JOURNAL OF CELLULAR PHYSIOLOGY 142.284-292 (1990)

Modulation of Tyrosine, Serine, and Threonine Phosphorylation and lntracellular Processing of the Epidermal Growth Factor Receptor by Antireceptor Monoclonal Antibody HIRONOBU SUNADA,* PETER YU, JEFFREY S. PEACOCK, AND JOHNMENDELSOHN Memorial S/odn-Kettering Cancer Center (t f.S., P.Y., J.S.P., J.M.),dnd Cornell University Medical Uoliege (j.M.1, New York, New York I002 I To investigate the functional significance of epidermal growth factor ( E L F ) receptor phosphorylation, experimental systems were explored in which receptor phosphorylation on tyrosine and serindthreonine could be differentially stimulated. Exposure of A431 cells to 20 nM EGF at 37°C results in phosphorylation of serine, threonine, and tyrosine sites on the receptor. Monoclonal antibody (mAb) 22.5 binds to the EGF receptor with affinity comparable to EGF and competes with the binding of EGF. Exposure of A431 cells to 20 n M EGF in the presence of 300 n M anti-EGF receptor mAb 225 (1 5-fold excess) selectively activated serine and threm i n e phosphorylation of the receptor, but not tyrosine phosphorylation. This observation indicates that EGF-mediated receptor phosphorylation on tyrosine and on serinekhreonine residues is dissociable. The intracellular fate of the EGF receptor was examined under conditions that produce different phosphorylation states of receptor amino acids. Exposure of A431 cells to ECF decreased the half-life (T,,2) of the receptor from 17.8 h to 5.6 h, with activation of tyrosine, serine, and threonine phosphorylation. Incubation with mAb 225 augmentcd the degradation rate (T, iL = 8.5 h) without activation of receptor phosphorylation. Concurrent exposure to ECF (20 n M j and mAb 225 (300 nM) resulted in cumparable enhanced degradation (T,,, = 9.5 h), with increased phosphorylation only on serine and threonine residues. These results suggest that serine/threonine phosphorylation is irrelevant to the augmentation of receptor degradation. Methylamine, an inhibitor of lysosomal function that did not affect phosphorylation of the E L F receptor, completely protected EGF receptors from rapid degradation induced by EGF, but it only slightly altered the rate of EGF receptor degradation elicited by mAb 225 or by EGF plus 15-fold excess mAb 225. In contrast, mAb 455, which binds to the receptor but does not inhibit EGF binding and ELF-induced activation of phosphorylation on tyrosine, serine, and threonine residues, did not influence EGF-induced rapid, methylamine sensitive degradation of E L F receptor. The results suggest that when EGF receptors are internalized under conditions that do not activate the receptor tyrosine kinase, they are sorted into a nonlysosomal pathway that differs from the methylamine-sensitive lysosoma1 pathway traversed following activation by EGF. The data indicate the possibility of a function for tyrosine kinase activation and tyrosine autophosphorylation in determining the lysosomal intracellular pathway of EGF receptor processing and degradation.

Epidermal growth factor (EGF) is a polypeptide growth factor that usually enhances cell growth in culture (Carpenter and Cohen, 1979), although the proliferation of some cell lines with high numbers of EGF receptors is inhibited (Gill and Lazar, 1981; Barnes, 1982; Kawamoto et al., 1983). The binding of EGF to its specific receptor elicits rapid autophosphorylation of the receptor on tyrosine (Tyr) residues (Ushiro and Cohen, 1980; Downward et al., 19841, due to stimulation of a n intrinsic Tyr protein kinase $1 1990 WILEY-LISS, INC.

Received March 3, 1989; accepted September 22, 1989.

"To whom reprint requestsicorrespondence should be addressed. This work was supported by Grants CA 42060 and CA 37641 from the National Institutes of Health. Abbreviations used: EGF, epidermal growth factor; EGTA, ethy lenebis (oxyethylenenitrile) tetraactic acid; mAb, monoclonal antibody; DMEM, Dulbecco's modified Eagle's medium; SDS, sodium dodecyl sulfate; Ser, serine; Thr, threonine; TPA, 12-0-tetradecanoylphorbol-13-acetate;Tyr, tyrosine.

EGF RECEPTOR PIIOSPHORY‘LATION AND PROCESSlNG

activity (Hunter and Cooper, 1985). EGF-induced autophosphorylation on Tyr residues stimulates the EGF receptorikinase activity for exogenous substrates (Bertics and Gill, 1985). The EGF receptor is also phosphorylated on serine (Ser) and threonine (Thr) residues following binding of EGF to the receptor (Hunter and Cooper, 1981; Iwashita and Fox, 1984). An EGF-associated increase in diacylgycerol has been implicated as a mechanism by which SeriThr-specific protein kinase C might be activated, with subsequent phosphorylation of the activated EGF receptor on SeriThr sites (Cochet et al., 1984; McCaffrey et al., 1984; Smith et al., 1983). Tumor-promoting phorbol esters, which activate protein kinase C, have the capacity to elevate phosphorylation of Ser and Thr residues on the EGF receptor. This is accompanied by a shift in a subpopulation of receptors from high affinity to low affinity for EGF and a partial block of receptor auto hosphorylation on Tyr. Phosphorylation of the Thr‘?! residue appears to be associated with this reduction in EGF-dependent receptor autophosphorylation on Tyr (King and Cooper, 1986). This observation is supported by the finding that phorbol ester fails to inhibit EGF-dependent autophosphorylation of the product of a point-mutated EGF receptor transcription unit, which encodes alanine instead of threonine at position 654 (Davis, 1988). Nevertheless, the functional significance of the affinity shift and the reduced Tyr autophosphorylation remains unclear. Following binding of EGF to its receptor, there is rapid clustering and internalization of both receptor and ligand, followed by lysosomal degradation (Schlessinger e t al., 1978; Haigler et al., 1978, 1979; Willingham et al., 1983). Studies on the transferrin receptor (May et al., 1984; Cox et al., 1985) and on the type I1 insulin-like growth factor receptor (Corvera and Czech, 1985) have suggested that increased phosphorylation of those receptors might affect the process of internalization. We have shown, however, that a n anti-EGF receptor mAb elicited rapid receptor internalization into endosomes without stimulating receptor phosphorylation (Sunada et al., 1986). This suggested that elevated phosphorylation of the EGF receptor is not essential for its internalization. In the present study, we explore possible differences in the intracellular fate of EGF receptors, following internalization mediated by EGF and by anti-EGF receptor mAb and examine the possibility that phosphorylation of the EGF receptor may provide a signal determining the pathway of intracellular trafficking. To test this hypothesis, we devised a n experimental system employing two ligands, EGF and anti-EGF receptor mAb 225, in which the phosphorylation of EGF receptors on Tyr and on SeriThr residues was modified separately. The degradation rate of EGF receptors and the sensitivity of receptor degradation to methylamine, a n inhibitor of lysosomal function, were determined in this system. Based on the quantitative analyses of the phosphorylation state and the degradation rate of EGF receptors, a possible functional significance of tyrosine phosphorylation on the intracellular pathway for processing of internalized EGF receptors is described.

