THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 257, No. 6, Issue of March 25, pp. 3053-3060, 1982 Printed In U.S.A.

Resolution of High and Low Affinity Epidermal Growth Factor Receptors INHIBITION OF HIGH AFFINITY COMPONENT BY LOW TEMPERATURE, CYCLOHEXIMIDE, AND PHORBOL ESTERS*

(Received for publication, July 22, 1981)

A. Christie King and Pedro Cuatrecasas From the Wellcome Research Laboratories. Denartment of Molecular Biology, Research Triangle Park, . - North Carolina 27709

Receptors for the mitogen epidermal growth factor (EGF) have been implicated as mediators of cellular transformation and tumor promotion. 12-0-Tetradeca- noylphorbol-13-myristate (PMA) causes immediate but transient reductions in the ability of human KB cells to internalize EGF by decreasing the affinity of cell sur- face EGF receptors 7- to 12-fold. After 90 min of PMA exposure, complete recovery of the high affinity appa- ratus occurs. Evidence is presented that the decreased binding capacity to "%EGF is caused by delaying the appearance at the plasma membrane of cryptic recep- tor sites of high affinity. Exposure of these high affinity binding sites is temperature dependent and is blocked by pretreatment of cells with inhibitors of de nouo protein synthesis.

After short incubation periods (0 to 60 min) with '''I- EGF, up to 90% of the internalized '261-EGF can be rapidly dissociated from cells by the addition of PMA. This treatment kinetically reveals a trypsin-insensi- tive, nondissociating component of cell-associated Iz6I- EGF. With PMA, both the rate (maximal at 4 h with PMA as compared to 24 h) and extent (&fold greater with PMA) of transfer of '"I-EGF into this trypsin- stable compartment is enhanced and accounts for the severalfold stimulation in ''%EGF accumulation. This evidence suggests that the internalization of '"I-EGF can be resolved into two components, one into which EGF immediately partitions and is freely exchangeable with the extracellular medium and that is disrupted by PMA. A second and previously unknown compartment retains '"I-EGF in the presence of PMA after a short delay to a considerably greater extent than that of untreated cells. Thus, without PMA, EGF is internalized into this nondissociating intracellular compartment, but the transfer is only 5-10% as efficient. These results suggest that the accumulation of stable intracellular mitogen-receptor complexes may be directed by a pop- ulation of very high affinity receptors which may be an element involved in the control of cellular growth and tumor promotion.

Epidermal growth factor promotes the growth and differ- entiation of epithelial and fibroblastic cells both in vivo and in vitro (1-3). Although it is generally accepted that EGF'

* The costs of publication of this 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.

' The abbreviations used are: EGF, epidermal growth factor; PMA, 12-0-tetradecanoylphorbol-13-myristate; DMEM, Dulbecco's modi- fied Eagle's medium; FCS, fetal calf serum; BSA, bovine serum albumin.

and other mitogenic peptides initiate a cascade of intracellular anabolic events through specific interactions with cell surface receptors (1-6), it is not known what secondary processes are required for establishing their ultimate mitogenic effect. I t is clear that a critical exposure period of 6 to 8 h is required before cells are committed to synthesize DNA in response to EGF (2, 3). During this time, EGF-receptor complexes are internalized (4) into cells via a receptor-mediated process (2- 6) and are delivered to lysosomes after a delay of about 1 h (5, 7-9). Therein, EGF is degraded into low molecular weight fragments or component amino acids (2-6, 10). Affinity-label- ing studies suggest that the receptor for EGF may only be partially degraded (11, 12). Even so, EGF receptors are inac- tivated and do not recycle back to the plasma membrane (8). This results in a reduction of up to 90% of the cell surface EGF receptors within the fist 2 to 4 h of EGF exposure. However, internalization and degradation of EGF continue to be mediated by the residual receptor population (13).

Recently, it has been appreciated that a variety of agents may modulate the interaction of EGF with cells and produce characteristic changes in mitogenic activity. To some extent, the alterations in receptor interaction produce paradoxical effects on the ability of EGF to induce DNA synthesis in growth-arrested cultures of cells. Thus, agents that stimulate the intracellular accumulation of EGF-receptor complexes 3- to 12-fold by inhibiting degradation in lysosomes (4, 7, 8, 10, 11, 14-16) either prevent (17, 18) or only slightly inhibit (15) the ability of EGF to induce mitogenesis. These results may indicate that a step subsequent to receptor binding and inter- nalization could play a critical role in mediation of the mito- genic activity of EGF (10, 11, 19).

The possible importance of distinct high affinity binding sites for the mitogenic activity of EGF has been suggested previously (20). Recently, it has been found that certain agents cause profound reductions in the affinity of surface EGF receptors and thereby decrease the binding capacity of cells for EGF. Interestingly, these compounds act to enhance the ability of EGF to stimulate mitogenesis in cells (21-25). These include the phorbol ester tumor promotors (21-24), the neu- rohypophyseal nonapeptide, vasopressin (25), quinone deriv- atives like vitamin K3 (26), and the toxic alkaloid of Strepto- myces, teleocidin B (27). The mechanisms leading to enhanced mitogenic activity are not known, but it seems clear that the inhibition of receptor binding capacity produced by these diverse agents takes place at some site distal to the EGF receptor (28, 29). It has been suggested that the reduction in affinity of EGF receptors by phorbol esters is transient and may serve to enhance the mitogenic activity of EGF by decreasing the overall extent of its degradation (24). Since the mitogenic response to EGF requires 6 to 8 h of continuous exposure to cells (2, 5, 20), and degradation of ''>I-EGF over

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this time frame is not appreciably altered in the presence of phorbol esters (uide infra), the short lived (1-2 h) alterations in EGF receptor affinity caused by phorbol esters and vaso- pressin (24, 25) cannot explain their potentiating effects.

