EGF receptor down-regulation attenuates ligand-induced second messenger formation

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EGF Receptor Down-Regulation Attenuates Ligand-Induced Second Messenger Formation ANN GILLIGAN,’ MARC PRENTKI,**’ AND BARBARA B. KNOWLES Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104; and *The Department of Biochemistry Biophysics and Diabetes Research Center, University of Pennsylvania, Philadelphia, Pennsylvania 19104

thereby activating an array of second messenger systems: the tyrosine-specific protein kinase of the receptor [l, 21; the Naf/Hf antiport [3, 41; plasma membrane Ca2+ channels [3,5,6]; and phosphoinositidase C hydrolysis of phosphatidylinositol 4,Sbisphosphate (PtdsIns(4,5)Pz) [6-131. The EGF signal is attenuated by down-regulation of surface receptors after selective internalization and degradation of EGF-receptor complexes [14-161 and by desensitization of receptor function as a consequence of protein kinase C phosphorylation of receptor residues, particularly at threonine 654 [8,9, 12,17, 181. Despite these mechanisms for limiting cellular responsiveness to EGF, deregulation of EGF receptor function, as evidenced by acquisition of a transformed phenotype, occurs in some cell types that constituitively produce EGF-like proteins [19-211. The demonstration that cells expressing the retroviral oncogene product, v-erb B, a truncated form of the EGF receptor which exhibits constitutive tyrosine-specific protein kinase activity [22, 231, acquire a transformed phenotype [24,25] suggests that unregulated second messenger formation confers the transformed phenotype. EGF receptor gene amplification with concomitant receptor protein overexpression is frequently observed in human tumors and tumor cell lines [26-291. The observation that NIH 3T3 mutants in which EGF receptor levels have been increased from 3 X lo5 acquire a transformed phenotype in the presence of EGF indicates that receptor overexpression is sufficient for abrogation of growth control [30]. Paradoxically, tumor cell lines of epithelial origin which overexpress the EGF receptor are growth inhibited by nanomolar EGF [26,3133]. EGF is a primary mitogen for liver cells, both in uivo and in vitro [34,35], and coupling between EGF receptor activation and PtdsIns(4,5)P, hydrolysis appears to be especially strong in epithelial cells of liver origin, including hepatocytes [7-91. We have previously determined that a human hepatocellular carcinoma-derived cell line, PLC/PRF/5, while tumor-derived, retains the differentiated functions of hepatocytes in vitro [36] and expresses, as do untransformed rat hepatocytes, approximately 300,000 EGF receptors per cell [31, 371. These cells proliferate in the presence of EGF [31] and respond

Epidermal growth factor (EGF)-induced increases in cytosolic Ca2+ and inositol polyphosphate production were compared in a human hepatocellular carcinomaderived cell line, PLC/PRF/5, and in an EGF receptoroverexpressing subline, NPLC/PRF/5. Formation of these second messengers was correlated to EGF receptor display at the cell surface by monitoring ligand-induced EGF receptor down-regulation. Both cell lines exhibited a strikingly similar cytosolic Ca2+ increase upon exposure to EGF. The initial inositol phosphate responses were also similar in the two cell lines; inositol 1,4,5-trisphosphate increased within lo-15 s and returned to prestimulatory values after 2 min in both cell lines, while inositol tetrakisphosphate and inositol 1,3,4-trisphosphate were elevated after a 2-min exposure to EGF. At later times the responses were markedly different; NPLC/PRF/5 cells exhibited prolonged production of inositol 1,3,4-trisphosphate and inositol tetrakisphosphate (maximum at l-3 h) but PLC/PRF/5 cells showed decreased levels of these isomers after 10 min and a return to basal values by 1 h. Exposure of PLC/PRF/B cells to EGF caused a progressive decrease in the amount of EGF receptor at the cell surface whereas such treatment did not change the surface receptor levels in NPLC/PRF/B cells. Kinetic analysis of EGF receptor down-regulation showed that receptor internalization was rapid enough to account for the transient nature of the inositol phosphate response in PLC/PRF/5 cells. Thus, the divergent patterns of signaling exhibited by the two cell lines may reflect differences in the efficiency of EGF-induced down-regulation of surface receptors. 0 is90 Academic Pre38.1~.