285

MATERIALS AND METHODS Materials 32P-orthophosphate (carrier-free), Nal”I, and L-”Smethionine (1,108 CiIm mol) were purchased from New England Nuclear Research Products (Boston, MA). Mouse EGF was purchased from Collaborative Research (Waltham, MA). Phosphate-free modified Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Irvine Scientific (Santa Ana, CAI. Monoclonal antibodies 225,528, and 455 raised against human EGF receptors were purified from ascites fluid obtained from mice injected with the respective hybrydomas (Masui et al., 1984). Rabbit immunoglobulin to mouse IgGs was purchased from Accurate Chemicals (Westbury, NY). The A431 human epidermoid carcinoma cell line was obtained from Dr. G. Sato (Cell Sciences Center, Lake Placid, NY). Cell culture Cells were grown at 37°C in a 1:1 (volivol) mixture of DMEM and Ham’s F-12 nutrient medium (DMEMIF12) containing 5% newborn calf serum in a n atmosphere of 5%’C02195% air, unless otherwise stated.

32Pphosphorylation of the EGF receptor in intact cells A431 cells were plated a t 1.3 x 10’ cellsI35 mm plate and incubated for 2 days in DMEMIF-12 containing 5% newborn calf serum. Medium was switched to phosphate-free DMEM supplemented with L-glutamine (48.4 mg per 100 ml), sodium pyruvate (11.0 mg per 100 ml), and 4% newborn calf serum, and the cells were incubated with ”P-orthophosphate (0.75 m Ciiml) a t 37°C for 15 h. EGF was added for the last 5-60 min, a s indicated in the figure legends or tables. After removing medium, the cell layer was rinsed with phosphatebuffered saline (PBS),lysed with 500 ~1 of RIPA buffer (0.05 M Tris-HC1, pH 7.710.15 M NaC110.8% Triton X10010.8% Na deoxycholate/O.OB%SDSi10 mM EDTA1 100 M Na3V0,/50 mM NaF10.3 mM phenylmethylsulfonyl flouridei5 mM iodoacetic acid) on ice for 10 min, and scraped with a rubber policeman. After removal of nuclei and insoluble material by centrifugation a t 15,000 xg for 20 min, the supernatant was used for immunoprecipitation. Metabolic labeling of the EGF receptor with “S-methionine A431 cells (5 x lo4 cells135 mm plate) were plated in DMEMIF-12 containing 5% newborn calf serum. Twenty-four hours later, cells were refed with 2 ml of the labeling medium (DMEMIF-12 containing 5% newborn calf serum and 58 pCi 35S-Met)and incubated for 2 days at 37°C. Then the labeling medium was removed, and the cell layer was rinsed with PBS, followed by a rinse with chase medium (DMEMIF-12 containing 5% newborn calf serum, supplemented with unlabeled L-Met, 54.7 mgilOO ml). The chase incubations at 37°C were started by adding 1 ml of the chase medium with or without EGF (20 nM) and/or 225 mAb (300 nM). At varying time points, cells were harvested in 500 ~1 RIPA buffer as described above.

286

SUNADA ET AL

Immunoprecipitation As described previously (Sunada et al., 1985) 300 pl of the RIPA lysate containing 32P-labeled or 35S-Met labeled EGF receptors was subjected to immunoprecipitation in a 1.5 ml microcentrifuge tube, with 4.5 g anti-EGF receptor mAb 52816.6 pg rabbit antimouse antibody60 p1 Pansorbin (20% suspension in PBS). The pelleted immune complex was rinsed with 500 p1 RIPA buffer two times, then with 500 pl of 0.5 M LiC1/ 0.1% SDS for 5 min a t room temperature to remove nonspecifically coprecipitated labeled materials. For 32P-labeled samples, a 1:l mixture (viv) of 0.5 M LiCli 0.1% SDS and RIPA buffer was used for the last wash, instead of using a n undiluted solution of 0.5 M LiC1/ 0.1% SDS, in order to increase the recovery of 32P-labeled EGF receptor in a n immunoprecipitated pellet.

ing sites and dissociation constant were measured on intact cells o r on cells fixed with 0.2% paraformaldehyde by Scatchard analysis as previously described (Kawamoto et al., 1983). To determine the percent of receptors occupied b EGF, paraformaldehyde fixed cells were exposed to '251-labeled EGF (0.025, 0.05,0.1,

0.2,0.5,1.0,2.0,4.0,8.0,10,and20nM)inthepresence of simultaneously added 300 nM mAb 225, or in the absence of mAb, for 120 min a t 37°C and processed as described (Kawamoto et al., 1983).

