Epidermal Growth Factor · culminates in cell division. Epidermal growth factor, a low molecular...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 253, No. 11, Issue of June 10, pp. 39’70-3977, 1978

Prmted in U.S.A.

Epidermal Growth Factor RELATIONSHIP BETWEEN RECEPTOR REGULATION AND MITOGENESIS IN 3T3 CELLS*

(Received for publication, July 25, 1977)

AHARON AHARONOV,~ REBECCA M. PRUSS,~ AND HARVEY R. HERSCHMAN

From the Department of Biological Chemistry and Laboratory of Nuclear Medicine and Radiation Biology, UCLA School of Medicine, Los Angeles, California 90024

Exposure of confluent nondividing 3T3 cells to 10 nn mouse epidermal growth factor (EGF) at 37”, followed by incubation for 45 h at 37”. leads to a 70 to 8.5% reduction in the binding capacity for ‘“,BI-EGF. Scatchard analysis of the binding data indicate that the reduction of ‘“‘)I-EGF binding is due to a decrease in the number of available EGF- receptors per cell, without any change in the affinity of the receptors for EGF. This modulation of the EGF-receptor by the growth factor. termed “down regulation.” is dependent on temperature, EGF concentration. time, and the physiological state of the cell. Receptor loss occurs at physiological EGF concentrations (0.1 to 10 nm) which span the concentration range which is mitogenic for 3T3 cells. Maxi- mal stimulation of either 1Wlthymidine uptake or cell division occurs at 1 nn EGF. a concentration at which only 20% of the EGF-receptor sites are occupied and down regulation is only 55% complete. Low EGF concentrations (~1 nn) result in down regulation of unoccupied EGF- receptors. Uown regulation of the EGF-receptor also occurs in SV40-transformed 3T3 cells. Growing 3T3 cells exposed to EGF also loose available EGF-receptors. In contrast to confluent cells, dividing 3T3 cells rapidly replace EGF- receptors on the surface of the cell, in the presence of EGF.

When EGF is removed from the medium the EGF-receptor is quickly replenished; by 13 h one-half the “down regulated” receptors are restored. All EGF binding capacity returns by 20 h after removal of the growth factor. The half- life of the EGF-receptor. estimated by blocking protein synthesis with cycloheximide. is approximately 6 h.

The initial massive EGF internalization and concomitant receptor down regulation do not appear to be sufficient for EGF mitogenesis. The mitogenic response of the cells to EGF requires the continuous presence of EGF over a 3- to 4-day period; in contrast. down regulation is completed within 4 h.

Continued occupancy of a portion of the remaining and restored EGF surface receptors is essential for mitogenesis.

* This work was supported by Grants CA-15276 and GM24797 from the National Institutes of Health and Contract EY-76-03-0012 between The Deuartment of Enerav and the University of Califor- nia. The costs of-publication of thii”article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Chaim Weizmann Fellow. ?+ Supported by Training Grants GM-00364 and T32-CA-09056.

The major role of the reduction of EGF-receptors by receptor down regulation may be to adjust the cell’s subsequent sensitivity to EGF.

Mitogens are thought to interact with specific receptors prior to the initiation of a series of biochemical events, the so- called “positive pleiotypic response” (l), which eventually culminates in cell division. Epidermal growth factor, a low molecular weight polypeptide (M,. = 6045). is a potent mitogen for cultured human fibroblasts (2. 3) and for the clonal murine cell line 3T3 (4). A sensitive and specific radioreceptor assay has been developed to measure EGF-receptors on the surface of human fibroblasts (5, 6) and 3T3 cells (7, 8).

A number of ligands are able to regulate the concentration of the specific cell surface receptors to which they bind. The first observation of this phenomenon was the demonstration that membrane antigens could be removed from the cell surface after reaction with their specific .antibodies (9-11). “Antigenic modulation,” the term used to describe this phenomenon, was later found in some cases to be due to endocytosis of the antigen. antibody complex. The /3-adrenergic ago- nists (12) and a number of peptide hormones including insulin (13), growth hormone (14), and thyrotropin-releasing hormone (15) are able to modulate the level of their specific cell surface receptors. The ability of a ligand to negatively modulate the level of its own receptor has been given the more general term of “down regulation” (16). Receptor down regulation is dependent on ligand concentration. The time required for receptor loss varies in different systems from minutes to hours. Regu- lation of the insulin and /3-adrenergic receptors has been demonstrated both in viva and in vitro (17, 18).

Carpenter et al. (5) demonstrated that mouse ‘“‘I-EGF binding to human fibroblasts at 37” reached a peak at 30 min and then declined over the next 4 to 5 h. Subsequently. Carpenter and Cohen (19) demonstrated that bound human “,‘I-EGF is internalized and degraded by human fibroblasts at 37”. They also observed that cells that had bound and then degraded EGF were unable to rebind labeled EGF. These data suggested that (i) binding of EGF leads to the internalization and degradation of both EGF and (possibly) receptor and that (ii) as a consequence of this interaction the number of EGF- receptors on the cell surface was reduced. i.e. down regulation of the EGF-receptor occurred.