INTRODUCTION Epidermal growth factor (EGF) binds to a specific transmembrane receptor protein at the cell surface, 1 To whom reprint requests should be addressed at Wistar Institute of Anatomy and Biology, Room 123,36th St. at Spruce, Philadelphia, PA 19104. 2 Present address: Institut de Biochimie Clinique, University of Geneva, CH-1211 Geneva, Switzerland.

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strongly to EGF with increased cytosolic Ca2+ and inosito1 1,4,5trisphosphate (Ins(1,4,5)PJ production [7]. Proliferation of NPLC/PRF/5 cells, a subline of PLC/ PRF/5 which expresses approximately 10 times more EGF receptors at its surface, is inhibited by nanomolar EGF [31]. In this study we examine the alterations in EGF-induced PtdsIns(4,5)P2 hydrolysis which occur upon receptor overexpression and test the hypothesis that EGF receptor overexpression alters EGF signaling by rendering ineffective the regulatory mechanisms which control the extent of second messenger formation. EGF-induced Ins(1,4,5)P, production, production of Ins(1,4,5)P3 metabolites, and cytosolic Ca2+ fluxes were measured in both the PLC/PRF/5 and NPLC/PRF/5 cell lines and correlated with cell surface EGF receptor levels. Initially, both lines respond similarly to EGF with increased cytosolic Ca2+ and inositol polyphosphate production. Upon prolonged exposure to EGF, the receptoroverexpressing cell line generates a sustained inositol phosphate response while the response in PLC/PRF/5 cells is transient. EGF-induced receptor down-regulation appears to limit the responsiveness of PLC/PRF/5 cells to EGF but is ineffective for NPLC/PRF/5 cells. EXPERIMENTAL Cell culture. PLC/PRF/5, a human hepatocellular carcinoma-derived cell line established by Alexander et al. [38], and NPLC/PRF/5, a subline derived in our laboratory from explants of a tumor which arose in an athymic mouse injected with PLC/PRF/5 cells [31], were passaged at split ratios of 1:2 by trypsinization with 0.25% trypsin/ 0.1% EDTA in Dulbecco’s modified phosphate-buffered saline (PBS) and dilution in Eagle’s minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum and 2 mM glutamine (complete MEM). Cells were maintained at 37°C in a humidified CO* (5%) incubator. NPLC/PRF/5 and the parental line are closely related as determined by karyotype analysis, patterns of hepatitis B virus integration into the host genome, and processing of oligosaccharides of the EGF receptor [31]. Znositol phosphate measurements. Cells were seeded at 2-4 X 10s cells per loo-mm petri plate (Falcon 3003) and labeled 5 days later for 24 h with 10 ml of complete MEM containing 2.5-3.0 &i/ml of myo[23H]inositol (Amersham, 20 Ci/mmol) and then with 2.5-3.0 &i/ml of myo[2-3H]inositol in serum-free MEM for another 18-24 h. Cells were washed three times with PBS, preincubated in serum-free MEM containing 0.1% bovine serum albumin (BSA) (incubation medium) for 30-45 min, and washed three more times with PBS. Cells were exposed to incubation medium alone or to medium supplemented with EGF (Collaborative Research, receptor grade) or with angiotensin II (Boehringer-Mannheim Biochemicals) for various times at 37°C. Reactions were terminated by rapid aspiration of the reaction medium and immediate addition of 0.8 ml of ice-cold 10% trichloroacetic acid. Precipitated material was scraped from plates and transferred to 15 ml conical centrifuge tubes along with the acid extract. Plates were washed once with 0.8 ml of 10% trichloroacetic acid, the wash was combined with the initial extract, and the protein precipitate was removed by centrifugation. Acid-soluble extracts were prepared for high-performance liquid chromatography (HPLC) analysis by four washes with water-saturated diethylether, drying by lyophilization, and reconstituting with water. NPLC/PRF/5 extracts often contained several-fold more label per milligram of cell protein than did PLC/