RESULTS

Modulation of EGF-mediated phosphorylation of the E G F receptor b y anti-receptor m A b Monoclonal antibody 225 competitively inhibits binding of EGF to its receptor by occupying the reccpSDS-gel electrophoresis and a u t o r a d i o g r a p h y tor with a dissociation constant (K, = 1-2 nM), which Immunoprecipitated "P-labeled EGF receptor was is similar to the K, for EGF (Sato et al., 1983). During extracted with 50 pl sample buffer (0.01 M sodium the first 60 rnin following exposure to either mAb o r phosphate, pH 7.0, 1%SDS, 10% glycerol, 0.001% bro- EGF, receptor internalization is elicited with comparamophenol blue, 5 mM EDTA, 0.2%2-mercaptoethanol) ble kinetics and to a similar extent (Sunada et al., a t 100°C for 2 min. After centrifugation in a microcen- 1986). However, mAb 225 has no intrinsic capacity to trifuge (12,OOOg, 3 m i d , 35 pl of the supernatant was activate phosphorylation of the EGF receptor (Sunada loaded on a 7%! polyacrylamide slab gel and electro- et al., 1986). Thus, mAb 225 provides a useful reagent phoresed (Sunada et al., 1985). The EGF receptor pro- with which EGF-mediated phosphorylation of the EGF tein separated in a gel was located by presoaking the receptor can be modulated without altering ligand-induced receptor internalization. gel with 25%) isopropanolilO9i acetic acid for 30-60 The effect of mAb 225 on EGF-mediated phosphorymin, then staining in 0.01% Coomassie brilliant blue/ 25%'isopropanolilO% acetic acid for 2-3 h, followed by lation of the EGF receptor was closely examined in destaining with a destaining solution (10% isopro- cultured A431 cells. Monoclonal antibody 225 at a conpanoli7%' acetic acid). The gel was dried on a Whatman centration 15-fold higher than EGF (allowing less than 3M filter paper and the radioactivity located by expos- 10% of receptors to bind EGF) was found to inhibit ing the gel to a Kodak x-ray film XAR-5 a t room tem- selectively EGF-dependent Tyr-phosphorylation, but perature for 15 min with a n intensifying screen. 35S- there was little effect on EGF-induced phosphorylation Met-labeled EGP receptor was clectrophoresed as of the receptor on Ser and Thr residues (Fig. 1).Quanabove and visualized on a n x-ray film by fluorography titative analysis of phosphoamino acid content of the using sodium salicylate as a n enhancer (Chamberlain, EGF receptor in the absence and presence of the two ligands is presented in Table 1. This analysis reveals 1979). that, in the absence of exogenous EGF, a baseline level Analysis of phosphoamino acids of Tyr phosphorylation accounting for 6% of total phosThe gel band corresponding to 170 kDa EGF recep- phoamino acid content is present. In a series of ten tor, located as above, was excised out, and the backing experiments, the baseline figure varied from 2 to 6%. piece of filter paper was removed. The 32P-labeled EGF This low level of Tyr phosphorylation is also evident on receptor in a gel piece was partially hydrolized with autoradiographs of the two-dimensional thin-layer 200 pl of 6 M HC1 at 110°C for 1 h. Two portions of 10 electrophoresis analysis, if autoradiograph exposure pl of the hydrolyzate were taken for measurement of times are prolonged. We further investigated the selective augmentation the radioactivity in a liquid scintillation counter, to determine the total incorporation of 32Plinto the EGF of SeriThr phosphorylation observed in the presence of receptor. The rest of the hydrolyzate was dried with a EGF and excess mAb 225. A selective inhibition of Tyr Savant Speed Vac centrifuge, resuspended in a small phosphorylation could occur, if the kinase system revolume of H,O, and applied onto a Dowex AG1-X8 col- sponsible for Ser and Thr phosphorylation is activated umn. The column was washed with H20, and the ab- by low occupancy of the EGF receptor by EGF, whereas sorbed 32P-labeled materials were eluted with 0.5 M high occupancy is required to activate Tyr phosphoryHC1 and lypholyzed. The recovery of radioactivity lation (autophosphorylation) to detectable levels. This possibility was examined by experiments measuring through the procedures was 7 8 4 5 % . "P-phosphoamino acids were analyzed by thin-layer electro- the level of EGF receptor phosphorylation in response phoresis as described (Cooper et al., 1983). The radio- to varying concentrations of EGF, in the presence activity associated with individual phosphoamino acids or absence of 300 nM mAb 225. A431 cells pre-equiliwas measured in a scintillation counter by scraping brated with "P-orthophosphate were incubated with varying concentrations of EGF for 30 min a t 37"C, and respective spots from a thin layer plate. the phosphorylation state of the EGF receptor was delz5I-EGF binding a s s a y termined (Fig. 2). Half-maximal responses were obEGF was labeled with lz5I by the chloramine-T served for both SeriThr phosphorylation and Tyr phosmethod (Hunter and Greenwood, 1962). l2'1-EGF bind- phorylation a t about 5 nM EGF (Fig. 2A, B). In the

287

EGF RECEPTOR PHOSPHORYLATION AND PROCESSING

1 2 3 4

2 EGF

1

CONT

1.5

z

t

3 225

4 225lEGF

2

I

1.0

0.5

P P

2.0 1.8

[

1.6

-

A

0.5

0

-

*-

4

I

1

1

I

1

5

10

20

B

Treatment A. DMEM 225 EGF 225lEGF B. DMEM TPA TPAl225 C. DMEM kethylamine EGF Methylamine/EGF

P-Tyr 4.7 4.1 32.8 7.3

22.4 26.3 51.9 52.8

48.8 41.5 154.5 105.1

75.9 71.9 239.2 165.2

3.6 2.9 2.4

33.3 58.1 53.7

75.7 200.7 141.3

112.6 261.7 197.3

2.4 2.5 37.4 31.5

19 8 22.8 43.7 51.5

49.9 ~. 43.7 136.3 134.4

72.1 69.1 217.4 217.4 ~~

'Near-confluent A431 cells were preincuhated in phosphale-free Dulbecco's modified Eagle's medium supplemented with 4% newburn calf ~ e r u i nand "Porthophosphate a t 37°C for 15 h to establish equilibrium conditions. After further incubation a t 37'C in the presence of the various ligands and modulating a ents, the cells were solubilized and EGF receptors were immunoprecipitated. 3$-phosphoarnino acids in Lhe EGF receptors were analyzed as described in Experimental Procedures. Data are averages of two samples and are representative of three experiments. A: CeIB were incubated for 30 min in the presence oC20 nhl EGF, or 300 UM mAb 225, or 300 nM mAb 225 plus 20 nM KGY, added as described in Figure 1. B: Cells wcre incuhated at 37°C for 30 min in the presence of 200 ng'ml TP.4. or 300 nM mAb 225, or to the two ligands added simultaneously. C Cells were incubated a t 37°C for 20 min in the presence o f 3 0 mM methylamine, followed by addition of 20 nM EGF for 30 min.

presence of excess mAb 225 (300 nM) against the EGF receptor, autophosphorylation of the receptor on Tyr residues was blocked, even when 20 nM EGF was

'

1.o 0.5

0

'fl

'1

:225

I

I

2

5

1

10

20

h

C

3 0

Total

CONT

- /6

X

TABLE 1. Phosphoamino acid content of A431 cell EGF receptors' 3ZPcpm x 10 ' P-Thr P-Ser

+ 225

*A

2

10

Fig. 1. Modulation of the EGF receptor phosphorylation by mAb 225. Near-confluent A431 cells prelabeled with ""P-orthophosphate at 37°C for 15 h, were incubated with 1) no addition; 2) 20 nM EGF (37"C, 30 min); 3) 300 nM mAb 225 ( O T , 30 min followed by 37"C, 30 min); and 4) 300 nM mAb (O"C, 30 m i d , then together with 20 nM EGF (37"C, 30 rnin). To maintain consistency in the conditions, every culture was placed on ice for 30 min either in the presence or absence of mAb 225, prior to the addition of EGF. Left, autoradiography of immunoprecipitated 32P-EGFreceptor (arrowhead) separated in a 7% polyacrylamide gel in the presence of SDS. Right, two-dimensional thin-layer electrophoresis patterns of 32P-phosphoaminoacids associated with the EGF receptor. S, phosphoserine; T, phosphothreonine; Y, phosphotyrosine. With longer autoradiography exposure time, baseline tyrosine phosphorylation in control cells becomes evident.