We have determined that incubation of 3T3 cells with

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EGF-Receptor Regulation and Mitogenesis in 3T3 Cells

murine EGF leads to a reduction of EGF-receptors at 37”, but not at 0”. The modulation of the 3T3 EGF-receptor by the growth factor is dependent on concentration of EGF. time. and physiological state of the cell. in addition to temperature. The receptors remaining on the surface of the cell after EGF- induced receptors loss have the same affinity as receptors present in the absence of EGF; there is only one class of receptors by this criterion.

We have compared the time course and concentration dependence of EGF-induced 1 “Hlthymidine uptake and cell division with that required for receptor binding and down regulation. It appears that down regulation of EGF-receptor occurs much quicker than the time necessary to commit cells to divide.

EXPERIMENTAL PROCEDURES

Materials- Dulbecco’s modified Eagle’s medium (DME medium)’ was obtained from Gibco, Grand Island, N. Y.; trypsin was obtained from either Gibco or from ICN, Cleveland, Ohio. Fetal calf serum (FCS) was obtained from Reheis, Phoenix, Ariz. Carrier-free lBiI and Imethyl-:‘Hlthymidine were purchased from New England Nuclear, Boston, Mass. Cycloheximide was purchased from Sigma, St. Louis, MO., actinomycin D from Calbiochem, Los Angeles, Ca., and bovine serum albumin from Miles, Kankakee, Ill.

Cells and Cell Cultures~Swiss 3T3 cells (clone 42) and SV40 transformed Swiss 3T3 cells (SV3T3) were obtained from Dr. C. Fred Fox (Department of Bacteriology, UCLA). Cell stocks were cultured in a 10% CO, atmosphere at 37” in loo-mm Falcon tissue culture plates in DME medium containing 10% FCS. For subculturing, subconfluent cultures were dissociated with 0.02% EDTA plus 0.05% trypsin solution as previously described (4) and seeded in plates containing fresh medium. For experiments, cells were grown in 35- mm Falcon tissue culture plates containing 2 ml of DME medium and 5% FCS. Duplicate plates of cells were counted by hemocytome- ter or using an ElectrozoneiCelloscope model 112 TH manufactured by Particle Data, Inc., Elmhurst, Ill.

Epidermal Growth Factor- EGF was purified from the submaxil- lary glands of adult male Swiss Webster mice by the method of Savage and Cohen (20).

EGF was labeled with lsiI according to the chloramine-T method of Hunter (21), with slight modifications. Two millicuries of carrier- free Ipi1 were added to 5 to 20 pg of EGF in 0.1 ml of 1 M potassium phosphate buffer, pH 7.5. Iodination was initiated by the addition of 0.02 ml of chloramine-T (10 mgiml in 0.05 M potassium phosphate buffer, pH 7.5). The reaction was carried out for 1 min at 22” with gentle shaking, then 0.02 ml of sodium metabisulfite (20 mg/ml in 0.05 M potassium phosphate buffer, pH 7.5) and 0.1 ml of KI (30 mg/ ml in the same buffer) were added. Albumin (0.1 ml, 10%) was added and the labeled protein was separated from unreacted iodine by passing the mixture over a Sephadex G-25 (0.8 x 30 cm) column. The specific activity of ‘“.>I-EGF was 1100 to 2000 Ciimmol. Labeled EGF (specific activity = 1200 Ciimmol) and unlabeled EGF had identical dose response curves for stimulation of cell division. The labeled EGF was kept at -20” until used.

EGF-Receptor Assa,? - The EGF-receptor assay was performed at o”, except where indicated. Monolayers of cells grown in 35.mm tissue culture dishes containing DME medium and 5% fetal calf serum were washed three times with ice-cold “binding solution” composed of saline A (22) and 0.1% albumin. When binding was performed at 37”, we used either DME medium + 0.1% albumin or DME medium + 5% FCS. One milliliter of the binding solution was added to each plate and then ‘“‘I-EGF was added to initiate the receptor assay. The plates were incubated on ice (except where indicated). Unbound radioactivity was removed by washing the plates five times with ice-cold binding solution (total 10 ml). The washed monolayers were solubilized with 1 ml of 0.5 N NaOH for 1 h at 22” and counted in a Beckman Biogamma counter. Specific binding was determined by measuring the difference in cell-bound radioactivity in the presence and absence of 5 pg of unlabeled EGF

’ The abbreviations used are: DME medium, Dulbecco’s modified Eagle’s medium; EGF, epidermal growth factor; FCS, fetal calf serum; albumin, bovine serum albumin; Hepes, (2-hydroxyethyl)-l- piperazineethanesulfonic acid.

(preincubated for 30 min at 0”). Nonspecific binding was less than 10% of the total binding. All experiments were performed in duplicate or triplicate.

Thymidine Incorporation - Stimulation of [“Hlthymidine uptake was determined by measuring the amount of [“Hlthymidine (1 &i/ plate) incorporated into confluent 60-mm 3T3 cell monolayers grown in DME medium + 5% FCS during a 2-h pulse 20 to 22 h after treatment of the cultures with EGF. At the end of 2 h, cultures were placed on ice, washed three times with 2 ml of ice-cold phosphate- buffered saline (NaClIP,, 0.15 M NaCl in 0.01 M potassium phosphate buffer, pH 7.41, and then precipitated for 15 min with 3 ml of ice-cold 10% trichloroacetic acid. The precipitate was washed three times with cold NaClIP, and solubilized with 1 ml of 0.5 N NaOH. A portion of the solubilized material was neutralized and 13H]thymidine incorporated was assayed by liquid scintillation counting.