Until recently, it was thought that '"I-EGF bound to a homogeneous class of high affinity cell surface receptors. Indeed this appears to be the case for human (HF) and murine (3T3) fibroblastic cell lines (4-6). However, human epithelial cells derived from neoplastic tissues (HeLa, KB) and rat cell lines exhibit heterogeneous binding at physiologic tempera- tures (22, 24, 26). We now present evidence that, prior to exposure of cells to EGF, cell surface receptors may exist in a low affinity state. Time, temperature, and de nouo protein synthesis play a critical role in the subsequent appearance of a small population of high affinity binding sites. Thus, expres- sion of high affinity receptors for EGF may be similar to that proposed for nerve growth factor receptors where sequential conversion of a portion of low affinity sites to sites of higher affinity appears to occur (30). We have used the phorbol esters as tools to resolve kinetically the pinocytosis of EGF into two components of different affinities. In this report, we describe some of the properties of a subpopulation of PMA-sensitive, high affinity receptors and present evidence that these recep- tors may be important in mediation of the control of cell growth and tumor promotion.

EXPERIMENTAL PROCEDURES

Materials and Cell Culture-KB cells, derived from an oral epi- dermoid carcinoma, were grown in DMEM containing Earle's bal- anced salts and 2 mM glutamine, with streptomycin and penicillin each at 100 units/ml, and 10% FCS (Gibco), and equilibrated with 5% co2/95% air. Mouse EGF was purified (31) from adult male submax- illary glands (Pel-Freeze) and iodinated with chloramine-?' (1) to a specific activity of 2.5 to 4.5 X lo5 cpm/ng. PMA and cycloheximide were purchased from Sigma. Trypsin and soybean trypsin inhibitor were from Worthington.

Iz5I-EGF Binding Assay-KB cells were removed from culture flasks with two washes of 0.05% EDTA in phosphate-buffered saline, washed twice, and resuspended into DMEM with or without 2% FCS. Iz5I-EGF was added, and, at the indicated times, samples of cells were removed and filtered through 1.0-micron pore Millipore fiiters with two consecutive 10-ml washes. The conditions for dissociation and trypsinization are given in the figures. KB cells have a maximal binding capacity of approximately 360 pg of EGF/106 cells.

Preparation of Cell Lysates-KB cells were harvested in 0.05% EDTA, washed three times in harvest buffer (0.05 M borate, 0.15 M NaCl, 1 mM MgC12, 1 mM CaC12, pH 7.2), resuspended into 2 pellet volumes of lysis buffer (0.02 M sodium borate, 0.2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, pH 10.2), stirred with a Vortex mixer, pelleted at 50,000 X g for 20 min, and resuspended in Krebs' buffer containing 0.1% bovine serum albumin for '"I-EGF binding assays. Alternatively, cells were harvested, washed, and resuspended into 10 volumes of 0.02 M 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, 0.2 mM EDTA, 2 m ~ phenylmethylsulfonyl fluoride, pH 7.2. After cells were allowed to swell on ice for 30 min, they were then homog- enized by Polytron for two 30-s intervals on ice. Membranes were then prepared by differential centrifugation by removing nuclei and unbroken cells at 10,OOO X gfor 10 min. The supernatant was decanted and membranes were pelleted by centrifugation at 50,000 X g for 20 min. The results of '"1-EGF binding shown in Fig. 10 were identical using both membrane preparations.

RESULTS

Effect of Phorbol Myristate on the Time Course of Asso- ciation of '"I-EGF with KB Cells-Phorbol esters may act to lower the affinity (22-24) or number (32) of EGF receptors in cultured human and murine cell lines. Confusion over the exact nature of the effect stems from the transient inhibition by PMA of EGF receptor binding capacity (24 and below). To characterize the inhibitory effect; of PMA on '"I-EGF binding, internalization, and loss of '"I-tyrosine in KB cells, a time course of lz5I-EGF (2.5 ng/ml) association was performed at

37 "C (Fig. 1). The presence of lo-@ M PMA causes an immediate inhibition of the interaction of '"I-EGF with cells. This inhibition persists for up to 120 min, but the extent of inhibition decreases over time, until the inhibitory effect is reversed. Thus, exposure of KB cells to PMA after longer incubation periods (2-6 h) results in an enhanced association of the growth factor with cells. The initial inhibitory effect of phorbol esters on '"I1-EGF association at 37 "C has been previously documented (24). However, our results differ, since we show that with PMA the maximal accumulation of I2'I-

EGF is attained but is shifted to later times of incubation. The reversal of the initial inhibitory effect is not prevented by addition of a second dose of lo-* M PMA and indicates that recovery is not due to metabolism of the phorbol analogue (not shown).

Nutrient-containing medium and low concentrations of se- rum are required to restore completely '2'1-EGF binding ca- pacity and may explain why complete recovery was not ob- served previously (24). In the absence of serum (see Fig. 4) or in the presence of 2% serum and 10 pg/ml cycloheximide (Fig. 1, closed triangles), the maximal association of '"'I-EGF with cells equals the steady state level obtained subsequent to the usual clearance of surface receptors and lZ5I-EGF degradation products ("down regulation") in untreated cells. Thus, the results obtained when protein synthesis was inhibited are similar to those observed in a recent publication (24). The time course of '251-EGF accumulation in the presence of lo-@ M PMA, 10 p g / d cycloheximide, 2% serum was identical with that with M PMA and no serum supplement (data not shown). These results suggest that "'I-EGF accumulates equally as well in KB cells treated with PMA after a short delay only if serum is present and that de nouo protein synthesis may be required for maximal association but not for the initial inhibitory effect of phorbol esters on lZ5l-EGF association with cells.