J

CONT

:I X X

60

0

+ 225 U

0

0.5

2

5

10

20

EGF (nM) Fig. 2. Concentration dependency of EGP-mediated phosphorylation of the EGF receptor in the presence or absence of mAb 225. Nearconfluent A431 cells were prelabeled with 32P-orthophospbateat 37°C for 15 h. Cells were incubated on ice for 30 min either in the absence ( C ) or presence ( 0 ) of 300 nM mAb 225, then with varying concentrations of EGF at 37°C f o r another 30 min. A: 32P-phosphotyrosine,and B: 3'P-phosphoserine plus "'P-phosphothreonine associated with the EGF receptor were measured as described in Fig. 1; C: Receptor occupancy by EGF in the absence (0) or presence ( 0 ) of300 nM mAb 225 was measured as described in Experimental Procedures.

added, whereas SeriThr phosphorylation of the EGF receptor was reduced by only 30%. The selective inhibition of Tyr phosphorylation by mAb 225 was observed when cells were preincubated with the mAb a t 0°C for 30 min prior to addition of EGF a t 37°C (Fig. 1, Table 1) and when the mAb and EGF were added simultaneously a t 37°C (data not shown). The data sug-

288

SUNADA ET AL

5-

5

h

4 -

a cv m b

ul

0 -

3 -

2 1

2

urating amounts of mAb, low occupancy of the EGF receptor by EGF can generate the signal leading to activation of a SeriThr kinase system at a nearly maximal level, whereas a similar degree of receptor occupancy by EGF in the absence of mAb is associated with minimal SerjThr phosphorylation. We considered the possibility that crosslinking of EGF receptors by bivalent mAb could activate phosphorylation on Ser/Thr. However, in our experimental conditions, there was a 100- to 150-fold molar excess of mAb over antigen (EGF receptor) present in the culture, a condition that typically does not produce crossbridges between Ags. Furthermore, a n experiment employing Fab fragment of mAb 225, with or without the addition of a secondary rabbit antimurine antibody to the mAb, showed that bivalent immunoglobulin was not required to elicit the effect of the mAb upon EGF-mediated receptor phosphorylation on Ser and Thr residues (data not shown). Another possibility we considered was that binding of mAb might enhance the capacity of the EGF receptor to serve a s a substrate for protein kinase C. However, this is unlikely, as concurrent exposure to mAb did not result in a n increase in TPA-induced phosphorylation of receptor SeriThr (Table IB).

Modulation of the catabolic rate and the intracellular processing of the EGF receptor with anti-receptor mAb 225 Fig. 3 . Hill plot of the EGF receptor phosphorylation vs. receptoroccupancy by EGF. Net increase of ""P-phosphotyrosine ( 0 ) and The observation that mAb 225 elicits rapid internal"P-phosphoserine plus "'P-phosphothreonine ( 0 ) were calculated by ization of the EGF receptor, without stimulating recepsubtracting Lhe resuective control values found in the absence of ex- tor phosphorylation and with kinetics comparable to ogenously ldded EkF If n molecules of EGF-bound receptors interact to yield tyrosine au- those observed with EGF, suggests that phosphorylatophosphorylation, the equation is tion of the EGF receptor is not required for ligandn (EGF) + n (receptor) n (EGF-bound receptor) induced receptor internalization (Sunada et al., 1986). (phosphotyrosine-receptor),, To explore the possible role of phosphorylation on subThe slope of'the plot of log (receptor occupancy by EGFl vs. log iphossequent processing of the internalized EGF receptor in photyrosine content) gives the reaction order n. A431 cells, we investigated the degradation rate of 35SMet labeled receptor following internalization induced by EGF, by mAb 225, and by EGF plus excess mAb 225. gest that activation of the EGF receptor!Tyr kinase can Figure 4 shows SDS-gel electrophoresis patterns of imbe independent of the activation of kinases (or phos- munoprecipitated 35S-Met-labeled EGF receptors. The phatases) that regulate phosphorylation of the EGF re- relative amount of EGF receptor was estimated by scanning densitometry of the fluorogram, and the avceptor on Ser and Thr residues. Figure 2 shows receptor occupancy by EGF a t differ- erage values from two to six experiments were used to under varying conent concentrations, in the absence and presence of 300 calculate receptor half-lives nM mAb 225. For example, the addition of 20 nM EGF ditions (Table 2). Parallel studies were performed with a control mAb, resulted in approximately 90% receptor occupancy without the addition of mAb, but less than 10% in the 455, which binds to the EGF receptor with a weaker presence of mAb. These findings are consistent with affinity than mAb 225 (Kd = 20nM vs. 2nM) (Sato et our previous observations that the K, for EGF and for al., 1983). Unlike mAb 225, which binds to the polymAb is 2 nM and that they compete stoichiometrically peptide backbone of the EGF receptor, mAb 455 recogfor binding to the EGF receptor (Sato et al., 1983). Re- nizes carbohydrate residues on the receptor (Gooi et al., ceptor Tyr phosphorylation was further analyzed in 1985). In addition, mAb 455 does not block EGF bindterms of the reaction order by plotting the net increase ing, unlike mAb 225, so that cells exposed to mAb 455 in receptor phosphotyrosine content a s a function of can also bind EGF, which will activate phosphorylation receptor occupancy by EGF (Fig. 3). A straight line of Tyr a s well as SeriThr on the receptor. Like mAb with a slope of 2.3 suggests that intermolecular (most 225, mAb 455 lacks intrinsic capacity to activate tylikely bimolecular) interaction of EGF-bound receptors rosine kinase (or other kinases) in intact cells (Sato et is involved in a n activation mechanism. Similar anal- al., 1983). Furthermore, mAbs 225 and 455 both cause ysis for SeriThr phosphorylation, on the other hand, downregulation of receptor expression, resulting in a yielded a nonlinear relationship. This indicates that 90% reduction of EGF binding capacity, with 1 h of the extent of EGF receptor phosphorylation on SeriThr exposure to either antireceptor mAb (Sunada et al., residues does not simply correlate with the extent of 1986). The T1,2 of EGF receptors in cells unexposed to receptor occupancy by EGF. At present, it is unclear why, in the presence of sat- ligand was 17.8 h (Table 2 A:i). Incubation of A431 log (% RECEPTOR OCCURSNCV)

-

289

EGF RECEPTOR PHOSPHORYLATION AND PROCESSING

0

1

2

3

DMEM

+EGF

+mAb225

4

8

12

0

Fig. 4. Kinetics of the EGF receptor degradation. A431 cells were labeled with 35S-Met for 2 days, The labeled medium was replaced with fresh medium, and the cells were incubated in the absence or presence of 30 nM methylamine for 30 min. The following additions

4

812

0

4

8 1 2

then were made: none, 20 nM EGF, or 300 nM mAb 225. The cells were then incubated at 37°C for indicated periods. "S-Met-labeled EGF receptor was immunoprecipitated and subjected to SDS-polyacrylamide gel electrophoresis, and autoradiography was performed.