RESULTS

Binding of EGF at 0” and 37”-Our initial experiments were designed to investigate the binding to and subsequent fate of EGF in 3T3 cells under various conditions. Confluent monolayers of 3T3 cells grown in 35-mm plates were incubated with 60 ngiml (10 nM) of ‘“‘>I-EGF at 0” or at 37” (Fig. 1). Maximal binding was achieved after incubation for 30 to 40 min at 37” and 90 to 120 min at 0”. The maximal amount of EGF bound at 0” was 63% of that bound at 37”. At 37” the amount of cell-bound radioactivity decreased (after 30 min) with increasing time of incubation, reaching about 40% of the maximal binding at 6 h of exposure to EGF. The remaining ““I-EGF found associated with the cells after exposure for 6 h at 37” would include growth factor both bound to the outside of the cell and growth factor internalized in endocytic vesicles. according to the model of Carpenter and Cohen (19). These results are similar to the binding kinetics observed by Carpen- ter et al. (5).

Effect of EGF Concentration on the Binding Reaction-The concentration dependence of “,,I-EGF binding is the same at 0 and 37” in the range of 0 to 50 ngiml (the solid and open

symbols overlap at the 10 and 25 rig/ml points), but saturation subsequently occurs at different concentrations (Fig. 2). Max- imal binding at 0” is at an EGF concentration of 60 ngiml (10 nM), whereas maximal binding at 37” occurs at approximately 100 ngiml (17 nM).

Fate of Bound EGF- We next carried out a series of experiments designed to determine what happens to EGF after it is bound to the surface of the 3T3 cell. Confluent plates of 3T3 cells were incubated for 1 h with lZ’I-EGF (60 ngiml), either at 0” or 37”. Following binding, control plates were

,: 0 I 2 3 4 5 6

2 TIME, (hr) -

FIG. 1. Time course of ‘2”I-EGF binding to 3T3 cells at 37” and 0”. To each confluent 35.mm culture plate, 1 ml of binding medium containing 60 ng of ““I-EGF was added. Dishes were incubated at the indicated temperatures. At each of the time intervals the cell- bound radioactivity was determined in duplicate dishes as described under “Experimental Procedures.” 0, binding at 37”; 0, binding at 0”.


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3972 EGF-Receptor Regulation and Mitogenesis in 3T3 Cells

:k A 0 50 100 150

: - ‘251-EGF, (rig/ml)

FIG. 2. Effect of ‘*“I-EGF concentration on binding to 3T3 cells. Indicated concentrations of ‘*“I-EGF were added to confluent culture plates. Specific binding was determined after 30 min at 37” or 90 min at 0” as described under “Experimental Procedures.” Data are expressed as the average of duplicate determinations. 0, binding at 37”; 0, binding at 0”.

washed quickly and used to measure the total ‘““I-EGF bound. Additional plates were further incubated either at 0” or 37” with fresh DME medium + 5% FCS (without EGF) for 4.5 h, with medium changes every 1.5 h. After these incubations the radioactivity associated with the cells was measured.

Initial binding at 0” was, once again. 63% of that bound at 37” (Fig. 3). When cells initially exposed to EGF at 37” were

further incubated at 0” without EGF, only 20% of the bound EGF was eliminated. In contrast, 78% of the radioactivity bound to the cells at 0” was removed by subsequent incubation at 0” without EGF. When cells are exposed to EGF at either temperature, washed, and further incubated at 37”, greater than 95% of the labeled EGF is removed from the cells at the end of the 4.5-h incubation.

To clarify this situation further we followed the kinetics of loss of EGF from the cells at the two temperatures. ‘“;I-EGF was bound to cells at 37” for 1 h. After a rapid wash, the loss of radioactivity from the cells into the supernatant was followed at both temperatures. The small amount (about 30%) of labeled EGF released at 0” after binding at 37” (Fig. 3) came off the cells within the first few minutes of washing (Fig. 4). At 37”, most of the radioactivity was released from the cells within 2 h. The radioactive material released from the cells at 0” and 37” was analyzed by chromatography on a Sephadex G- 25 column. The labeled material released at 0” was eluted nearly exclusively in the excluded volume. In contrast, over 65% of the material released over a l-h period at 37” eluted in the included volume of the column (data not shown). These experiments suggest that, after washing, the loss of EGF from 3T3 cells appears to be a combination of simple dissociation from the cell surface and a temperature-sensitive event(s) which first rapidly prevents loss of EGF by diffusion and then leads to breakdown and elimination of EGF.

