less pronounced (7-fold). The hormone-mediated increase in cytotoxicity of EGF labeled poly-L-ornithine-beta-amanitin conjugates, combined with their moderate toxicity in the mouse, encourages further examination of such compounds in tumor model systems in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterologous regulation of EGF receptors in fibroblastic cells

Platelet-derived growth factor (PDGF) increases the mitogenic activity of epidermal growth factor (EGF) in several cells lines, including BALB/C-3T3. PDGF-treated BALB/C-3T3 cells manifest a reduced capacity to bind 125I-labeled EGF due to a loss of high affinity EGF receptors. Cholera toxin potentiates the ability of PDGF to both decrease EGF binding and initiate mitogenesis. Whether PDGF increases EGF sensitivity via its effects on EGF receptors is not known and requires a more complete understanding of the mechanism by which PDGF decreases EGF binding. 12-O-tetradecanoylphorbol 13-acetate (TPA) also reduces EGF binding in BALB/C-3T3 and other cells, presumably by activating protein kinase C and, consequently, inducing the phosphorylation of EGF receptors at threonine-654. PDGF indirectly activates protein kinase C, and EGF receptors in PDGF-treated WI-38 cells are phosphorylated at threonine-654. Thus, the effects of PDGF on EGF binding may also be mediated by protein kinase C. We investigated this hypothesis by comparing the actions of PDGF and TPA on EGF binding in density-arrested BALB/C-3T3 cells. Both PDGF and TPA caused a rapid, transient, cycloheximide-independent loss of 125I-EGF binding capacity. The actions of both agents were potentiated by cholera toxin. However, whereas TPA allowed EGF binding to recover, PDGF induced a secondary and cycloheximide-dependent loss of binding capacity. Most importantly, PDGF effectively reduced binding in cells refractory to TPA and devoid of detectable protein kinase C activity. These findings indicate that PDGF decreases EGF binding by a mechanism that involves protein synthesis and is distinct from that of TPA.     

Toxicity of ligand and antibody-directed ricin A-chain conjugates recognizing the epidermal growth factor receptor

Approximately equal amounts of 125I-mAb 225 (a monoclonal antibody recognizing the human epidermal growth factor receptor) and 125I-labeled epidermal growth factor (125I-EGF) were bound by HeLa cells. However, these two EGF receptor binding moieties had different fates after binding. Sixty percent of cell-associated 125I-EGF was internalized. The majority of internalized 125I was released from the cell within 2 hr. In contrast, whereas only 30% of bound 125I-mAb 225 was internalized by HeLa cells, the internalized radioactivity remained cell-associated. EGF and mAb 225 were used to construct ricin A-chain (RTA) conjugates. The two chimeric molecules, EGF-RTA and mAb 225-RTA, were equally toxic to human HeLa cells. EGF-RTA was also toxic to murine 3T3 cells. In contrast, mAb 225-RTA was not toxic to 3T3 cells, consistent with the human EGF-receptor specificity of mAb 225. Neither conjugate was cytotoxic to EGF receptor-deficient 3T3-NR6 cells. Rapidity and potency of protein synthesis inhibition of HeLa cells were equivalent for the two chimeric conjugates, as was the degree to which colony-forming ability was reduced. However, ammonium chloride enhanced the toxicity of EGF-RTA but not mAb 225-RTA, suggesting that the two toxic chimeric toxins--like the unconjugated receptor-binding moieties--are processed differently by HeLa cells.     

Differential regulation of glucose transporter 1 and 2 mRNA expression by epidermal growth factor and transforming growth factor-beta in rat hepatocytes.