PMA Causes a Delay in Receptor-mediated Internaliza- tion of '251-EGF-Since PMA drastically reduces the initial association of lz5I-EGF with KB cells (Fig. 1) during the period

time. hours time. hours

FIG. 1 (left). Time course of the interaction of "'I-EGF with KB cells. KB cells were harvested with 0.05% EDTA in phosphate- buffered saline and washed 2 times with Krebs' bicarbonate, pH 7.4, 0.1% BSA. Cells were resuspended into DMEM, 2% FCS (IO' cells/ ml) and incubated at 37 "C with 2.5 ng/ml '"I-EGF under three different incubation conditions: (a) untreated cells, ( b ) PMA, and (c) M PMA with 10 pg/ml cycloheximide. At different times, 2004 aliquots of cells were vacuum fdtered through LO+ pore Millipore fiiters and washed twice with 5 ml Krebs', 0.1% BSA. All values are corrected for nonspecific binding by including 3 pg/ml unlabeled EGF.

FIG. 2 (right). Trypsin sensitivity of "'1-EGF associated with KB cells over time with and without PMA. The experimental protocol is identical with that of Fig. 1 except that after the indicated incubation with '*'I-EGF, the cells were exposed to 200 pg/ml of trypsin for 10 min at 37 "C. This treatment removes 80% of surface EGF receptors (Table I), and we have previously demonstrated that

I-EGF insensitive to trypsin exposure has an intracellular location (IO). The percentage of "'1-EGF insensitive to trypsin in the presence of IO-* M PMA is shown (M), as is the per cent difference in trypsin sensitivity between untreated cells and lo-* M PMA-treated cells (A- - -A).

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of time (0-60 min) when internalization and lysosomal trans- location of surface EGF-receptor complexes occurs (7-9), it seemed possible that PMA might be interfering with the internalization process itself. To study this, the sensitivity of cell-associated '"I-EGF to trypsin was determined. Trypsin treatment (200 pg/ml) at 37 "C for 10 min inhibits -80% of '2511-EGF binding to KB cells (Table I). Therefore, any "'1- EGF which remained associated with cells subsequent to this brief trypsin exposure, was considered to be protected and was inferred to be compartmentalized intracellularly (10). In this experiment, cells were incubated with M PMA and 10 ng/ml '251-EGF for the indicated incubation times; then the total '"I-EGF which was insensitive to trypsinization was determined. During the time course of the transitory PMA inhibition (0-90 min), the extent of trypsin-insensitive I2'I- EGF increases over time with PMA and reaches a plateau after about 4 h (Fig. 2). These results demonstrate that EGF becomes resistant to trypsinization even in the presence of PMA. However, a direct consequence of the PMA-induced lag in lZ51-EGF accumulation (Fig. 1) is that at early times of incubation there is a greater percentage of surface-bound (i.e. trypsin-sensitive) '"I-EGF compared to untreated cells. Thus, surface-bound radioactivity is about 35% greater in cells treated with PMA after 30 min of incubation, and this per- centage decreases over time so that after 2 to 4 h the difference between surface-bound '"I-EGF found on PMA treated and untreated cells is less than 10% (Fig. 2). These results indicate that the trypsin sensitivity of "'I-EGF associated with cells decreases over time and suggests that radiolabeled EGF is internalized into KB cells treated with PMA with a delay consistent with the time course shown in Fig. 1. Even though

TABLE I Effect of trypsinization of KB cells on "'I-EGF binding capacity The trypsin sensitivity of EGF receptors was determined by pre-

treating cells with 200 pg/ml trypsin at 37 "C for the indicated times. Trypsin was inactivated by the addition of soybean trypsin inhibitor

pg/ml) and the KB cell suspensions were incubated at 4 "C for I-EGF binding assays at a saturating concentration of "'I-EGF (40

Time of trypsin expo- Specifically bound 12'1- Percentage of control ng/ml).

sure EGF rnin cpm X 10"

0 25,632 100 23,016 89

2 20,509 79 5 22,164 86 7.5

10 13,612 52 5,200 20

30 3,297 13

1

the total accumulation of l2'1-EGF is delayed by PMA, there does not appear to be any significant differences in the ability of '"I-EGF to be degraded after early periods of exposure (Table 11). However, the extent of dissociation of "'I-EGF with PMA may be slightly less after 0 to 10 min of incubation.

PMA Transiently Inhibits a High Affinity Component of Pinocytosis of "'I-EGF-Many reports have suggested that active analogues of phorbol esters may inhibit "'I-EGF bind- ing to a variety of cell types by decreasing the apparent affinity or number of cell surface EGF receptors (22-24), and one report suggested that this inhibition may be reversible (24). We have found similar results with human KB cells. The association of '"I-EGF after a 1-h incubation with lo-' M PMA is reduced at all concentrations of EGF tested (Fig. 3, left) but is either completely eliminated or drastically reduced at the lowest concentrations (0.05-5 ng/ml). Since "'I-EGF is internalized into cells in the presence of PMA (Fig. 2), albeit with a temporal delay (Figs. 1 and 2), the reduced capacity of KB cells to accumulate l2'1-EGF must be caused by an inhi- bition of the pinocytosis of EGF. Consistent with the recovery observed in Fig. 1, after 6 h of exposure of cells to M PMA in the presence of DMEM, 2% serum, the capacity of cells to bind and internalize l2'1-EGF at 37 "C is fully recovered at all concentrations of '"I-EGF tested and is even slightly en- hanced (Fig. 3, left). These results confirm previous findings that the ability of EGF to interact with surface receptors is reduced by PMA treatment after early incubation periods (22-24). Furthermore, these results extend the observation that EGF-receptor interactions may be partially recovered after several hours of PMA exposure (24) and demonstrate that full recovery of '"I-EGF pinocytosis can be obtained in the presence of complete nutrient medium and serum (Fig. 3, left).