In order to investigate whether the temperature-sensitive event which prevents subsequent dissociation at 0” might be internalization of EGF, we attempted to determine whether the material remaining associated with the cells after washing was sensitive to tryptic digestion. ‘““I-EGF was bound to cells, either at 37” or o”, for various lengths of time. At the end of each period of exposure to EGF the cells were transferred to O”, quickly washed, and treated with trypsin. After a 30-min incubation the radioactivity both in the incubation medium and associated with the cells was measured. If the initial binding is carried out at O”, the majority of the labeled EGF is susceptible to proteolysis; i.e. the bulk of the counts are found

in the supernatant, regardless of the time of incubation with EGF (Fig. 5B). In contrast, at 37” the susceptibility of cell- associated EGF to proteolysis varies with the time of exposure to EGF (Fig. 5A). At early time points much of the EGF bound at 37” is susceptible to subsequent proteolysis at 0”. With

- FIG. 3. Dissociation of cell-bound ‘““I-EGF at different tempera-

tures. Confluent plates of 3T3 cells were incubated for 1 h with 60 rig/ml of “>I-EGF at 37” (A) or 0” (B). Following binding at either temperature, control plates were washed quickly and radioactivity was determined to measure the total ““I-EGF bound. Additional plates were further incubated either at 0” or 37” with fresh DME medium + 5% FCS without EGF for 4.5 h, with medium changes every 1.5 h. After these incubations the radioactivity associated with the cells was measured.

“I L I -J

~‘“~-c-/ j I I I I I I I I I Ii I I I

0 30 60 90 120

TIME, (MN)

FIG. 4. Time course for dissociation of cell-bound ‘““I-EGF. Con- fluent plates containing DME medium + 0.02 M Hepes + 5% FCS were incubated for 1 h with ‘““I-EGF at 37”. After a rapid wash with DME medium + 0.02 M Hepes + 0.1% albumin, one-half of the plates were incubated with 1 ml of the latter medium at 37” and one-half at 0”. At the indicated times, the cells were removed from duplicate plates with a rubber policeman and centrifuged in a microfuge (Beckman) for 1 min. The radioactivity in the supernatants was measured. 0, incubation at 37”; 0, incubation at 0”.


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EGF-Receptor Regulation and Mitogenesis in 3T3 Cells 3973

0 50 100 TIME-,-(min) ‘- -

FIG. 5. Susceptibility of cell-bound ‘““I-EGF to proteolysis. Con- fluent cultures of 3T3 cells were exposed to 60 rig/ml of 12”1-EGF in DME medium + 0.02 M Hepes + 5% FCS for different times, either at 37” (A) or at 0” (B). At each time point the cells were chilled on ice and quickly washed. To each dish, 1 ml of 0.25% trypsin (in saline A containing 0.1% albumin) was added. After 30-min incubation on ice, 0.5 ml of DME medium + 5% FCS was added and the cells were centrifuged for 1 min in a microfuge. The radioactivity both in the supernatants and associated with the cells was measured. Data are plotted as the percentage of radioactivity in pellet and supernatant in each tube. 0, 0, radioactivity in cell pellets; A, A, radioactivity in the supernatants.

progressive incubation, however, an increasingly larger frac- tion of the associated radioactivity is rendered resistant to proteolysis. All of these data are consistent with the sugges- tion of Carpenter and Cohen (19) that bound EGF is internalized, degraded, and eliminated from the cell by reactions which require metabolic activity.

“Down Regulation” of the EGF-Receptor -If loss of cell- associated EGF at 37” is due in part to the internalization of the ligand. receptor complex, we might expect to observe reduced receptor availability following exposure to EGF; i.e. “down regulation” of the EGF-receptor. We consequently measured the number of available EGF-receptors at various times after exposure of 3T3 cells to EGF. Confluent 3T3 cells were incubated in DME medium + 5% FCS at 37” with 10 nM EGF. Experiments were designed so that all incubation periods with EGF terminated at the same time. After various periods of exposure to EGF, plates were incubated at 37” with three successive 1.5-h washes of 2 ml of DME medium + 5% FCS without EGF to remove bound EGF (c.f. Fig. 3A). Plates were then put on ice, and the usual EGF-receptor binding assay was performed. A dramatic reduction in the subsequent binding of lZ”I-EGF to the cells occurs following prior incubation with 10 nM EGF. After 4 h of exposure to EGF and appropriate washing, the capacity to bind ““I-EGF was reduced by 85% (Fig. 6). This decreased binding of 12”I-EGF indicates a loss of EGF-receptors in response to EGF exposure. In a number of experiments using 10 nM EGF, receptor loss varied between 70 and 85%.

It is possible that, despite the observation that our 12’)I-EGF

and unlabeled EGF have the same biological activity, the affinity of 12”I-EGF is altered as a result of the labeling procedure. The apparent down regulation of the EGF-receptor could be interpreted as persistent binding of unmodified EGF. To eliminate this possibility we compared the efficacy of labeled and native EGF in reducing subsequent rebinding of labeled EGF. Confluent 35-mm dishes of 3T3 cells were exposed to 10 nM either native or lZ;I-EGF for 30 min at 37”. The cells were then washed for 4 h at 37” in three changes of medium without EGF. Cells exposed to ‘““I-EGF lost 96% of the label during this incubation period. After the wash, 12.rI- EGF binding to control cells, cells exposed previously to unlabeled EGF, and cells exposed to ““I-EGF was assayed by the standard binding procedure. Cells exposed to unlabeled EGF bound only 60% of EGF found on control cells; i.e. a 40% down regulation. Cells previously exposed to 12”1-EGF bound only 50% of the counts found on control cells; i.e. a 50% down regulation. There is no significant difference in the ability of saturating amounts of native and iodinated EGF to reduce rebinding of 12:I-EGF.