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PRF/5 cells. However, basal levels of inositol polyphosphates were similar between the two cell lines so this enhanced labeling may not be relevant to substrate labeling. High-performance liquid chromatography. Trichloroacetic acid extracts derived from an equivalent of 2 mg of cell protein were reconstituted with 250 pl of water and the highly phosphorylated isomers of inositol phosphate separated by HPLC. Cell extracts were separated on a Partisil Sax 10 column as outlined [7] using a variation of the method of Irvine et al. [39]. Fractions (0.6 ml) were collected, 0.9 ml of 50% methanol and then 4.6 ml of ACS II scintillation fluid (Amersham) added, and samples counted on the tritium channel of a Beckman scintillation counter. To avoid quenching of radioactivity due to high salt, 0.3 ml of each fraction above fraction 39 was removed and 0.3 ml of water added before addition of 50% methanol. Protein analysis. Protein pellets collected after trichloroacetic acid extraction were resuspended in l-4 ml of Lowry reagent (Sigma protein assay kit) and the protein content of 100 to 400 pl aliquots was measured by the method of Lowry [40]. Cytosolic calcium measurements. Near confluent cultures were switched to serum-free MEM 16 to 20 h before harvest. Cells were detached from T-75 flasks by trypsinization, diluted with serum-free MEM containing 0.20% BSA and 15 mM NaHepes, pH 7.4, and collected by centrifugation. After one wash, cells were loaded with the Gas+ indicator dye fura 2 by incubation with 2 PM fura 2/AM (Calbiothem-Behring) as described [7]. EGF- and angiotensin II-induced increases in cytosolic Gas+ were determined by monitoring fluorescence of suspensions (5-10 X 10’ cells/2 ml) of fura a-loaded cells using an MB-2 four-filter air turbine fluorescence spectrometer (Johnson Foundation, Philadelphia, PA) at excitation wavelengths of 340 nm (CaZf-fura 2 complex) and 385 nm (free fura 2) and at the emission wavelength of 510 nm. Cell surface iodination, immunoprecipitation, and gel electrophoresis. Near-confluent monolayers of cells in loo-mm petri plates were incubated in serum-free MEM for 18 to 22 h and then in serum-free MEM containing 25 rig/ml EGF and 0.1% BSA for 1 or 4 h at 37°C. Control plates were incubated in serum-free MEM containing 0.1% BSA for 1 h. After three washes with Ca’+-MgZC-free PBS, cells were subjected to cell surface iodination by the lactoperoxidase/glucose oxidase reaction [41]. After iodination, cells were harvested by scraping into 135 mM NaI/8.1 mM Na,HP0&.5 mM KHzP04/2.7 mM KC1 supplemented with 2 mM phenylmethylsuifonylfluoride and 50 pg/ml leupeptin, collected by centrifugation, and resuspended in 0.8 ml of 0.1 M Tris-HCl, pH 6.8/2 mM EDTA/lS% glycerol containing the same protease inhibitors. Cells were then solubilized by addition of nonidet P-40 (NP-40) to a final concentration of 1% and incubated at 4°C for 1 h. After centrifugation (Eppendorf) for 15 min at 4°C to remove insoluble debris, cell extracts were quick-frozen and stored at -70°C. Sepharose-protein A beads (Pharmacia) (25% [v/v] suspension in STN [0.15 M NaCl/O.Ol M Tris-HCl, pH 7.4/0.25% NP-401) were armed with EGF receptor Rl antibody [42] by constant mixing at 4’C for 1.5 h. After four washes of the beads with STN, aliquots of iodinated cell extracts (lo7 cpm) were incubated with 100 pl of the 25% suspension of armed beads. Samples were incubated at 4°C with frequent vortexing for 1.5 h, washed once with 0.45 MNaCl/O.Ol MTrisHCl, pH 7.4/0.25% NP-40, and then washed nine times with STN. Bead pellets were boiled for 5 min in 50 pl of (0.08 M Tris-HCl, pH 6.8/1.6 mMEDTA/lZ% glycerol/2% SDS/O.1 M dithiothreitol/0.02% bromophenol blue), and supernatants separated by SDS-polyacrylamide gel electrophoresis using a 7.5% resolving gel and a 5% stacking gel [31]. Fluorescence-actiuated cell sorting. Cells shifted to serum-free MEM 16-20 h previously were incubated at 37°C with 25 rig/ml EGF in 0.1% BSA for 15 min, 30 min, 1 h, and 4 h or with 500 rig/ml EGF for 1 or 4 h. Controls were incubated with 0.1% BSA for 1 h. After incubation cells were detached by brief trypsinization, washed twice with cold complete MEM, and plated in triplicate in 96-well plates. The single cell suspensions were incubated in duplicate (1 h, 4”C, 5-