It is not clear whether EGF is internalized into cells by a single homogeneous population of high affinity surface recep- tors (2-6) or whether multiple receptor types may exist (22- 24). Scatchard analysis of "'I-EGF binding data obtained at 37 "C demonstrates a homogeneous class of receptors on human and murine fibroblastic cell lines (2-6) while epithelial cells may express two populations of receptors of different affinities (22-24). Since Scatchard analysis of data obtained at 37 "C may be invalid due to the nonequilibrium conditions imposed by the process of internalization, we wished to ana- lyze the parameters of pinocytosis of '"I-EGF with PMA by double reciprocal plots. The uptake of "'I-EGF into KB cells with and without PMA is linear for the first 30 min of incubation (not shown); therefore, "'I-EGF pinocytosis was accessed at different EGF concentrations at 37 "C after a 15- min incubation. At this time, the surface EGF receptor con-

TABLE I1 Effect of PMA on intracellular degradation of 12'I-EGF

Cells were incubated with "'1-EGF (10 ng/ml) in DMEM, 2% FCS for the indicated incubation times and washed free of unbound '"I-EGF with 1 ml of Krebs', 0.1% BSA. The amount of '"I-EGF associated with cells at the time of the washing step is shown in the fvst column. The radioactivity was then allowed to dissociate from celis for 1 h at 37 "C in 1 ml of DMEM, 2% FCS (second column). The extent of degradation of '''I-EGF was determined by precipitating "'I-EGF with 10% trichloroacetic acid at 4 "C as described in Ref. 10 (third column).

Specific cell-associated l a s I -

EGF "'I-EGF dissociated after 1 h Trichloroacetic acid soluble '''1 dissociated

Time -

-PMA +PMA -PMA dissoci- +PMA dissoci- Per cent

ated

Per cent

ated Per cent degraded degraded

Per cent

min 0 5,722 2,234 490 9 485 22

1,375 19 208 5

660 170

32 7

10 7,496 2,316 1,745 23 534 7 326 16

20 8,326 2,710 785 34 766 10 388

3,630 44 16

30 9,847 1,125

3,035 42

4,695 1,978 24 530 19

48 60 19,584 4,306 41 1,800

1,430 8,000

47 3,044 42

31 756 25 6,542 33 1,179 27

5 7,212 2,091

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3056 Epidermal Growth Factor High Affinity Receptors

I 'Ot /'

6 hours

E 10 20

EGF. n g p l

FIG. 3. Concentration dependence of the interaction of laaI- EGF with KB cells exposed to PMA. Because the association of cells with ""I-EGF in the presence of M PMA is inhibited after early incubation periods and enhanced at later times, we investigated the EGF concentration dependence of this interaction after cells were exposed to lo-& M PMA for 1 or 6 h. After exposure, KB cells were

centration is not limiting (5, 10) and there is no degradation of '"I-EGF or release of radiolabeled breakdown products (7, 8).

Double reciprocal plots of data collected in this manner are linear (Fig. 3, right) and therefore are unlike Scatchard plots of data obtained at 37 "C (not shown). The half-maximal concentration for "'I-EGF accumulation (&in) is substantially reduced after 1 h with PMA (13 nM) when compared to its untreated counterpart (2.2 nM). The maximal velocity ( Vmax) with PMA is also reduced about %fold (Fig. 3, right). The absolute values of Kpin or VmaX will differ depending on the length of the incubation, since the data suggest that the inhibitory effect of PMA is transient. The apparent decrease in the affinity of the pinocytic apparatus (z.e. the sum of surface binding and internalization) caused by PMA cannot be due solely to the inhibitory effect of PMA on internalization of '"I-EGF (Fig. 2 ) . Scatchard analysis of I2'I-EGF binding data obtained a t 37 "C and previously published (22-24) cannot rule out this possibility because of the inherent prob- lems of calculating bound/free ratios in a nonequitibrium system where internalization and degradation occur (33, 34). Thus, the high affinity binding sites for lZ5I-EGF on the surface of KB cells may represent a unique subpopulation of EGF receptors, and PMA may interfere with the binding of "'I-EGF only to this subclass of very high affinity EGF receptors. After the cells were incubated with PMA and EGF for 6 h in the presence of 2% serum to allow for recovery from the PMA treatment (Fig. I), the apparent Kpin in both PMA- treated and untreated cells again exhibits a high affinity (1.9 nM), and the maximal pinocytic capacity (Vmax) for each condition is similar (Fig. 3, right). The V,,, for pinocytosis after 6 h of exposure to EGF is reduced about 4- to 5-fold from the initial value, and this is consistent with the clearance of surface receptors known to occur after incubation of cells with EGF for prolonged periods (2-6). Thus, PMA does not prevent the receptor internalization process, but the time course (Fig. 1) suggests that this process is delayed.

Effect of PMA and Trypsin on the Temporal Association of lZ5I-EGF with KB Cells-From the previous results, it seemed possible to resolve the temporal association of "'1- EGF with KB cells into two kinetically distinct components if more than one receptor population directed the internali-

EGF. nM"

harvested as described in Fig. 1 and incubated with varying concen- trations of carrier-free lZ5I-EGF (0.1 to 50 ng/ml) for 15 min. The cells were then fittered and washed as described. Left, concentration de- pendence of '"I-EGF interaction with KB cells at 37 "C. Right, double reciprocal plots of the data shown at the left.

,O PMA +trypsin

8 12 I

16 time, minutes

FIG. 4. Effect of PMA on dissociation of lZ51-EGF from KB cells. Cells were incubated with I2'I-EGF (2.5 ng/ml) for the indicated times in Krebs', pH 7.4, 0.1% BSA. The temporal interaction of 12'1- EGF was monitored as described in Fig. 1 (t".). A t the times indicated by tbe arrows, PMA was added to the incubation to a final concentration of M. The extent of dissociation of I2'1-EGF was then monitored by removing a 200-1.11 aliquot of cells from the incu- bation and filtering as described (O---O). Cells were also treated throughout the incubation with IO-" M PMA ( D " c I ) or IO-' M PMA with a brief trypsin exposure as described in Fig. 2 (H).

zation of "'1-EGF. Cells were incubated with lZ5I-EGF for varying times and then treated with PMA to induce rapid dissociation of lZ5I-EGF (Fig. 4). A nondissociating component of '251-EGF results (open circles) which has an absolute mag- nitude slightly less than that observed when the incubation with "'I-EGF was initiated with PMA in the medium (open triangles). However, mild trypsinization of cells exposed to both '"1-EGF and lo-' M PMA removes the surface bound radioligand and results in a trypsin-resistant, nondissociating component of cell-associated '"1-EGF.