Treatment of 3T3 cells with cycloheximide had no effect on the initial binding of lZ-‘I-EGF or on the subsequent loss of EGF-receptors, indicating that these phenomena do not require protein synthesis (Table 1). NaF. an inhibitor of meta-

PREINCUBATION WITH EGF, (hr)

FIG. 6. Time course for down regulation of EGF-receptors. Con- fluent 3T3 cells in 35-mm plates containing DME medium + 5% FCS were incubated at 37” with 10 nM EGF. After various periods of exposure to EGF, triplicate plates were incubated at 37” with fresh DME medium + 5% FCS for 4.5 h, with medium changes every 1.5 h. Plates were then put on ice, and lZ”I-EGF binding was determined as described under “Experimental Procedures,” using 60 rig/ml of labeled growth factor.

TABLE I

Effect of cycloheximide and NaF on binding of ‘“‘I-EGF to 3T3 cells at37

Confluent 3T3 cells in 35-mm plates in DME medium + 0.1% albumin were exposed for 0.5 h at 37” to cycloheximide (20 Fgiml) or NaF (0.02 M). Then lzzI-EGF (60 ngiml) was added and plates were incubated at 37” for different time periods. At the indicated times, cell-bound radioactivity was determined on duplicate plates as described under “Experimental Procedures.” ““I-EGF binding is expressed as nanograms of EGF bound per 10” cells. Protein synthesis was inhibited 87% bv 20 &ml of cvcloheximide.

Treatment

” .- ‘Z”*-E@ % of ‘“q-EGF % of bound at con-

30 min tro1 bound at co;,-

4h

‘=I-EGF (10 nM) l”;I-EGF (10 nM) + cyclo-

heximide (20 pg/ml) 12”I-EGF (10 nM) + NaF

(0.02 M)

nkz w

0.80 100 0.37 46 0.80 100 0.36 45

0.80 100 0.56 70


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bolic energy production, also had no effect on the initial binding of 12,‘I-EGF to 3T3 cells. However. NaF did inhibit the subsequent loss of cell-bound ligand following prolonged incubation at 37”. These data indicate that ligand-induced receptor loss is an energy-requiring process, in contrast to ligand binding.

Effect of EGF Concentration on Receptor Loss-In order to determine concentrations of EGF effective in bringing about loss of receptor, we exposed cells to different EGF concentrations for 4 h at 37” and then incubated for 4.5 h in the absence of EGF (to remove bound growth factor). Binding assays were then carried out at O”, and the number of available receptors and the affinity of the available receptors were calculated from Scatchard plots (23). Loss of EGF-receptors occurs in the range from 0.1 to 10 nM (Fig. 7). This spans the EGF concentration range which is both present in biological fluids (24) and mitogenic for 3T3 cells (4).

Binding of EGF might be reduced as a result of an EGF- induced reduction in receptor affinity, rather than loss or inactivation of a portion of the EGF-receptors. Scatchard plot analysis of the data (Fig. 7, inset), however, demonstrates that the EGF-receptors remaining after down regulation have the same K,, (5.8 nM) as receptors present on the unexposed cell. Reduced binding of EGF is thus the result of reduction of available EGF-receptors, without a change in affinity of the remaining receptors. The K,, for the EGF-receptor of 3T3 cells is about one order of magnitude lower than that observed for human fibroblasts; the number of EGF-receptors on the two cells is about the same (2, 5, 19).

Recovery of EGF-Receptors - 3T3 cells in which the EGF- receptor has been “down regulated” recover their receptors

r 0 03r

0 IO” IO9 a7 EGF ADDED, (Ml

FIG. 7. Effect of EGF concentration on down regulation of EGF- receptors. Confluent 3T3 cells in 35mm plates containing DME medium + 5% FCS were incubated for 4 h at 37” with different concentrations of EGF. Triplicate plates were then incubated at 37” with fresh DME medium + 5% FCS for 4.5 h, with medium changes every 1.5 h. Plates were then put on ice and lL’I-EGF binding was determined as described in Fig. 6 and “Experimental Procedures.” Inset, Scatchard plots of binding data after down regulation with different concentrations of EGF. Data are expressed as bound/free EGF as a function of EGF bound in picomoles per 10” cells. EGF concentrations used: 0, 10 nM; A, 1 n&r; A, 0.1 nM; 0, 10 PM; n , 1 PM. The per cent of available sites for the different treatments was calculated from extrapolation of the Scatchard curves to the horizon-

fully within approximately 20 h following removal of EGF from the medium (Fig. 8). Incubation for 13 h in fresh medium is required to restore 50% of the initial EGF binding capacity.