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10 X lo5 cells per 0.1 ml of FACS buffer [PBS containing 5% fetal calf serum and 0.1% NaN,]) with Rl monoclonal antibody to the EGF receptor at a dilution previously determined to give maximum binding. Cells were washed three times and then incubated at 4°C for 1 h with FITC-tagged goat immunoglobulin G (IgG) anti-mouse IgG Fc fragment y-chain-specific (Cappel, West Chester, PA) diluted $ in the FACS buffer. Negative controls (one well/treatment group) were incubated with FITC-labeled antibody alone. Cells were washed three times and resuspended in 400 pl, and immunofluorescence was measured by flow cytometry using an Ortho Instruments Cytofluorograf. RESULTS

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To measure the extent of activation of PtdsIns(4,5)Pz hydrolysis by EGF, production of inositol tetrakisphosphates (InsP,) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P,) were measured, in addition to monitoring increases in Ins(1,4,5)P3. Phosphorylation of Ins(1,4,5)P3 to form inositol 1,3,4,5tetrakisphosphate (Ins(1,3,4,5)P,) occurs rapidly in cells possessing a Ca2+-activated 3-kinase [43, 441 and activation of a 5-phosphatase by protein kinase C may stimulate rapid conversion of Ins(1,3,4,5)P, to Ins(1,3,4)P3 [45]. Hydrolysis of PtdsIns(4,5)P, is, therefore, accompanied by formation of Ins(1,3,4,5)P4 and Ins(1,3,4)P3, as well as its initial product Ins( 1,4,5)P3. The time courses of appearance of these inositol polyphosphates after exposure to EGF were compared in PLC/PRF/5 and its EGF receptor-overexpressing variant NPLC/PRF/5. These experiments were performed without LiCl supplementation so that the long-term kinetics of PtdsIns(4,5)P, hydrolysis could be measured. At 12-15 s after addition of EGF, Ins(1,4,5)P3 levels were substantially elevated (2.67-fold) in PLC/PRF/5 cells while Ins( 1,3,4)P3 and InsP, levels remained close to basal values (Fig. 1A). Within 2 min, Ins(1,4,5)P3 levels had returned to prestimulatory values, but the levels of Ins(1,3,4)P3 and InsP, were greatly increased. The early response of NPLC/PRF/5 cells to EGF was similar to that observed in PLC/PRF/5 cells except that Ins(1,4,5)P3 levels were only slightly increased (1.76fold) at 12 to 15 s after EGF addition (Fig. 1B). While the initial responses in the two cell lines were quite similar, the later responses were markedly divergent. In PLC/PRF/5 cells, Ins(1,3,4)P, and InsPl levels have decreased from peak values by 10 min and are close to basal levels by 1 h (Fig. 1A). By contrast, Ins(1,3,4)P, and InsP, levels continued to increase in NPLC/PRF/ 5 cells, reaching a maximum value by l-3 h after EGF addition (Fig. 1B). Increases in InsP, were observed in both cell lines even after Ins(1,3,4)P3 levels were no longer increasing. The 5-phosphatase which produces Ins(1,3,4)P3 may not be as active at later times, or some of the Ins(1,3,4)P3 may be converted to inositol 1,3,4,6tetrakisphosphate, a reaction known to occur in liver homogenates [46].