We have examined by Northern blot analysis the expression of two members of the glucose transporter family of genes (GLUT-1 and GLUT-2) in regenerating liver and in hepatocytes cultured under various conditions. GLUT-1, although thought to be a growth-associated gene, is not expressed in normal or regenerating liver, whereas GLUT-2, a liver-specific gene, is abundant in normal liver and gradually up-regulated during liver regeneration. Conversely, in hepatocytes cultured conventionally on dried rat tail collagen (RTC) in the presence of EGF and insulin, which potentiate proliferation, GLUT-1 mRNA is rapidly and abundantly expressed, whereas GLUT-2 is depressed. To investigate the causes of this "switch" in glucose transporter expression seen when hepatocytes are removed from the liver and cultured under the conventional proliferative conditions, we examined the effects of specific growth factors and extracellular matrices on cultured hepatocytes. EGF, a potent liver mitogen, although causing a threefold induction of GLUT-1, was found to have no effect on GLUT-2 expression, suggesting that the increase in GLUT-2 seen in regenerating liver is not due to EGF. Inhibition of protein synthesis by cycloheximide in cultured hepatocytes does not prevent the induction of GLUT-1 mRNA. In addition, treatment of cells with cycloheximide appears to stabilize the GLUT-2 mRNA, preventing the usual down-regulation of this gene in cultured hepatocytes. The expression of the two glucose transporter mRNAs also differed when the hepatocytes were adherent to particular cell matrices. Culture of hepatocytes on a reconstituted basement membrane gel matrix (EHS) is known to restrain their growth and mediate high levels of differentiated hepatocytic functions that are lost under conventional culture conditions. Unlike cells on RTC, hepatocytes on EHS expressed low levels of GLUT-1 mRNA, and decreased GLUT-2 mRNA. TGF-beta, an attenuator of DNA synthesis, when added to cultures on RTC, substantially down-regulated GLUT-2 but had no effect on GLUT-1. We propose that the effectors, EGF, TGF-beta and basement membrane components, play a significant role in the regulation of expression of GLUT-1 and GLUT-2 in hepatocytes.     

Epidermal growth factor stimulates formation of inositol phosphates in BALB/c/3T3 cells pretreated with cholera toxin and isobutylmethylxanthine

Epidermal growth factor (EGF) stimulated the formation of inositol trisphosphate, inositol bisphosphate, and inositol phosphate in density-arrested BALB/c/3T3 cells pretreated for 1.5-4 h with cholera toxin, a potent activator of adenyl cyclase, and isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor. Concomitant addition of cholera toxin, IBMX, and EGF to cells did not increase inositol phosphate levels, and pretreatment with both agents was more effective than pretreatment with either alone. Pre-exposure of cells to cholera toxin and IBMX also enhanced the increase in inositol phosphates occurring in response to platelet-derived growth factor (PDGF). Preincubation of cells with cholera toxin and IBMX in the presence of cycloheximide abolished the effects of these agents on EGF- and PDGF-stimulated inositol phosphate production as well as the lesser increase in inositol phosphate formation produced by cholera toxin and IBMX in the absence of hormone. Preincubation of cells with cycloheximide did not affect EGF binding or the ability of PDGF to stimulate inositol phosphate formation. Cycloheximide also precluded EGF-induced inositol phosphate production when presented to cells 3 h after addition of cholera toxin and IBMX. These findings show that, under the appropriate conditions, EGF is capable of stimulating inositol phosphate formation in a nontransformed cell line.     

Involvement of insulin receptor substrates in epidermal growth factor induced activation of phosphatidylinositol 3-kinase in rat hepatocyte primary culture.

Short-term incubation of adult rat hepatocytes with epidermal growth factor (EGF) caused tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 when the cells had been submitted to primary culture from 1-18 h. Tyrosine-phosphorylated IRS-1 and IRS-2 bound to the regulatory subunit (p85) of phosphatidylinositol (PtdIns) 3-kinase, thereby activating the enzymic activity. Tyrosine phosphorylation of the IRSs and activation of PtdIns 3-kinase in 3 h cultured hepatocytes both proceeded similarly to the same actions of insulin; the activation was rapid and transient, with peak values at 15-30 s and with similar EC(50)s in the nM range in both cases. A possible involvement of insulin receptors in these insulin-like actions of EGF was excluded by the following three lines of evidence. Insulin caused tyrosine phosphorylation of the insulin receptor beta-subunit but EGF did not. In contrast, the EGF receptor was phosphorylated by EGF, but the insulin receptor was not. The actions of EGF, but not those of insulin, were inhibited by AG1478, a selective inhibitor of EGF receptor tyrosine kinase. Cultured hepatocytes exposed to insulin or insulin-like growth factor-I (IGF-I) for a short period responded to the subsequent addition of EGF, whereas EGF-treated cells responded to insulin. The cells, however, displayed receptor desensitization under the same conditions, that is, no response was observed upon repeated addition of the same agonist, EGF, insulin or IGF-I. Thus, the EGF receptor-initiated signalling was mediated by PtdIns 3-kinase associated with tyrosine-phosphorylated IRSs in short-term cultured rat hepatocytes.     