Down Regulation of EGF-Receptors in Growing versus Confluent 3T3 Cells - For these experiments the “growing” 3T3 cells were approximately 40% of saturation density. Con- fluent and growing cells were exposed to 10 nM EGF at 37” for different times and then incubated for three 1.5-h periods in the absence of EGF and assayed for “,‘I-EGF binding. The initial loss of EGF-receptors in growing cells is only slightly smaller than that observed in confluent, nondividing cells (Fig. 9). In contrast to confluent cells, growing cells appear to put receptors back onto the cell surface in the presence of EGF and subsequently keep these receptors available. After an initial 70% reduction in receptors, the growing cells rapidly (5 h) replaced over 40% of the lost receptor activity and eventually established, in the presence of EGF, a receptor level 75 to 80% of that found in unexposed cells. These data suggest that in confluent cells internalization of EGF leads to a diminished steady-state receptor concentration, while in cy- cling cells lost receptor is restored in the membrane after an

initial, transient down regulation. The number of EGF-receptors per cell in the growing cultures is about the same as confluent cells.

DOUJ~ Regulation of EGF-Receptors in Normal versus

FIG. 8. Recovery of EGF binding capacity after down regulation. Confluent 3T3 cells in 35.mm plates containing DME medium + 5% FCS were incubated for 4 h at 37” with 10 nM EGF. Plates were then incubated at 37” with fresh DME medium + 5% FCS for 4 h (with medium changes ever” 80 min) in order to remove bound EGF. Plates were f&her incubated at 37” with DME medium + 5% FCS for different periods of time. At the indicated times triplicate plates were put on ice and “‘I-EGF binding was determined.


FIG. 9. Down regulation of EGF-receptors in growing ucrsus confluent 3T3 cells. Confluent 3T3 cells and growing cells (at 40% of saturation density) in 35.mm plates containing DME medium + 5% FCS were exposed to 10 nM EGF at 37” for different times. EGF binding was measured as described under “Experimental Proce- dures.” The slight rise in binding after 8 h for confluent cells occurred occasionally, but did not seem to be significant (c.f. Figs. 6 and 10). 0, confluent cells; 0, growing cells. tal axis.


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Transfbrmcd 3T3 Cells - 3T3 cells were compared to SV40- transformed 3T3 cells (SV3T3) for ability to down regulate EGF-receptors. There was no basic difference between these two cell types in the EGF-induced loss of EGF-receptor (Fig. lo), despite the fact that the 3T3 cells were nondividing. There are about 1.9 x lo” EGF molecules bound/SVSTJ cell at 0”. 3- fold higher than 3T3 cells.

Degradation of EGF-Receptors - We measured binding of l”“I-EGF to confluent 3T3 cells in 35mm plates containing cycloheximide (20 Fgiml) or actinomycin D (2 pgiml) for different time periods, in order to determine the rate of disappearance of the EGF-receptor. There is a decrease in the number of receptors with time in both cases. The effect with cycloheximide, however, is faster. The half-life of the receptor obtained with cycloheximide is 6 h, whereas the half-life of EGF-receptor in the presence of actinomycin D is approximately 15 h (Fig. 11). The viability of the cells after 15 h, as assayed by trypan blue exclusion, was more than 80%.

EGF Concentration DrJpendence for DNA Synthesis. CPU Division, and Down R<Jgulation - We characterized the concentration dependence of EGF stimulation of DNA synthesis by measuring the amount of ]:‘H]thymidine incorporated into trichloroacetic acid-precipitable material during a 2-h pulse 20

to 22 h after addition of varying concentrations of EGF to confluent 60-mm cultures (maximal incorporation of 13H]thymidine occurs in confluent 3T3 cells at 20 to 22 h after addition of EGF). The concentration dependence of EGF-induced ]:‘H]thymidine incorporation and cell division was compared to the concentration dependence of EGF-receptor occupation and EGF-receptor down regulation (Fig. 12).

EGF stimulates both DNA synthesis and cell division max- imally at a concentration far below that required for complete receptor occupancy. In order for EGF to stimulate a full biological response, only about 20% of the total EGF-receptor sites available at 0” need to be occupied. (Binding of EGF at 0 is far more easily interpreted than is binding at 37”, where “binding” includes receptor occupation, internalization, and degradation of labeled EGF. If we use 37” binding data for our calculation (see Fig. 2) only about 13% of the receptors need be occupied in order to stimulate a full biological response.)

Time Course for EGF Stimulation of [“HjThymidine Incor- poration and Cell Division- We next investigated the time

course of EGF stimulation of I 3H]thymidine incorporation and cell division in 3T3 cells. Confluent 60-mm cultures were treated with EGF (10 ngiml). After various times of exposure the EGF-containing medium was withdrawn and the cells

I I I I

I I 1 I 5 IO 15 20


FIG. 10. Down regulation of EGF-receptors in normal versus transformed 3T3 cells. Confluent 3T3 cells and 3T3 cells transformed by SV40 virus (SV3T3), grown in 35-mm plates containing DME medium + 5% FCS, were exposed to 10 nM EGF at 37” for different times. EGF binding was measured as described under “Experimen- tal Procedures.” 0, 3T3 cells; 0, SV3T3 cells.

TIME, (hr)

FIG. 11. Effect of preincubation with cycloheximide and actinomycin D on “‘I-EGF binding. Confluent 3T3 cells grown in 35mm plates containing DME medium + 5% FCS were exposed at 37” to cycloheximide (20 pg/ml) or actinomycin D (2 Kg/ml) for different time periods. Triplicate dishes were then used for ““I-EGF binding as described under “Experimental Procedures.” 0, binding after exposure to cycloheximide; 0, binding after exposure to actinomycin D.