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These divergent patterns of inositol phosphate production were also observed for the less highly phosphorylated inositol phosphates. After 1 h incubation with EGF, inositol mono- and bisphosphate levels were greatly elevated in NPLC/PRF/5 cells but were close to basal values in PLC/PRF/5 cells (data not shown). Cytosolic Ca” Fluxes To ascertain whether the levels of Ins(1,4,5)P3 attained elicit mobilization of intracellular Ca’+, cytosolic Ca2+ fluxes in the two cell lines were measured upon addition of EGF in the presence or absence of extracellular Ca’+. These responses were compared to cytosolic Ca2+ responses elicited by exposure to angiotensin II, a peptide hormone known to stimulate PtdsIns(4,5)P, hydrolysis in liver [47]. At lo-20 s after EGF addition to PLC/ PRF/5 cells, cytosolic Ca2+ increased from an average basal value of 174 nM to an average peak value of 470 nM. Peak Ca2+ levels were attained within 45 s to 1 min but decreased to 26% (SE = 2.3%, n = 8) of the peak by 10 min (Fig. 2A). Increasing extracellular Ca2+ from 2 to 7 mM at 10 min after EGF addition caused a further elevation in cytoplasmic Ca2+, presumably because of augmented Ca2+ influx. Chelation of extracellular Ca2+ only slightly decreased the initial peak but the late sustained component of the response was no longer observed (Fig. 2B). These cells responded within seconds to angiotensin II (50 pg/ml) with increased cytosolic Ca2+; addition of 5 mM CaC12 after a lo-min exposure elicited only a small elevation in cytosolic Ca2+ (Fig. 2C). After chelation of extracellular Ca2+ the sustained response to angiotensin II, which was smaller than that after EGF, was no longer observed (Fig. 2D). The cytosolic Ca2+ response to EGF in NPLC/PRF/5 cells was strikingly similar to that obtained in PLC/ PRF/5 cells. NPLC/PRF/5 cells responded somewhat faster to EGF; cytosolic Ca2+ increased from an average basal value of 98 nM to an average peak value of 349 nM within 20-30 s (Fig. 3A). This increased rate of response may underlie the relatively small accumulation of Ins( 1,4,5)P3 observed in these cells as Ca2+-activated reactions which metabolize that isomer may be stimulated more rapidly. Comparable increases in cytosolic Ca2+ were observed at the peak in both cell types, although the sustained component in NPLC/PRF/5 cells appeared to be greater than that in PLC/PRF/5 cells, persisting at 52% (SE = 3.4%, n = 8) of the peak at 10 min (Fig. 3A). Addition of 5 mM Ca2+ at 10 min after EGF addition also resulted in an augmentation of cytosolic Ca2+ in these cells. The intracellular component of the Ca2+ response to angiotensin II predominated in both cell lines (Figs. 2C, 2D, 3C, and 3D). Inositol Phosphate Responses to Combinations of EGF and Angiotensin II The inositol phosphate responses to combinations of EGF and angiotensin II were compared in the two cell

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FIG. 1. Inositol phosphate responses to EGF in PLC/PRF/5 and NPLC/PRF/5 cells. PLC/PRF/5 cells (A) were exposed to 25 rig/ml of EGF for 0,12-15 s, 2 min, 10 min, and 1 h and NPLC/PRF/5 cells (B) were exposed to 25 rig/ml EGF for 0,12-15 set, 2 min, 1 h, and 3 h. Cell extracts were separated by HPLC; counts in Ins(1,3,4)P,, Ins(1,4,5)P3, and InsPl fractions were measured, corrected for background and dilution (fractions 40 and above), and expressed as counts per minute/milligram of cell protein. Values shown are means + SEM of three to nine separate experiments, except for those for 3 h, which were repeated twice.

lines to determine whether the prolonged PtdsIns(4,5)P2 hydrolysis observed in NPLC/PRF/5 cells exposed to EGF might result from factors other than EGF receptor overexpression, e.g., alterations in enzymes of the inosito1 phospholipid signaling pathway or enhanced labeling of PtdsIns(4,5)P, in NPLC/PRF/5 cells. Continuous exposure of PLC/PRF/5 cells to 25 rig/ml of EGF for 1 h did not cause a sustained increase in InsPB and InsP4, but addition of 50 pg/ml of angiotensin II for 1 min immediately after the l-h incubation led to an inositol phosphate response comparable to that observed in cells exposed to angiotensin II alone (Fig. 4, top). NPLC/ PRF/5 cells exposed to EGF for 1 h or to angiotensin II for 1 min generated large increases in inositol trisphosphates (InsPs) and InsPl (Fig. 4, bottom). Cells incubated with EGF for 1 h followed by angiotensin II for 1 min generated inositol phosphates close to levels expected for an additive response in both cell lines. The

levels of InsPs and InsPl after incubation of NPLC/ PRF/5 cells with angiotensin II alone for 1 min or 1 h were of the same magnitude as those observed in PLC/ PRF/5 cells after the same treatment. EGF-Induced

EGF Receptor Down-Regulation

To analyze the relationship between EGF receptor down-regulation and the time course of inositol phosphate production in the two cell lines, the EGF surface receptor levels were examined as a function of time of exposure to EGF. The surface receptor population was selected by iodination of intact cells followed by immunoprecipitation of EGF receptors from cell extracts. Representative autoradiograms of SDS gels of the immunoprecipitates are shown in Fig. 5. As expected, the intensity of the receptor band in unstimulated NPLC/ PRF/5 cells was much greater than that in PLC/PRF/5