TABLE 2. Modulation of phosphorylation state and half-life of the EGF receptor' Culture Condition Ligand A. Control R. 455 C1. 225 C2. 225lEGF D1. EGF D2. 455iEGF 'All values are mean 'Hours.

EGF receptor phosphorylati on TYr SerlThr ? ?

2 ~ T

+++ +++ 2

++ ++ ++ +++ ++++ ++++

of EGFR' ligand-induced changes (ii) TlpLwith ligand (i) T,,,-EGFR TL,2control 17.8 ? 1.0 0.85 i 0.09 15.2 ? 1.6 8.5 ? 0.5 0.48t 0.03 9.5 i 0.5 0.53 5 0.03 0.31 i 0.06 5.6 k 1.0 7.7 2 0.8 0.43 i 0.04

Tlr2 of EGFR'

methylamine-induced changes (iii) TI,,-EGFR (iv) TI), with ligand Methylamine TI,, control 20.2 I 1.5 18.5 i 5.1 .92 * 0.25 12.5 ? 0.5 .62 0.02 .69 i 0.05 14.0 i 1.0 18.6 i 3.4 .92 0.17 3 3 2 0.03 16.7 i 0.6 +

+

SD.

cells with mAb 455 had a modest effect on the EGF TI,, to 48% of control, and a comparable reduction was receptor Tli2, reducing it to 15.2 h, which is 85% of the observed with a 15-fold excess of mAb 225 plus EGF T,,, with control cells (Table 2B, i and ii). Consistent (Table 2C, i and ii). This demonstrates the capacity of with previously reported data (Krupp et al., 1982; Sto- antireceptor mAb to increase markedly the catabolism scheck and Carpenter, 19811, EGF caused rapid degra- of internalized EGF receptors, with a degradation rate dation of the EGF receptor, with a T,,, of 5.6 h in these approaching but not reaching that observed with the cells, a decrease to 31% as compared with controls (Ta- natural ligand, EGF. We have reported that the cytotoxicity of ricin-conble ZD,i and ii). Interestingly, in the presence of EGF plus mAb 455 (which can bind simultaneously to EGF jugated EGF was potentiated by NH4C1, a lysosomal receptors on A431 cells), the receptor was degraded inhibitor (Vollmar et al., 1987). However, unlike the rapidly (Tl,z -7.7 h), with a decrease in half-life to 43% natural ligand, EGF, the cytotoxicity of ricin-conof control, which is comparable to the decrease induced jugated mAb 225 was not altered by the presence of by EGF (Table 2D, i and ii). This suggests that the NH4C1 in the culture medium (Vollmar et al., 1987). effect of EGF on receptor processing is dominant in This suggested that the observed shortened half-life of spite of the presence of a noncompeting antireceptor receptor internalized after EGF binding, compared mAb. with the half-life of receptor internalized after mAb Monoclonal antibody 225 also activated the catabo- binding, might be due to differences in lysosomal-delism of the EGF receptor, resulting in a reduction of pendent processing. To explore this possibility, the ef-

290

SUNADA ET AL

fect of methylamine on the degradation rate of the EGF recept,or was examined. Methylamine does not disturb initial cellular responses to EGF, namely binding and internalization of EGF (data not shown; Yarden et al., 1981) and receptor phosphorylation (Table 1).Methylamine, however, inhibits lysosomal function by elevating the intralysosomal pH and by causing a release of lysosomal enzymes into the medium (Ohkuma and Poole, 1978; Riches and Stanworth, 1980). It also appears to block directly passage of EGF receptors from endosomes to lysosomes (Schaudies et al., 1987; Matrisian et al., 1987). If the internalized EGP receptor is transported to lysosomes, degradation of the receptor would be delayed by initially exposing cells t o methylamine, followed by the addition of ligand. As expected, methylamine completely abrogated the rapid degradation of the EGF receptor elicited by EGF, resulting in a halflife (18.6 h j similar to the control value (20.2 h) (Fig. 4, Table 2D, iii). This is 92% of the Tl12in control cells, representing a marked increase compared with the relative observed in the absence of methylamine (92% cf. 31%) (Table 2D:ii and iv). On the other hand, inhibition of lysosomal function by prior exposure to methylamine only partially affected the increased rate of EGF receptor degradation induced by mAb 225; unlike the result with EGF, exposure to mAb 225 or to EGF plus 15 x excess mAb 225, in the presence of methylamine, induced EGF receptor degradation a t a far more rapid rate (Tl12= 12.5 h and 14.0 h, respectively) compared with the control (T,,, = 20.2 h) (Fig. 4, Table 2C, iii). This is 62 and 69%' of the Tl12in control cells, representing only a modest change compared with the relative TlI2 observed in the absence of methylamine (48 and 53%) (Table 2C, ii and ivj. These observations indicate t.hat mAb-elicited EGF receptor internalizat,ion is followed by movement through a n alternative intracellular pathway that differs from the processing of the receptor elicited by EGF, and this alternate pathway is less dependent on lysosomal function. The selection for entry into the different intracellular processing pathways could be related to differences in phosphoamino acid content (Table 2). Experimental conditions leading t o increased phosphotyrosine content (exposure to EGF alone or to EGF and mAb 445) are correlated with a greater methylamine ef'f'ect on receptor TI!, (Table 2D, ii cf. iv) than those conditions (mAb 445, mAb 225, or mAb 225 and EGF) in which phosphotyrosine content is unchanged (Table 2B, ii cf. iv; Table 2C, ii cf. iv). This suggests that phosphorylation on Tyr may target receptor for more rapid entry into the methyalminesensitive, lysosomal pathway. In contrast, experimental conditions leading t o increased phosphorylation on Ser and Thr without increase phosphorylation on Tyr (mAb 225 and EGF) did not appear to facilitate more rapid entry ofthe internalized EGF receptor into a methylamine sensitive pathway.