2 k? 60 6 a 40

01 1 IO 100 [EGF], (rig/ml)

FIG. 12. Concentration dependence for EGF stimulation of l:‘H]thymidine uptake, cell division, ‘““I-EGF receptor binding, and EGF-receptor down regulation. Stimulation of i”H]thymidine uptake (0) was determined by measuring the amount of [“Hlthymidine (1 &i/plate) incorporated into confluent 60-mm 3T3 cell monolayers during a 2-h pulse at the peak of DNA synthesis, 20 to 22 h after treatment of the cultures with varying concentrations of EGF. Results are plotted as per cent of the maximum response over the untreated control. Stimulation of cell division (0) was determined by measuring the increase in cell number at the time of maximal response (3 days) after treatment of confluent 3T3 cell cultures with varying concentrations of EGF. Data are plotted as per cent of the maximum response over the untreated controls. Binding of ““I-EGF to confluent 3T3 cell monolayers in 35mm culture dishes (m) was determined after incubation of the cultures at 0” for 90 min with varying concentrations of ““I-EGF. Results are plotted as the per cent of the maximum specific binding. The EGF concentration dependence for EGF-receptor down regulation (0) was determined as described in Fig. 7.

were washed twice with 2 ml of conditioned medium taken from parallel untreated cultures. The cells were then refed with conditioned medium. Control plates, not exposed to EGF, were treated similarly. The increase in cell number following limited times of interactions with EGF was measured by cell count 3 days after the initiation of exposure to EGF. We also assayed stimulation of incorporation of I 3H]thymidine into trichloroacetic acid-precipitable material during a 2-h pulse 20 to 22 h after initiation of EGF treatment. The results of these experiments (Fig. 13) demonstrate the need for continuous EGF presence in order to provoke a full biological response. Treatment of 3T3 cells with EGF for less than 20 h is not sufficient to produce maximal I “Hlthymidine incorporation. Likewise, treatment with EGF for 24 h, by which time the cells have gone through a peak in the rate of ]“H]thymidine incorporation, is able to stimulate only a 30% increase in the density of 3T3 cells after 3 days. In contrast, maximal binding of EGF to 3T3 cells occurs rapidly (90 to 120 min at O”, 30 to 40


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0 6 12 IS 24

TIME WITH EGF. (hr)

TIME WITH EGF. (days)

FIG. 13. Time dependence of EGF stimulation of 13H]thymidine incorporation and Eel1 division. A, stimulation of [3H]thymidine incorporation (0) in confluent 3T3 cells in 60-mm culture dishes after various time periods of exposure to EGF (10 rig/ml). Cells were treated with EGF for various time periods, washed, and incubated in conditioned medium (see text). 13HlThvmidine incorporation was measured during a Z-h pulse at the peak bf DNA synthesis, 20 to 22 h after initial EGF treatment. Data shown are the amount of 13H]thymidine (counts per min) incorporated over the untreated control (20,000 cpm). B, stimulation of cell division (0) in confluent 3T3 cell cultures, grown in 60-mm culture dishes, after various time periods of exposure to EGF. Cells were treated with EGF (10 rig/ml) for various time periods, washed, and incubated in conditioned medium (see text). The number of cells per plate was measured at the time of maximal response, 3 days after initial treatment with EGF. Data are plotted as the per cent increase in cell number over the untreated control (1.72 x 10” cells/plate).

min at 37”, Fig. 1). It is clear from these data that within a brief period of time the great majority of cells have bound adequate amounts of EGF to initiate the biological response, yet removal of EGF from the medium results in the loss of the full final biological response. Our results are consistent with the findings that the continued presence of EGF is necessary for mitogenesis in cultured human fibroblasts (25) and glial cells (26).

DISCUSSION

We have shown that reduction in binding of ““I-EGF to 3T3 cells after exposure to EGF is due to a loss or inactivation of receptors rather than a change in receptor affinity. The loss of receptors is also concentration- and time-dependent. Scat- chard analysis (Fig. 7) has demonstrated that these remaining receptors have the same affinity as the initial population of receptors. That there is but one class of surface EGF-receptor on 3T3 cells is substantiated by our ability to select 3T3 variants lacking both a functional and a ligand binding EGF- receptor (7). Also, we have identified a single membrane receptor for EGF using a photoactivatable derivative of IshI- EGF; this entity is absent in an EGF nonresponsive, nonbinding variant (8).

The rapid loss of 12”I-EGF binding activity does not require protein synthesis but is temperature-sensitive and requires

metabolic energy. 12”1-EGF bound to the surface of the cell at 37” rapidly becomes trypsin-insensitive. in contrast to EGF bound at 0”. In the latter case the growth factor binding remains trypsin-sensitive. Continued incubation at 37” results in loss of labeled EGF from the cells. These observations are consistent with the hypothesis of Carpenter and Cohen (19) that bound EGF and receptor are first internalized by a process of absorptive endocytosis, then degraded by lysosomal enzymes, in primary cultures of human fibroblasts.