These results suggest that after longer incubations (2-6 h), "'I-EGF may be transferred into a trypsin-stable compart- ment. To study this further, we exposed cells to "'I-EGF for times up to 24 h with and without lo-' M PMA and determined the '251-EGF associated with cells that was resistant to trypsin treatment (see Table I). For untreated cells, the association of "'I-EGF decreases to about 10% of the maximal level after 24

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h. This is consistent with the known down regulation of surface receptors and degradation of internalized 12'I-EGF (2- 6). For these untreated cells, maximal transfer of "'I-EGF to a trypsin-stable compartment requires 24 h (Table 111). In contrast, exposure of cells to PMA results in a 15-fold en- hanced overall accumulation of lz5I-EGF into this trypsin- stable domain. Furthermore, the complete transfer of cell- associated I2'I-EGF into this compartment requires only 4 h when PMA is present rather than the 24-h period for un- treated cells.

Effect of Cycloheximide on lZ5I-EGF Accumulation in KB Cells ut 37 "C-Since the results of Fig. 1 suggest that the maximal uptake of lZ51-EGF into KB cells is sensitive to inhibitors of protein synthesis, we determined whether the inclusion of cycloheximide (10 pg/ml) has an effect on the "'1- EGF uptake capacity of KB cells in the absence of PMA. We found that full attainment of the high affinity state of surface EGF receptors at 37 "C is inhibited by the presence of 10 pg/ ml cycloheximide (Fig. 5). Thus, in untreated cells, a 1-h incubation with lz5I-EGF results in a higher apparent affinity of the receptors than that observed in the presence of inhibi- tors of de nouo protein synthesis. Puromycin (25 pg/ml) had the same effect on receptor affinity (data not shown), indicat- ing that binding of lZ5I-EGF is not inhibited nonspecifically by cycloheximide. The inhibitory effect of cycloheximide on

TABLE 111 Trypsin sensitivity of "'I-EGF associated with cells with and

without PMA

M PMA. After the times indicated, a 200-p1 aliquot of cells (IO6/&) was removed, treated with 200 pg/ml trypsin for 20 min at 37 "C, filtered, and washed as described in Fig. 2.

Cells were incubated with 20 ng/ml '*'I-EGF with and without

Untreated cells lo-' M PMA

2 10,246 6,147 60 22,216 10,470 47 4 14,990 10,974 73 24,190 23,308 97 6 10,044 8,202 82 27,846 27,012 97 8 10,575 9,155 87 24,220 26,420 100

24 1,305 1,310 100 17,430 17,210 100

FIG. 5 (left). Effect of cycloheximide on the concentration dependence of "'1-EGF interaction with KB cells. KB cells were harvested and then incubated with (0) and without (A) 10 pg/& cycloheximide for 1 h at 37 "C. Aliquots of cells were then vacuum fdtered and washed as described in Fig. 1. Cycloheximide inhibits the association of '2sI-EGF with KB cells a t all concentrations tested when included throughout the incubation. Notice in Fig. 6 that pretreatment of KB cells with cycloheximide for 4 h results in a greater degree of inhibition.

FIG. 6 (right). Effect of PMA and cycloheximide on high af- finity interaction of '"I-EGF with KB cells. KB cells were pre- treated with M PMA for 1 h at 37 "C (0) or with 10 pg/ml cycloheximide for 4.0 h (0) prior to harvesting as described in Fig. 1. Cells were then incubated with "'I-EGF (0 to 2.5 ng/ml) for 15 min at 37 "C to determine tbe initial rates of accumulation of Iz5I-EGF and then filtered and washed as described. The association of '"1- EGF in untreated cells is also shown.

the association of "'I-EGF with KB cells at 37 "C exhibits a lag of approximately 30-40 min (8). Prior to this time, there is no inhibition in the extent of lZ5I-EGF accumulation in the presence of cycloheximide at any concentration tested (0.1-20 ng/ml) (data not shown) which therefore differs from the immediate inhibition caused by phorbol esters (Fig. 1). More- over, the inhibition afforded by protein synthesis inhibitors is readily reversed by incubating the cells in medium containing low concentrations of serum for 1-3 h (not shown).

The difference in uptake with and without cycloheximide defines a de nouo synthesis-dependent, high affinity compo- nent of pinocytosis of Iz5I-EGF of low capacity which has heretofore been unrecognized (Table IV). Thus, EGF is inter- nalized into KB cells with high affinity in a manner which is highly dependent on de nouo protein synthesis. The results with phorbol esters (Fig. 3) and cycloheximide (Fig. 5) suggest that the internalization of "'I-EGF can be chemically resolved into at least two components with differing affinities.