DISCUSSION Exposure to a saturating concentration of EGF-induced receptor phosphorylation on Tyr, Ser, and Thr, and this w a s associated with a shortened receptor halflife of 5.6 h. When cells were exposed to saturating amounts of mAb 225, the half-life of the receptor was intermediately shortened to 8.5 h. A combination of

EGF and 15-fold excess mAb 225, was able t o stimulate selectively SeriThr phosphorylation of the EGF receptor without a n increase in Tyr phosphorylation, and this was accompanied by a reduced receptor half-life (9.5 h), similar to that observed with mAb 225 alone. The data indicate that receptor phosphorylation is not essential for increasing the rate of receptor degradation. This observation parallels our previous finding that EGF receptor internalization does not require phosphorylation (Sunada et al., 1986). Methylamine, a lysosomal inhibitor, abrogated the effect of EGF on the rate of receptor catabolism without changing EGF-induced receptor phosphorylation. However, the change in receptor half-life resulting from exposure t o mAb 225, in the presence or absence of EGF, was not substantially affected by methylamine a t a concentration at which near-maximal release of lysosomal enzymes was observed (Riches and Stanworht, 1980). These observations indicate the presence of a methylamine-insensitive pathway for internalized EGF receptor, distinct from a lysosomal, methylaminesensitive pathway. This is in agreement with the previous reports by Miskimins and Shimizu (1982, 19841, in which they proposed the existence of two distinct intracellular processing pathways following internalization of the EGF receptor. A novel pathway involving a nonlysosomal dense organelle and characterized by delayed ligand degradation was described. This was distinct from a lysosomal pathway, which involved early transport to lysosomes with rapid ligand degradation. It has been previously reported that leupeptin, another lysosomal inhibitor, also delays degradation of both EGF and EGF receptors in rat hepatocytes (Dunn et al., 1986). This finding supports our interpretation that the observed effects of methylamine may be attributed to its activity a s a lysosomal inhibitor. Our data on methylamine's effects on the receptor half-life suggest that the predominant processing pathway of internalized EGF receptors is lysosomal following exposure of A431 cells to EGF, which activates Tyr phosphorylation, whereas a nonlysosomal pathway becomes predominant when the receptor is internalized following exposure to mAb 225, which does not activate Tyr phosphorylation. This is in accordance with our previous observations that the toxicity of ricin A-conjugated EGF, but not ricin A-conjugated anti-receptor mAb 225, was enhanced by ammonium chloride, a lysosomal inhibitor, suggesting that EGF and mAb 225 are processed through different intracellular pathways (Vollmar et al., 1987). The question of the physiologic relevance of the mAb-induced pathway for receptor degradation must be addressed. Anti-EGF receptor mAb 225 inhibits both activation of receptor tyrosine kinase by EGF and EGF-induced changes in cell proliferation (Kawamoto e t al., 1983; Sat0 et al., 1983; Gill et al., 1984). It also directly inhibits the proliferation of some EGF receptor-bearing cells (Kawamoto et al., 1983; Sato et al., 1983; Masui et al., 1984). We believe that the mechanism of inhibition for these cells involves interruption of a n autocrine loop mediated by TGF-a (Mendelsohn et al., 1987). However a direct physiologic inhibitory effect mediated by the binding of mAb t o receptor is a possibility that cannot be dismissed. Routing the EGF receptor to a methylamine-sensi-

EGF RECEPTOR PHOSPHORYLATION AND PROCESSING

tive, lysosomal pathway, which provides the most rapid rate of receptor degradation, is not determined by the increased SeriThr phosphorylation of the receptor, a s the methylamine-induced changes in values are modest and comparable in the presence of mAb 225 (absence of SeriThr phosphorylation) or EGF plus excess mAb 225 (activation of SeriThr phosphorylation). This is consistent with the finding that the phorbol ester, TPA, which increases SeriThr phosphorylation and can transiently induce internalization of the EGF receptor in the absence of its ligand, does not elicit lysosomal degradation of internalized EGF receptor. Rather, the phorbol ester-treated EGF receptor is rapidly recycled to the cell surface (Beguinot et al., 1985). Similarly, although phorbol esters can increase serine phosphorylation of the insulin receptor and potentiate insulin-mediated receptor internalization, they fail to affect the degradation rate of internalized insulin in the lysosomes (Hachiya et al., 1957). Without elevated Tyr phosphorylation, the majority of EGF receptors did not enter into the methylaminesensitive pathway, regardless of the presence of EGF in the culture medium or the elevation of SeriThr phosphorylation on the EGF receptor. Recently, i t was reported that a point-mutated EGF receptor lacking tyrosine kinase activity underwent slower degradation than the wild type receptor upon exposure to EGF (Honegger et al., 1987). In addition, this kinase-deficient receptor appeared to be routed in a different intracellular pathway from the lysosomal pathway. Our results, together with the findings of Honegger et al. (19871, support the hypothesis that Tyr phosphorylation or tyrosine kinase activity may generate a signal to sort the internalized EGF receptor into a lysosmal, methylamine-sensitive pathway. Analysis of the reaction order for EGF-mediated receptor Tyr phosphorylation suggests a n intermolecular, possibly bimolecular, interaction of EGF-bound receptors as a n activation step for receptor kinase in intact cells. Recent reports have presented evidence for dimer formation by the EGF receptor molecules (Yarden and Schlessinger, 1957a, b; Boni-Schnetzler and Pilch, 1987; Cochet et al., 19581, and a dimer model for the mechanism of' Tyr phosphorylation of the EGF receptor was proposed. The results of our reaction order analysis are in agreement with this proposed mechanism. When 20 nM EGF was added t o cells in the presence of 15-fold excess mAb, EGF was bound to only about 5% of the receptors, and the remaining receptors (about 93%) were mAb-bound. This did not induce a detectable increase in Tyr phosphorylation, whereas nearly maximal levels of SeriThr phosphorylation were observed. Interestingly, a 5% level of receptor occupancy as a result of exposure to 0.5 nM EGF in the absence of mAb led to only a minimal increase in Ser/Thr phosphorylation. The reason for this difference in the presence of saturating concentrations of antireceptor mAb is unclear at present. However, a recent finding of Thr"' phosphorylation on the EGF receptor in protein kinase C deficient cells indicates that as-yet-undefined phosphorylating enzymatic systems may be associated with regulation of the EGF receptor (Davis and Czech, 1987; Yu, and Mendelsohn, manuscript in preparation). Thus, although mAb binding did not increase phos-

291

phorylation of the EGF receptor by TPA-activated protein kinase C, the possibility remains that the mAb may alter the susceptibility of the EGF receptor to serve as a substrate for other kinases or phosphatases that could be activated or deactivated by a low amount of EGF. It should be noted that the SeriThr residues phosphorylated after stimulation by EGF in saturating concentrations, or by EGF plus 15-fold excess mAb, may be either identical or different. The significance of the pathway utilized for intracytoplasmic translocation of the EGF receptor and the importance of the rapidity of its degradation are unknown. However, delivery of tyrosine kinase activity to selected intracellular substrates, in locations that are not necessarily perimembrane, may be critical in mediating the EGF growth signal. Similarly, the duration of tyrosine kinase-substrate interaction may be dependent on the enzyme degradation rate, and this could have physiologic significance. These potential mechanisms for regulating the growth factor mitogenic signal merit further investigation.