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FIG. 2. Cytosolic Ca2+ fluxes in PLC/PRF/5 cells exposed to EGF or angiotensin II. Suspensions of fura 2-loaded cells were exposed to 25 rig/ml of EGF in the presence of 2 mM Ca2+ (A) and after EGTA treatment to lower extracellular Ca” (B). Responses to angiotensin II (50 pg/ ml) were also monitored in the presence (C) and absence (D) of extracellular Ca ‘+ . In some experiments (A, C), extracellular Ca2+ was increased 10 min after ligand addition by addition of 5 mM CaCl,. Cellular fluorescence was continuously monitored at excitation wavelengths 385 and 340 nm and emission wavelength 510 nm. Results shown are typical of eight different experiments and are illustrated using the 385-nm signal. Increases in cytosolic Ca2+ are expressed as percentage of the maximum 385-nm signal. This maximum was determined by lysing cells with 0.4% Triton X-100 and measuring the change in the 385-nm signal upon addition of 5 mM EGTA. This initial value was corrected for extracellular Ca’+-fura 2 complexes by subtracting the change in the 385-nm signal that occurred upon addition of EGTA to intact cells.

cells (Fig. 5A, cf. lane 1 with lane 4). The receptor bands immunoprecipitated from PLC/PRF/5 cells incubated with 25 rig/ml of EGF for 1 h (Fig. 5A, lane 5) or 4 h (Fig. 5A, lane 6) showed a progressive decrease in intensity. By contrast, EGF receptor levels at the surface of NPLC/PRF/5 cells were apparently unchanged upon incubation with that dose of EGF (Fig. 5B, lanes l-3). Binding of the EGF receptor monoclonal antibody Rl to the surface of intact cells which had been previously exposed to EGF for various times is illustrated in Table 1. Challenge with 25 rig/ml EGF induced EGF receptor internalization in PLC/PRF/5 cells within 15 min incubation; the degree of internalization increased with time of incubation and dose of EGF (Table 1). Redistribution of EGF receptors from the plasma membrane to cytosolic vesicles is observed by immunofluorescent microscopy in those cells when they are exposed to saturating levels of ligand (Gilligan and Knowles, unpublished observations). By contrast, surface EGF receptor levels of NPLC/PRF/5 cells did not show any decrease upon ex-

posure to 25 rig/ml EGF; at a higher concentration of EGF where most of the EGF receptors will be occupied (500 rig/ml), there is but slight diminishment in antibody binding even after a 4-h incubation (Table 1). Thus, the difference in ligand-induced down-regulation observed for the two’ cell lines is not merely a consequence of different degrees of receptor occupancy. DISCUSSION

The present findings indicate that NPLC/PRF/5 cells, which overexpress the EGF receptor, respond to EGF with sustained PtdsIns(4,5)Pz hydrolysis and with maintenance of high surface levels of receptor. By contrast, PLC/PRF/5 cells, which express a level of receptor closer to that of untransformed hepatocytes, respond with a transient inositol phosphate signal whose diminishment correlates with ligand-induced receptor downregulation. The sustained response to EGF exhibited by NPLC/PRF/5 cells is not merely a consequence of more

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FIG. 3. Cytosolic Ca2+ fluxes in NPLC/PRF/B cells exposed to EGF or angiotensin II. Cytosolic Ca *+ levels were monitored as in Fig. 2 Cax+ (B), or after exposure to after exposing cells to 25 rig/ml of EGF in the presence of 2 mM Cax+ (A) and after chelation of extracellular 50 pg/ml of angiotensin II in the presence (C) or absence (D) of extracellular Cax+. These traces are representative of eight independent experiments.