To determine whether the initial inhibitory effect of PMA on internalization of '*'I-EGF was affecting a component of the pinocytic apparatus identical with that observed with cycloheximide, we determined the concentration dependence of 12'I-EGF association at 37 "C with KB cells pretreated with lo-' M PMA for 1 h or with 10 pg/ml cycloheximide for 4.0 h. The high affinity component of pinocytosis of 12'I-EGF was drastically reduced by both of these pretreatments to an identical extent (Fig. 6), and the inhibitory effects of these preincubations were not additive under these conditions (not shown). These data suggest that phorbol esters may be inter- fering with the expression of a distinct population of high affinity cell surface receptors which have an unusually high turnover rate or whose expression requires a rapidly turning over effector. The results presented thus far also indicate that the presence of phorbol esters may only cause a temporal delay in expression of these high affinity binding sites (Fig. 1). Moreover, full recovery of high affinity receptors (Fig. 3) does not occur in serum-containing medium after 4-6 h of PMA treatment if 10 pg/ml cycloheximide is present (data not shown). These results are consistent with those seen in Fig. 1 and with the possible interpretation that expression of high affinity EGF receptor sites may require protein synthesis.

Effect of Temperature on the Affinity of EGF Receptors- The inhibitory effects of phorbol esters, vasopressin, and quinones on It5I-EGF binding capacity are highly temperature dependent (22-26) and at least for that of phorbol esters do not occur in membranes prepared from sensitive cell lines (22). It is not clear how these diverse agents can induce identical changes in EGF receptors at presumably distinct

TABLE IV Cycloheximide treatment of cells defines low capacity, high affinity

interaction of '251-EGF Cells were incubated with and without 10 pg/ml cycloheximide for

1 h at 37 " c in the presence of various concentrations of "'I-EGF. The amount of radioactivity associated with cells was determined by vacuum filtration as described (IO" cells/assay). A represents the difference in the association of '"I-EGF between untreated apd cycloheximide-treated cells.

EGF Specific "'I-EGF association

Untreated cells 10 p g / d cy- A cloheximide w / m l cpm

0.2 550 80 470 0.5 1,460 560 1.0

900 3,081 1,455 1,626

2.5 5,603 2,809 2,794 5.0 7,754 5,306 2,448

10,448 2,213 13,967 11,443 2,535

10 12,661 20

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3058 Epidermal Growth Factor High Affinity Receptors

sites on cell membranes, or why reduced temperatures protect cells from their inhibitory effects. Carpenter and Cohen (4) fist demonstrated that the association of '251-EGF with cells was highly temperature dependent. Since lZ5I-EGF is not internalized at 0 "C, the temperature dependence observed could be explained by receptor recycling which has been suggested to occur for some nutrient carrier receptors (13,35, 36). However, we have recently reported that receptors for EGF on KB cells do not recycle (8). To determine the nature of the temperature dependence of 1251-EGF association with cells, we studied the concentration dependence of lZ5I-EGF interaction with KB cells at 37 and 0 "C (Fig. 7). The enhanced accumulation of lz5I-EGF at 37 "C is caused by a notable temperature-dependent shift in the affinity of EGF receptors. At 0 "C, the half-saturation for '251-EGF receptors is about 10-12 nM, while that observed at 37 'C occurs at a consider- ably lower concentration (1-2 nM). The maximal lZ5I-EGF accumulation is only slightly decreased at the lower temper- ature.

This observation may explain why there is no inhibition of lZ5I-EGF binding by vasopressin (25), quinones (26), or phor- bo1 esters (22-25) at 0 "C, since at this temperature, high affinity EGF receptors are not accessible to the ligand. We have observed identical temperature dependency for high affinity interaction of EGF in HeLa cells, murine 3T3 cells, and two human fibroblast cell lines (data not shown). Fur- thermore, lZ5I-EGF receptor affinity is essentially identical at low temperatures (Fig. 7) and in the presence of 10 pg/ml cycloheximide (Fig. 5). The addition of cycloheximide does not further reduce receptor affinity when assayed at 4 "C, and cells pretreated with 10 pg/ml cycloheximide for 2-3 h have identical receptor affinities at 37 and 4 "C (Fig. 8). These results suggest that the minimum affinity state of EGF recep- tors is within the range of 10-13 nM at reduced temperatures or with cells treated with cycloheximide or phorbol esters.

Are Cryptic lZ5I-EGF High Affinity Binding Sites Exposed at 37 "CZ-The preceding results indicate that there are binding sites on cells which direct the internalization of IZ5I- EGF and express different affinities for this process under physiologic conditions. These high affinity receptors are not accessible to the radioligand at 0 "C (Fig. 7) nor are they present on cells treated with PMA after 1 h (Fig. 3, left) or on cells treated with 10 pg/ml cycloheximide for 4.0 h (Fig. 6). In

- 2 4 6 8 1 0 1 2

EGF, ng/rnl EGF (ng/ml)

FIG. 7 (left). Effect of reduced temperature on lasI-EGF inter-

trations of '"I-EGF (0 to 20 ng/ml) at 4 "C for 3 h or at 37 "C for 1 action with KB cells. KB cells were incubated with varying concen-

h. The 3-h incubation period at 4 "C was sufficiently long to reach equilibrium at all concentrations tested. We have previously shown that Scatchard analysis of binding data obtained at 4 "C with KB cells is linear with about 20,000 binding sites/cell and a dissociation constant of 1 to 2 nM (8).

FIG. 8 (rzght). Effect of cycloheximide on '"I-EGF binding measured at 4 "C. KB cells were incubated with I2'I-EGF only in the high affinity range (0 to 12 ng/ml) to determine whether cyclo- heximide had a further inhibitory effect on this interaction at 4 "C. The incubation conditions were identical with those of Fig. 7 (0) or contained 10 pg/ml cycloheximide (A).

addition to the affinity alteration, cells treated under these conditions show a slightly reduced maximal binding capacity that may be consistent with exposure of cryptic binding sites. These cryptic sites would comprise only a minor portion (20 to 30%) of the total receptor population. Thus, the results can be best explained either by the existence of two different populations of receptors with different affinities with the higher affinity component being in a cryptic state, or by a single class of binding sites of which a portion may be con- verted from a low affinity state to one of a higher affinity as suggested for nerve growth factor receptors (30). To study this, we determined the trypsin sensitivity of lz5I-EGF binding sites similar to that of Fig. 2.