ACKNOWLEDGMENTS The authors are grateful t.o Ms. Frantzie Paul for preparation of the manuscript. Special thanks are given to Dr. Carol MacLeod for comments on the manuscript and t o Dr. Alec Goldenberg for helpful discussion. LITERATURE CITED Barnes. D.W. (1982)Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum-free cell culture. J Cell. Biol., 93tl-4. Beguinot, L., Hanover, J.A., Ito, S., Richert, N.D., Willingham M.C., and Pastan, I. (1985) Phorbol esters induce transient internalization without dcgradation of unoccupied epidermal growth factor receptors. Proc. Natl. Acad. Sci. U.S.A., 82:2774-2778. Bertics, P.J., and Gill, G.N. (1985) Self-phosphorylation enhances the protein-tyrosine kinase activity of the epidermal growth factor receptor. J. Biol. Chem.. 260:14642-14647. Boni-Schnetzler, M. and Pilch, P.F. (1987) Mechanism of epidermal growth factor receptor autophosphorylation and high-affinity binding. Proc. Natl. Acad. Sci. U.S.A., 84:7832-7836. Carpenter, G., and Cohen, S. (1979) Epidermal growth factor. Annu. Rev. Biochem., 48:193-216. Chambcrlain, J.P. (1979) Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal. Biochem., 98t132-135. Cochet, C., Gill, G. N., Meisenhelder, J., Cooper, J.A., and Hunter, T. (1984)C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity. J . Biol. Chem., 259t2563-2558. Cochet, C., Kashles, O., Chambaz, E.M., Borrello, I., King, C.R., and Schlessinger, J. (1988) Demonstration of epidermal growth factorinduced receptor dimerization in living cells using a chemical covalent cross-linking agent. J. Biol. Chem., 263:3290-3295. Cooper, J.A., Sefton, B.M., and Hunter, T. (1983) 1)etection and quantification of phosphotyrosine in proteins. Methods Enzymol., 99: 387-402. Corvera, S., and Czech, M.P. (1985) Mechanism of insulin action on membrane protein recycling: A selective decrease in the phosphorylation state of insulin-like growth factor-I1 receptors in the cell surface membrane. Proc. Natl. Acad. Sci. U.S.A., 825'314-7318. COX,T.M., O'Donnell, M.W.;Aisen; P., and London, I.M. (1985) Hemin inhibits internalization of transferrin by reticulocytes and promoles phosphorylation of the membrane transferrin receptor. Roc. Natl. Acad. Sci. U.S.A., 82t5170-5174. Davis, R.J. (1988) Independent mechanisms account for the regulation by arotein kinase-C of the eoidermal growth factor receotor affiniiy i n d tyrosine-protein kinase activity. J . Biol. Chem., i 6 3 : 9462-9469. Davis. R.J. and Czech, M.P. (1987) Stimulation of epidermal gruwth factor receptor threonine 654 phosphorylation by platelet-derived

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growth factor in protein kinase C-deficient human fibroblasts. J. Biol. Chem., 262t6832-6841. Downward, J., Parker, P., and Waterfield, M.D. (1984)Autophosphorylation sites on the epidermal growth factor receptor. Nature, 311; 483-485. Uunn, W.A., Connolly, T.P., and Hubbard, A.L. (1986) Receptor-mediated endocytosis of epidermal growth factor by rat hepotocytes: Receptor pathway. J. Cell. Biol., 102t24-36. Fava, R.A., and Cohen, S. (1984) Isolation of a calcium-dependent 35-kilodalton substrate for the epidermal growth factor receptor/ kinase from A431 cells. J. Biol. Chem., 25Yt2636-2645. Gill, G.N., and Lazar, C.S. (1981) Increased phosphotyrosine content and inhibition of proliferation in EGF-treated A431 cells. Nature, 293t305-307. Gill, G.N., Kawamoto, T., Cochet, C., Le, A., Sato, J.D., Masui, H., MacLeod, C.L., Mendelsohn, d. (1984) Monoclonal anti-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epidermal growth factorstimulated tyrosinc protein kinase activity. J. Biol. Chem., 259: 7755-7760. Gilmore, T., DeClue, J.E., and Martin, G.S. i1985) Protein phosphorylation a t tyrosine is induced by the v-erb B gene product in vivo and in vitro. Cell, 40r609-618. Gooi, H.C., Hounsell, E.F., Lax, I.? Kris, R.M., Libermann, T.A., Schlessinger, J., Sato, J.D., Kawamoto, T.; Mendelsohn, J.. and Feizi, T. (19851 The carbohydrate specificities of the monoclonal antibodies 29.1, 455 and 3ClB12 to thc epidermal growth factor receptor of A431 cells. Biosci. Rep., 5r83-94. Hachiya, H.L., Talrayama, S., White, M.F., and King, G.L. (1987) Regulation of insulin receptor internalization in vascular endothelial cells by insulin and phorhol ester. J. Biol. Chem., 262r64176424. Haigler, H., Ash, J.F., Singer, S.J., and Cohen, S. (1978)Visualization by fluorescence of the binding and internalization of epidermal growth factor in human carcinoma cells A431. Proc. Natl. Acad. Sci. U.S.A., 75:3317-3321. Haigler, H.T., McKanna, J.A., and Cohen, S. (1979) Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A431. J. Cell. Biol., 81t382-395. Honegger, A.M., Dull, T.J., Felder, S., Van Ohberghen, E., Bellot, F., Szapary, D., Schmidt, A,, Ullrich, A., and Schlessinger, J. 11987) Point mutation at the ATP binding site of EGF receptor abolishes protein-tyrosine kinase activity and alters cellular routing. Cell, 51:199-209. Hunter, T., and Cooper, J.A. (1981) Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor cells. Cell, 24341-752. Hunter, T., and Cooper, J.A. (198.5)Protein-tyrosine kinases. Annu. Rev. Biochem., 54t897-930. Hunter, W.M., and Greenwood, F.C. (1962) Preparation of Iodine-131 labelled human growth hormone of high specific activity. Nature, 194t495-496. Iwashita, S., and Fox, C.F. (1984) Epidermal growth factor and potent phorbol tumor promoters induce epidermal growth factor receptor phosphorylation in a similar hut distinctively different manner in human epidermoid carcinoma A431 cells. J. Biol. Chem., 259t25592567. Kamata, N., Chida, K., Rikimaru, K., Horikoshi, M., Enomoto, S., and Kuroki, T. (1986) Growth-inhibitory effects of epidermal growth factor and overexpression of its receptors on human squamous cell carcinomas in culture. Cancer Res., 46t1648-1653. Kawamoto, T., Sato, J.D., Le, A,, Polikoff, J., Sato, G.H., and Mendelsohn, J. (19831 Growth stimulation of A431 cells by epidermal growth factor: Identification of high affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody. Proc. Natl. Acad. Sci. U.S.A., 80t1337-1341. King, C.S., and Cooper, J.A. 11986) Effects of protein kinase C activation after epidermal growth factor binding on epidermal growth factor receptor phosphorylation. J. Biol. Chem., 261t10073-10078. Kris, R.M., Lax, I., Gullick, W., Waterfield, M.D., Ullrich, A., Fridkin, M., and Schlessinger, J. (19851Antibodies against a synthetic peptide as a probe for the kinase activity of the Avian EGF receptor and v-erb B protein. Cell, 40:619-625. Krupp, M.N., Connolly, D.T., and Lane, M.D. (1982) Synthesis, turnover, and down-regulation of epidermal growth factor receptors in human A431 epidermoid carcinoma cells and skin fibroblasts. J . Biol. Chem. 257:11489-11496. Masui, H., Kawamoto, T., Sato, J.D., Wolf, B., Sato, G., and Mendelsohn, J. (1984) Growth inhibition of human tumor cells in athymic