Conversion of adrenergic mechanism from an alpha- to a beta-type during primary culture of rat hepatocytes. Accompanying decreases in the function of the inhibitory guanine nucleotide regulatory component of adenylate cyclase identified as the substrate of islet-activating protein

Adrenergic mechanism for phosphorylase activation was gradually converted from an alpha 1- to a beta 2-type during primary culture of rat hepatocytes. beta 2-Receptor-mediated cAMP generation was also much greater in 8-h cultured cells than in fresh cells. Incubation of hepatocyte membranes with [alpha-32P]NAD and the preactivated A-protomer (an active component) of islet-activating protein (IAP), pertussis toxin, resulted in the ADP-ribosylation of a specific IAP substrate protein (Mr = 41,000). This ADP-ribosylation diminished progressively when the membrane-donor hepatocytes had been cultured. The early diminution was interfered with by the addition of nicotinamide or isonicotinamide, a potent inhibitor of ADP-ribosyltransferase, to the culture medium. The decrease of the IAP substrate was well correlated with the potentiation of beta-adrenergic functions under various conditions of culture. beta-Receptor-mediated activation of GTP-dependent membrane adenylate cyclase was, but glucagon-induced activation was not enhanced by either prior culture of hepatocytes or prior exposure of membranes to the A-protomer of IAP. There was no further enhancement, however, when membranes from cultured cells were exposed to the active toxin. Thus, the IAP-susceptible inhibitory guanine nucleotide-regulatory protein is coupled to beta-adrenergic receptors in such a manner as to reduce the degree of activation of cyclase, and the decrease in this IAP substrate may be responsible, at least partly, for development of beta-receptor functions during culture of hepatocytes. Its possible relation to accompanying inhibition of alpha 1-receptor functions is discussed.     

HeLa cell mutants resistant to epidermal growth factor ricin A-chain conjugate

Epidermal growth factor (EGF) was linked to the toxic A chain of ricin toxin (RTA) to produce an EGF-receptor-specific cytotoxic agent, EGF-RTA. Three EGF-RTA-resistant mutants of the human HeLa cell line were selected. These mutant cell lines are 10-fold to more than 100-fold more resistant to EGF-RTA when compared to HeLa cells. The EGF-RTA-resistant mutants have at least as many EGF receptors as parent cells; the basis for the EGF-RTA-resistant phenotype must be distal to EGF binding. The EGF-RTA-resistant cells are not cross-ressitant to ricin or to diphtheria toxin; their mutant phenotype appears to be EGF specific. The EGF-RTA-resistant mutants are able to internalize and degrade EGF. However, the mutants have altered EGF receptor down-regulation and phorbol 12-tetradecanoate 13-acetate modulation properties. EGF-RTA/ammonium chloride and EGF-RTA/adenovirus co-treatment data suggest that the mutant defect(s) which confers EGF-RTA resistance is either in the endosome or at a step(s) in the intracellular EGF processing pathway between the endosome and the lysosome.     