efficient labeling of the PtdsIns(4,5)Pz substrate pool or of alterations in enzymes controlling inositol phosphate metabolism as the inositol phosphate responses to angiotensin II were of the same magnitude in both cell types. EGF-induced inositol phosphate production is initially quite similar in the two cell lines as is the intracellular component of the Cazf response. Thus, initial coupling between EGF receptor activation and the phosphatidylinositol signaling pathway appears to be basically unaltered upon EGF receptor overexpression. There have been many previous analyses of EGF-induced inositol phosphate production; however, this paper is one of the few to examine long-term accumulation in the absence of LiCl. LiCl is frequently used to inhibit inositol-1 phosphatase, thereby allowing amplification of the inositol phosphate signal [48,49]. This treatment also alters the levels of physiologically important inosito1 polyphosphates and may potentially change interactions between GTP-binding regulatory proteins and activated receptors [50, 511. Since PLC/PRF/5 and NPLC/PRF/5 cells generate substantial inositol phosphate responses to EGF in the absence of LiCl, the prolonged response in cells that overexpress the EGF receptor could be distinguished from the transient response in PLC/PRF/5 cells. These data prompt the novel con-

elusion that receptor overexpression does not alter initial transmembrane signaling but rather affects the duration of the response. Another recent study of the inosito1 phosphate response to EGF in the absence of LiCl showed that EGF induced prolonged inositol phosphate responses in A431 cells, an epithelial cell line which expresses l-3 X lo6 EGF receptors per cell [12]. Although comparable data from A431 variants expressing lower levels of EGF receptor were not reported by these investigators, their initial observations are consistent with our contention that EGF receptor overexpression leads to prolonged stimulation of PtdsIns(4,5)P2 hydrolysis. The conclusions from our study contrast with those from studies of mouse fibroblastic cell lines in which cells expressing very low levels of endogenous receptors were compared with those overexpressing the EGF receptor [lo]. In this case the cells expressing “normal” levels of receptor do not exhibit coupling between EGF receptor activation and PtdsIns(4,5)P, hydrolysis while the overexpressing variants exhibit measurable responses, suggesting that coupling mechanisms were altered by EGF receptor overexpression. While the length of the inositol phosphate response was not measured, the Ca2+ response in the receptor-overexpressing fibroblasts is transient compared to that observed in epithe-

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FIG. 4. Inositol trisphosphate and inositol tetrakisphosphate levels in PLC/PRF/5 and NPLC/PRF/B cells exposed to EGF and/or angiotensin II. Cells (PLC/PRF/5, top, and NPLC/PRF/5, bottom) were labeled with [3H]myoinositol and exposed to combinations of EGF (25 rig/ml) and angiotensin II (50 pg/ml): 0 control (A); 1 h EGF (B); 1 h EGF, then 1 min angiotensin II (C); 1 min angiotensin II (D); and 1 h angiotensin II (E). Radioactivity levels were expressed as counts per minute/milligram of cell protein and were determined as in Fig. 1 except that Ins(1,3,4)P3 and Ins(1,4,5)P, were combined to obtain InsPs levels. The results from a single experiment are shown and are representative of three separate experiments.

lial lines which overexpress the EGF receptor (NPLC/ PRF/5, A431) [6, 10, Fig. 31. A cell line expressing the level of EGF receptor found on PLC/PRF/5 cells mimics that expressed by the EGF responsive parenchymal hepatocyte in uiuo [37] and therefore represents a more physiologically relevant model for the ligand-receptor interactions that occur upon stimulation of epithelial cell growth. The sustained responses after EGF binding observed in NPLC/PRF/5 cells suggested that the mechanisms which limit receptor function are ineffective in cells that overexpress the EGF receptor. EGF-induced down-regulation of its own receptor has been observed in several cell tvnes I14-161 and receptor levels are reduced at the

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surface of PLC/PRF/5 cells after exposure to EGF. By contrast, EGF receptor levels remain elevated in NPLC/ PRF/5 cells even after long-term exposure to high concentrations of EGF. In A431 cells, internalization of EGF-receptor complexes is inhibited by high receptor occupancy [52,53], possibly as a result of a Ca2+/calmodulin-mediated inhibition of receptor internalization [52]. In addition, recycling of internalized receptor to the cell surface, rather than immediate routing to lysosomes for degradation, has been reported for some cells [37,54,55]. EGF also appears to induce synthesis of its own receptor so that stimulated biosynthesis may compensate for ligand-induced degradation of receptor [56, 571. A combination of these mechanisms may allow large numbers of EGF receptors to persist at the surface of NPLC/PRF/5 cells exposed to EGF. We suggest that the prolonged inositol phosphate response to EGF exhibited by these cells is a consequence of their inability to decrease EGF receptor density after exposure to ligand. In rat liver epithelial cells, inhibition of protein kinase C-desensitization enhances the inositol phosphate response to EGF and prolongs the inositol phosphate response to angiotensin II [8]. That work together with the results from the present study suggest that differential effectiveness of attenuating mechanisms may be a general strategy employed by membrane receptors to generate diverse patterns of second messenger formation. These results have particular significance for examining the autocrine theory of cellular transformation. In some instances, cells that secrete growth factors acquire a transformed phenotype, presumably because these fac1 A