Cells were pretreated at 37 "C with 250 pg/ml trypsin for varying periods of time after which trypsin activity was halted by the addition of soybean trypsin inhibitor. Cells were then assayed either at 4 "C to determine lZ51-EGF binding to residual low affinity sites or at 37 "C for an estimate of the uptake capacity for '251-EGF (Fig. 9). The binding of '251-EGF to low affinity receptors (4 "C) decreased with increasing times of exposure of cells to trypsin and reached a minimum of about 10% of the initial binding activity after about 20 min. The estimate of total pinocytic activity (assayed at 37 "C) also decreases but to a considerably lesser extent (-50% of the initial value). These results indicate that while most of the binding sites assayed at 4 "C can be removed by trypsinization, a portion of the sites exposed at 37 "C resists trypsinization. These sites may be cryptic prior to the addition of EGF and not simply exposed as a result of the increased temperature. The sites which are exposed at 37 "C after the addition of EGF are also sensitive to trypsin digestion (not shown) and thus, these data suggest that there is not a subpopulation of receptor sites resistant to trypsin treatment. Since the major- ity of low affinity sites was removed from cells by the trypsin pretreatment, the cryptic lz5I-EGF receptors exposed are most likely composed of receptors of higher affinity.

Cycloheximide and Reduced Temperatures Do Not De- crease the Affinity of IZ5I-EGF Receptors in Membranes Prepared from KB Cells--In order to determine whether there may exist a distinct population of cryptic, high affinity lZ5I-EGF receptors, we investigated the binding of lZ5I-EGF to membranes prepared from KB cells, since disruption of the

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FIG. 9 (left). Effect of trypsin treatment of KB cells on the binding (4 "C) and uptake (37 "C) capacity of KB cells. Cells were harvested as described and incubated for varying times (0 to 20 min) with 250 pg/ml of trypsin at 37 "C. Trypsin action was halted by the addition of soybean trypsin inhibitor (500 pg/ml) in DMEM, 2% FCS. Cells were then incubated with 30 ng/ml of Iz5I-EGF at 4 "C for 2 h as a measure of maximal cell surface binding capacity (A) or at 37 "C for a 30 min as an indication of the initial rate of lz5I-EGF accumulation (0).

FIG. 10 (right). Effect of pretreatment of KB cells with cyclo- heximide on the concentration dependence of "'I-EGF inter- action in membranes. Cells were treated with 10 pg/ml cyclohexi- mide for 4 h prior to harvesting. Cells were then washed and lysates prepared as described under "Experimental Procedures." Membranes were incubated with varying concentrations of '"1-EGF at 37 "C for 1.5 h, which was sufficiently long to reach equilibrium at all concen- trations tested. B/F, bound/free.

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Epidermal Growth Factor High Affinity Receptors 3059

cell by hypotonic lysis and homogenization could expose these sites. Scatchard analysis of equilibrium binding data for KB membranes displays heterogeneous classes of binding sites at all temperatures tested (0, 24, and 37 "C). The dissociation constants at each temperature are identical for both the high and low affinity binding components (0.2 and 14 nM, respec- tively). The capacity of the low affinity component is un- changed at all temperatures, while that of the high affinity component is reduced as the temperature is decreased (not shown). These results differ from those of intact cells which exhibit a homogeneous class of low affinity sites at 4 "C (8). Hypotonic lysis of cells is therefore sufficient to expose a small portion of these high affinity sites even a t reduced tempera- tures (-30% of total binding assayed at 37 "C, not shown). Thus, most of the high affinity binding sites in KB membranes are absent or inaccessible to the radioligand at reduced tem- peratures. These results may indicate that, at physiologic temperatures in intact cells, the exposure of high affinity binding sites could be dependent on fractional receptor occu- pation by EGF (30, 39) and may involve conversion of low affinity EGF receptors to a small population of high affinity receptors.

Because the appearance of high affinity receptors on intact cells is sensitive to protein synthesis inhibitors, we pretreated cells with 10 pg/ml cycloheximide for 3 h, prepared mem- branes from these cells, and then assayed for I2'II-EGF binding. Pretreatment of intact cells with cycloheximide in this manner results in a complete loss of the high affinity component of '"'I-EGF association (Fig. 7 ) . Thus, if there is a distinct class of high affinity receptors which turn over rapidly and direct the uptake of EGF, cycloheximide treatment should prefer- entially reduce this component in membranes as well as intact cells. In contrast, we find that membranes prepared from cells pretreated with cycloheximide still display heterogeneous binding with affinities similar to those of membranes prepared from untreated cells (Fig. 10). Nonetheless, the capacities of both the low and high affinity populations were reduced to a similar extent. These results may be consistent with the interpretation that high affinity EGF receptors are generated from a single pool of rapidly turning over low affinity recep- tors. In this case, the interconversion would be temperature dependent, and a portion of the sites would exist in a cryptic state in membranes prior to the exposure of cells to EGF. The appearance of high affinity binding sites may also depend on a factor extrinsic to the receptor itself which turns over rapidly and which is either responsible for unmasking these sites a t the cell surface or alternatively for inducing a chemical mod- ification which alters their conformation in a manner that increases their affinity.

DISCUSSION

Phorbol esters are known to effect changes in mammalian cells in vitro that mimic transformation by oncogenic viruses or chemical carcinogens. However, phorbol esters are tumor promotors, not initiators, since they do not appear to interact directly with the cellular genome. They have been described to have growth-promoting activity and seem to enhance syn- ergistically the mitogenic activity of purified growth factors, like EGF. Recent evidence may indicate that phorbol esters do not have endogenous mitogenic activity but may act to potentiate subthreshold levels of growth factors in serum (21). It is therefore of interest to understand the mechanisms through which phorbol esters and other tumor-promoting agents may potentiate the hormone-mediated gonvth of oth- erwise quiescent cells. This process would ensure fixation of carcinogen-induced DNA damage through DNA replication and cell proliferation.