Effect of photo-immobilization of epidermal growth factor on the cellular behaviors

We constructed photo-reactive epidermal growth factor (EGF) bearing p-azido phenylalanine at the C-terminal (HEGFP) by genetic engineering to investigate the possibility of immobilized EGF as a novel artificial extracellular matrix (ECM). The constructed recombinant protein was immobilized to glass surface by ultraviolet irradiation. A431 cells adhered both to HEGFP-immobilized and collagen-coated surfaces. Interaction between immobilized HEGFP and EGF receptors in the A431 cells was independent of Mg(2+) although integrin-mediated cell adhesion to natural ECMs is dependent on Mg(2+). Phosphorylation of EGF receptors in A431 cells was induced by immobilized HEGFP as same as soluble EGF. DNA uptake of hepatocytes decreased by immobilized HEGFP whereas it increased by soluble EGF. Liver-specific functions of hepatocytes were maintained for 3 days by immobilized HEGFP whereas they were not maintained by soluble EGF, indicating that immobilized HEGFP follows different signal transduction pathway from soluble EGF.     

Regulation of the transmodulated epidermal growth factor receptor by cholera toxin and the protein phosphatase inhibitor okadaic acid.

Addition of tumor promoting phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), to many cell lines results in a decrease of 125I-epidermal growth factor (EGF) binding and increased serine/threonine phosphorylation of the EGF receptor in a process termed transmodulation. It is, however, unclear whether or not receptor phosphorylation is causally related to the inhibition of high affinity EGF binding. We have investigated the significance of phosphorylation/dephosphorylation events in the mechanism of PMA-induced transmodulation using the adenylate cyclase activator cholera toxin and the serine/threonine protein phosphatase inhibitor okadaic acid. In Rat-1 fibroblasts treated at 37 degrees C, PMA induced a rapid decrease in EGF binding which persisted for 3 hours. In contrast, cells exposed to PMA in the presence of cholera toxin exhibited a marked recovery of binding within 60 minutes. The PMA-stimulated decrease in binding correlated with a rapid increase in the phosphorylation state of the EGF receptor. While phosphorylation of the receptor was sustained at an elevated level for at least three hours in cells receiving PMA alone, EGF receptor phosphorylation decreased between 1 and 3 hours in cells treated with PMA and cholera toxin. Furthermore, the cholera toxin-stimulated return of EGF binding was inhibited by treatment with the phosphatase inhibitor okadaic acid. These results suggest that a cholera toxin-activated phosphatase can increase binding capacity of the transmodulated EGF receptor in Rat-1 cells. Cholera toxin treatment elicited a qualitatively similar response in cells transmodulated by platelet-derived growth factor (PDGF). Okadaic acid antagonized the natural return of binding observed in cells stimulated with PDGF alone, indicating that a dephosphorylation event may be required for the recovery of normal EGF binding after receptor transmodulation.     

cAMP-mediated modulation of signal transduction of epidermal growth factor (EGF) receptor systems in human epidermoid carcinoma A431 cells. Depression of EGF-dependent diacylglycerol production and EGF receptor phosphorylation.

While a cAMP-dependent protein kinase (protein kinase A) has been suggested to phosphorylate epidermal growth factor (EGF) receptor in vitro, both intrinsic and EGF- or potent phorbol tumor promoter-induced phosphorylation of EGF receptor were found to be depressed in human epidermoid carcinoma A431 cells by prior incubation of the cells with various protein kinase A activators (e.g. cholera toxin, forskolin, cAMP analogues, or a combination of prostaglandin E1 and 3-isobutyl-1-methylxanthine). Protein kinase A activators did not change significantly either the number of EGF receptors or their affinity for EGF. The tryptic phosphopeptide map of EGF receptors from cells treated with cholera toxin alone or cholera toxin followed by EGF revealed unique peptides whose serine phosphorylation was preferentially depressed. However, the catalytic subunit of protein kinase A phosphorylated no threonine and little serine in the EGF receptors in the plasma membranes of isolated A431 cells in vitro, while serine residues in an unidentified 170-kDa membrane protein(s) other than EGF receptor were heavily phosphorylated. Pretreatment of the cells with forskolin blocked 1,2-diacylglycerol induction by EGF; growth inhibition by nanomolar levels of EGF could be partially restored by the presence of forskolin. These results indicate that an increase in intracellular cAMP modulates the EGF receptor signal transduction system by reducing EGF-induced production of diacylglycerol without direct phosphorylation of EGF receptors by protein kinase A in A431 cells.