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6

200K 116K 92SK -

B 200K 116K 92SK FIG. 5. Ligand-induced down-regulation of the EGF receptor. NPLC/PRF/S (lanes l-3) and PLC/PRF/5 (lanes 4-6) cells were incubated with serum-free MEM containing 0.1% BSA for 1 h (lanes 1 and 4), or incubated with 25 rig/ml EGF for 1 h (lanes 2 and 5) or 4 h (lanes 3 and 6). After incubation, cell surface proteins were iodinated, EGF receptors specifically immunoprecipitated from iodinated cell extracts, and the immunoprecipitates separated by 7.5% SDS-polyacrylamide gel electrophoresis. Autoradiograms of a representative gel are shown; the top (A) and bottom (B) panels show a 3.3 h and 50 min exposure, respectively, of the same autoradiogram.

EGF SIGNALING

TABLE

AND

RECEPTOR

DOWN-REGULATION

1

Flow Cytofluorometric Analysis of EGF Receptor Levels Cellular Treatment 0 EGF lh 25 rig/ml EGF 15 min 30 min lh 4h 500 rig/ml EGF lh 4h

PLC/PRF/5

130’ (98.6) b 68 57 55 23

(96.3) (94.2) (86.9) (51.5)

25 (47.3) 15 (37.8)

fluorescence NPLC/PRF/5

upon alterations EGF stimulation

(99.6) (99.3) (99.7) (99.9)

1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12.

tors bind to receptors at the secreting cell’s surface and generate a constitutive proliferative signal [ 19-211. Cell vulnerability to autocrine transformation may require defects in receptor down-regulation and homologous desensitization such that the proliferative signal is sustained. Our results may also address the seemingly conflicting observations that EGF receptor overexpression correlates with the malignant state, yet growth in many of the transformed epithelial cell lines that overexpress the EGF receptor, including the NPLC/PRF/5 cell line, can be inhibited by nanomolar EGF [26, 31-331. EGF receptor overexpression may confer a selective advantage to cells grown in the limiting concentrations of EGF likely to be found in uiuo by facilitating the generation of the second messengers that signal proliferation. Doses of EGF which inhibit growth induce prolonged second messenger production, which in turn might lead to inappropriate enzyme activation and gene expression. A variant human squamous cell carcinoma cell line which overexpresses the EGF receptor appears to escape the growth inhibitory effects of EGF by decreasing its surface EGF binding capacity upon exposure to EGF [Ml. Thus, the functional role of EGF as a growth inhibitor or growth stimulator may depend not only upon circulating levels of ligand and cell surface receptor density, but also

in the mechanisms which modulate of intracellular signaling pathways.

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1207 (99.8) 906 (99.2)

Note. PLC/PRF/5 and NPLC/PRF/5 cells were treated with serum-free MEM containing 0.1% BSA which was supplemented or not with either 25 rig/ml or 500 rig/ml of EGF. After harvesting, cells were incubated in duplicate with suitably diluted Rl monoclonal antibody to the EGF receptor (5-10 X lo5 cells, 4”C, 1 h). Cells were washed three times and then incubated with FITC-tagged goat IgG antimouse IgG Fc fragment y-chain-specific antibody (4”C, 1 h). After washing, cells were resuspended in buffer and immunotluorescence measured by flow cytometry. Two thousand cells per sample were analyzed; results are expressed as: “Mean channel fluorescence after conversion from log to linear scale; bthe percentage of cells exhibiting fluorescence greater than fluorescence of cells incubated with FITC-labeled second antibody alone. A representative experiment is shown. Duplicate aliquots from each treatment group were analyzed by flow cytometry and results averaged. This experiment was repeated three times and essentially identical, independent results were obtained.

141

CELLS

This work was supported by NIH Grants DK 35914, DK 19525, CA 37225, and CA 10815. We thank Melissa Moody for excellent technical assistance in passaging cell lines and for performing some of the FACS analyses.

1068 (99.8) 990 1241 1076 936

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