There is increasing evidence that phorbol esters may me- diate their effects on cell growth at the plasma membrane, and specific high affinity receptors have been found for phor- bo1 ester analogues (28, 29). Interestingly, phorbol esters in- hibit the interaction of EGF to its cellular receptors (22-24, 32) and the potency of this inhibition by phorbol analogues correlates with their effectiveness as tumor promotors (32). The inhibition of EGF interaction with its receptor is not caused by direct competition between the phorbol tumor promotors and EGF for either the EGF receptor or for that of PMA (28, 29). A variety of other mitogenic and tumor-pro- moting agents has been shown to cause similar inhibitory effects on EGF-receptor interactions. These include saccharin and cyclamate (37), teleocidin B (27), quinones (26), and vasopressin (25). The data present,ed in this report offer for the first time a viable mechanistic explanation for modulation of the EGF-receptor interactions associated with viral trans- formation (38) and tumor promotion: the inhibitory effects of these agents on EGF-receptor interactions may involve a unique subclass of high affinity EGF receptors.

The results of this report show that the binding and inter- nalization of '""I-EGF in KB cells are characterized by a kinetically complex pinocytic process. Under physiologic con- ditions, there exist both high and low affinity receptor popu- lations, each contributing to the uptake of EGF into cells. For the first time, we demonstrate that the higher affinity com- ponent is regulated by temperature (Fig. 7) and de novo protein synthesis (Figs. 1, 5, and 9). We also show that as in Rat-1 cells, phorbol esters transiently interfere with the expression at the cell surface of high affinity receptors (Figs. 1, 3, and 4).

The two affinity states of the EGF receptor described herein could represent a single class of surface macromolecules of which only a portion of a large pool of low affinity receptors is converted to a higher affinity state. Alternatively, there may exist heterogeneous EGF receptor types. Interconversion of two affinity states has been proposed for both nerve growth factor (30) and insulin (39) receptors. The temperature de- pendence and reduced trypsin sensitivity of the high affinity site suggest that it is either absent or cryptic prior to exposure of cells to EGF. There is no high affinity interaction of I2'I- EGF with intact KB cells a t reduced temperatures. This suggests that the higher affinity sites are exposed via a tem- perature-dependent process. Furthermore, attainment of this higher affinity state can be prevented even a t physiologic temperatures either by inhibitors of protein synthesis or phor- bo1 esters. Since the inhibitory effects of cycloheximide and phorbol esters are not additive, these agents are most likely interfering with the same molecular component that regulates expression of cryptic high affinity EGF receptors. The inhib- itory effects of these agents may either be directly on a distinct high K d receptor population or an effector that regulates its expression at the cell surface. Nonetheless, it seems likely that these agents (cycloheximide and phorbol esters) interfere with the interaction of EGF with a high affinity receptor via different molecular pathways.

The results with cycloheximide clarify the possibilities that may occur. First, if distinct receptor populations exist, the de novo synthesis of high affinity receptors may be induced by the binding of EGF to low affinity receptors already present on cells. Second, the two putative receptor types may turn over at different rates, with the higher affinity population turning over more rapidly. Third, the expression of high affinity EGF receptors could require an effector which turns over rapidly. This effector could be required for the intercon- version of a single receptor type to multiple affinity forms or it could be required to unmask these sites at the cell surface.

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3060 Epidermal Growth Factor High Affinity Receptors

While pretreatment of cells with cycloheximide results in a preferential loss of the high affinity component in intact cells (2.e. that exposed at the plasma membrane), hypotonic lysis and homogenization expose at least a portion of these sites. Similarly, "'I-EGF interacts with both receptor types at re- duced temperatures when membranes are prepared by hypo- tonic lysis, while in intact cells, only low affinity binding is measured. These data with membranes and the differential trypsin sensitivity of EGF receptors suggest that cells express heterogeneous populations of EGF receptors of differing affin- ities. These receptor types have distinct defining molecular characteristics. An inference from the sum of this data is that there may exist a rapidly turning over effector which is re- quired for expression at the cell surface of the higher affinity receptor population.

It is known that EGF produces characteristic morphologic changes in some cell types, such as pseudopod extension and cell rounding (40, 41). This suggests that EGF may induce changes in membrane fluidity or the cellular cytoskeleton, or both. Alterations in lipid fluidity or membrane structure caused by cytoskeleton rearrangements could result in expo- sure of cryptic EGF binding sites, similar to the exposure known to occur for serotonin receptors and blood group anti- gens (42, 43). In addition, EGF binding to surface receptors could induce conformational changes which result in altered affinity. Such conformational changes may be induced by aggregation of surface receptors (7) or may require direct chemical modification by a distinct enzymatic effector. It is of interest that recent evidence suggests that EGF receptors may be phosphorylated by endogenous tyrosine protein kinases and that protein kinases like these may be largely responsible for the events associated with transformation processes and the resultant loss of mammalian growth control (44-46). Clearly, more studies are required in order to sort out all the molecular processes important for growth control and cellular transformation. The results from this study show that the interaction of EGF with KB cells is kinetically complex and that two distinct receptor populations of different affinities exist for EGF. Phorbol esters only affect the interaction of the mitogen with one single subtype of receptor. Each receptor type appears to mediate translocation of EGF into cells, but is regulated differentially. Continuous treatment of cells with PMA results in enhanced transfer of EGF into a trypsin- resistant, nondissociating compartment whose temporal ap- pearance correlates well with the appearance of high affinity receptors. These results suggest that the higher affinity recep- tor population could be involved in growth alterations asso- ciated with transformation and tumor promotion.

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A C King and P Cuatrecasasesters.

of high affinity component by low temperature, cycloheximide, and phorbol Resolution of high and low affinity epidermal growth factor receptors. Inhibition

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