mice by anti-epidermal growth factor receptor monoclonal antibodies. Cancer Res., 44r1002-1007. Matrison, L.M., and Rodland, K.D., and Magun, B.E. (1987) Disruption of intracellular processing of epidermal growth factor by methylamine inhibits epidermal growth factor-induced DNA synthesis hut not early morphological or transcriptional events. J. Biol. Chem., 262:6908-6913. May, W.S., Jacobs, and Cuatrecasas, P. (1984) Association of phorhol ester-induced hyperphosphorylation and reversible regulation of transferrin membrane receptors in HL60 cells. Proc. Natl. Acad. Sci. U.S.A., 81.2016-2020. McCaffrey, P.G.. Friedman, B., and Rosner, M.R. (19841 Diacylglycerol modulates hinding and phosphorylation of the epidermal growth factor receptor. J . Biol. Chem., 259:12502-12507. Mendelsohn, J., Masui, H., Goldenberg, A. (1987) Anti-epidermal growth factor receptor monoclonal antibodies may inhibit A431 tumor cell proliferation by blocking on autocrine pathway. Trans. Assoc. Am. Physicians Miskimins, W.K., and Shimizu, N. (19821 Dual pathways for epidermal growth factor processing after receptor-mediated cndocytosis. J. Cell. Physiol., 112:327-338. Miskimins, W.K., and Shimizu, N. (1984)Uptake of epidermal growth factor into a lysosomal enzyme-deficient Organelle: correlation with cells mitogenic response and evidence for ubiquitous existence in fibroblasts. J. Cell. Physiol., 11Xt305-306. Ohkuma, S., and Poole, B. (1978) Fluorescence probe measurement of the intralysosomal pH in living cells and the pertubation of pH by various agents. Proc. Natl. Acad. Sci. U.S.A., 75:3327-3331. Riches, D.W.H., and Stanworth, D.R. 11980) Primary amines induce selective release of lysosomal enzymes from mouse macrophages. Biochem. J. 188:933-936. Sato, J.D., Kawamoto, T., Le, A.D., Mendelsohn, J., Polikoff, J., and Sato, G.H. (1983)Biological effects in vitro of monoclonal antibodies to human epidermal growth factor receptors. Mol. Biol. Med., 1: 511-529. Schaudies, R.P., Gorman, R.M., Savage Jr., C.R., and Poretz, R.D. (1987) Proteolytic processing of epidermal growth factor within endosomes. Biochem. Biophys. Res. Commun., 143r710-715. Schlessinger, J., Shechter, Y ., Cuatrecasas, P., Willingham, M.C., and Pastan, I. (1978) Direct visualization of binding, aggregation and internalization of insulin and epidermal growth factor on living fibroblastic cells. Proc. Natl. Acad. Sci. U.S.A., 75t2659-2663. Smith, K.B., Losonczy, I., Sahai, A,, Pannerselvam, M., Fehnel, P., and Salomon, D.S. 11983) Effect of 12-O-tetradecanoylphorbol-13acetate (TPA) on thc growth inhibitory and increased phosphatidylinositol (PI)responses induced by epidermal growth factor (EGF) in A431 cells. J. Cell. Physiol., 117:91-100. Stoscheck, C.M., and Carpenter, G. (1981) Characterization of the metabolic turnover of epidermal growth factor receptor protein in A-431 cells. J. Cell. Physiol., 120t296-302. Sunada, H., MacLeod. C., and Mendelsohn, J. (1985) A direct radioimmunoassay for human epidermal growth factor receptor using 32P-autophosphorylatedreceptor. Anal. Riochem., 149:438-447. Sunada, H., Magun, B.E., Mendelsohn, J., and MacLeod, C.L. (1986) Monoclonal antibody against epidermal growth factor receptor is internalized without stimulating receptor phosphorylation. Proc. Nat,l. Acad. Sci. U.S.A., 83t3825-3829. Tanner, L.I., and Leinhard, G.E. (1987) Insulin elicits a redistribution of transferrin receptors in 3T3-Ll adipocytes through a n increase in the rate constant for receptor externalization. tJ. Biol. Chem., 262: 8975-8980. TJshiro, H., and Cohen, S. (1980) Identification of phosphotyrosine as a product of epidermal growth factor-activated protein kinase in A431 cell membranes. J. Bid. Chem., 255:8363-8365. Vollmar, A.M., Banker, D.E., Mendelsohn, J . , and Herschman, H.R. (1987) The toxicity of ligand and antibody-directed Rican A chain conjugates recognizing the epidermal growth factor receptor. J. Cell. Physiol., 131:418-425. Willingham,M.C., Haigler, H.T.,E’itzgerald,D.J.P., Gallo, M.G.,Rutherford, A.V., and Pastan, I.H. (1983) The morphologic pathway of binding and internalization of epidermal growth factor in cultured cells. Studies on A431, KB, and 3T3 cells, using multiple methods of labelling. Exp. Cell Res., 146:163-175. Yarden, D.Y., Gabbay? M., and Schlessinger, J . (1981) Primary amines do not prevent the endocytosis of epidermal growth factor into 3T3 fibroblasts. Biochem. Biophys. Acta, 674r188-203. Yarden, Y. and Schlessinger, J. (1987) Epidermal growth factor induces rapid, reversible aggregation of the purified epidermal growth factor receptor. Biochemistry 26t1443-1451.