One striking observation we have made is the difference in the down regulation of the EGF-receptor observed in growing uersus confluent 3T3 cells. In the former cells, although initial loss of receptors occurs as it does in confluent cells, the level of EGF binding rapidly returns to nearly its initial value. Moreover, the EGF binding activity found on the surface of the growing cell after continuous exposure to EGF exhibits qualitatively different features; this re-established EGF-binding activity does not diminish in the presence of EGF. The slower rate of protein degradation and the more rapid rate of protein synthesis which occurs in growing cells (1) may be factors in this difference. Both the mechanism and the signif- icance of this contrast between growing and resting cells remain to be elucidated, however.

Down regulation of the EGF-receptor also occurs in 3T3 cells transformed by SV40 virus (SV3T3) with about the same characteristics as in normal 3T3 cells. Clearly. an intact growth control mechanism is not required in order for a cell to exhibit receptor down regulation in response to EGF.

“Spare” EGF-receptors apparently exist both for the mito- genie response and for receptor down regulation. Only 20% of the sites on a 3T3 cell are occupied after exposure to 1 nM EGF (Fig. 2). This concentration of EGF, sufficient for maximal biological response (4, and Fig. 12). leads to a 55% reduction of EGF-receptors (Fig. 7). Unoccupied receptors are apparently inactivated as a result of ligand binding to only a minor portion of the cell surface EGF-receptors, either through direct interaction between occupied and unoccupied receptors or conversely through membrane-mediated or other indirect events. The down regulation of unoccupied receptors has also been noted for the growth hormone receptor of human lympho- cytes (14).

When EGF was removed from the medium the EGF-receptor was restored relatively rapidly; one-half of the loss was replaced by 13 h and the full complement of receptors was restored by 20 h following removal of the growth factor. This half-time of receptor restoration is similar to that observed for human growth hormone (14) and human EGF (19), although the time course for full restoration is somewhat different. In contrast, the restoration of the thyrotropin-releasing hormone-receptor appears to take much longer (15).

In the present experiments the t,,z for EGF-receptor degradation is approximately 6 h. The t,,, for growth hormone receptors is 10 h (14). while insulin receptors have a t,,, of 30 to 40 h (27). The thyrotropin-releasing hormone-receptor is much less sensitive to inhibition of protein synthesis (1.5).

The concentration of a receptor, like other membrane com- ponents, is not fixed but is a reflection of a state of continual synthesis and degradation. Any change in the rate of synthesis or degradation will result in a change in plasma membrane receptor concentration. Gavin et al. (13) have suggested that the change in plasma membrane receptor concentration brought about by down regulation provides a potential mechanism for regulation of target cell sensitivity to the ligand (hormone, growth factor). This hypothesis appears to be born


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out by studies on the insulin receptors of both patients and experimental animals (cf. Kahn, Ref. 27).

It is unclear what role (i) down regulation of EGF-receptors and (ii) internalization of the ligand receptor complex play in the final biological (mitogenic) response to EGF. The correla- tion between the concentrations required for biological activity and both loss of receptor and internalization of the EGF. receptor complex suggests that both of these phenomena (receptor removal from the surface of the cell and ligand- receptor internalization) may play a fundamental role in mitogenic stimulation. Even at saturating concentrations of EGF, however, approximately 20% of the total number of EGF-receptors remain on the cell surface. Moreover, maximal EGF-receptor down regulation and the initial massive internalization of EGF require only 4 h in the presence of EGF (Fig. 6). In contrast, it is clear from the time course experiments for 13H]thymidine incorporation and cell division (Fig. 13) that (i) the occupation of at least a portion of the EGF- receptors remaining on the cell surface following the 4 h required for receptor binding and down regulation is necessary for the mitogenic response, and (ii) massive internalization of EGF-receptor complex in the first 4 h, while perhaps necessary, is not sufficient for mitogenesis.

Our data suggest that the role of receptor down regulation may be to alter the cell’s sensitivity to the growth factor. Because EGF is required continuously for mitogenic activity, not just during the time required for down regulation and initial internalization of the ligand receptor complex, it is clear that the receptor sites still present on the cell surface after these initial events are important and essential media- tors of EGF’s mitogenic action.

We have recently found that EGF increases binding of 3T3 cells to concanavalin A-coated nylon fibers and reduces concanavalin A-mediated hemadsorption to 3T3 cells (28). This EGF-induced membrane change occurs with a concentration and time dependence similar to the EGF concentration and time dependence for receptor down regulation and does not occur in an EGF nonresponsive, nonbinding 3T3 variant. Whether down regulation and the change in concanavalin A- receptor mobility represent a common cell surface membrane alteration by EGF remains to be determined. It may be that EGF internalization, EGF-receptor down regulation, and these surface membrane changes are characteristics corre- lated with, but not necessary for, the mitogenic response of 3T3 cells to EGF. We are presently attempting to determine whether EGF internalization and receptor down regulation

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can be dissociated from the mitogenic response by physiological manipulations and by the selection of additional nonresponsive variants.

REFERENCES


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A Aharonov, R M Pruss and H R Herschmanmitogenesis in 3T3 cells.

Epidermal growth factor. Relationship between receptor regulation and

1978, 253:3970-3977.J. Biol. Chem.

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