The EMBO Journal vol.3 no.3 pp.531-537, 1984

Biosynthesis of the epidermal growth factor receptor in A431 cells

E.L.V. Mayes* and M.D. WaterfieldDepartment of Protein Chemistry, Imperial Cancer Research Fund,P.O. Box 123, Lincoln's Inn Fields, London WC2A 3PX, UK

*To whom reprint requests should be sentCommunicated by M.F. Greaves

A monoclonal antibody RI against the human epidermalgrowth factor receptor has been used to study biosynthesis inthe carcinoma cell line A431. Two glycoproteins of apparentmol. wts. 95 000 and 160 000 were immunoprecipitated fromcells labelled for short times with [35S]methionine or [3H]man-nose. Pulse-chase studies show the 160 000 mol .wt. glyco-protein to be a precursor of the 175 000 mol. wt. receptor,but do not establish a precursor role for the 95 000 mol. wt.glycoprotein. Limited proteolysis, peptide mapping, endo-glycosidase digestion and the use of monensin andtunicamycin show that the 95 000 mol. wt. glycoprotein isstructurally related to the 160 000 mol. wt. glycoprotein andthat both glycoproteins have -22 000-28 000 mol. wt. ofoligosaccharide side chains. Monensin blocks conversion ofthe 160 000 to the 175 000 mol. wt. mature receptor, a pro-cess which involves complexing several of its N-linkedoligosaccharide chains. Pulse-chase studies showed that animmunoprecipitable polypeptide of 115 000 mol. wt., or95 000 mol. wt., in the presence of monensin, was secreted in-to the medium at late chase times. The possible mechanismsfor the origins of all the receptor-related polypeptides arediscussed.Key words: A43 1 cells/biosynthesis/EGF receptor/membraneglycoprotein/N-linked glycosylation

IntroductionIn recent years biochemical and biophysical studies haveshown that a dynamic equilibrium exists between the surfaceand internal membranes of the cell which allows internal-isation of membrane components and also mediates de novosynthesis, recycling and insertion of surface membrane com-ponents (for a recent review, see Brown et al., 1983). Select-ivity in signalling or internalisation at the surface membraneis in part achieved through glycoprotein receptors which canact as signal transducers or as carriers for the internalisationof a variety of compounds. The acetylcholine receptor(Anholt et al., 1984) and low density lipoprotein (LDL) recep-tor (Goldstein and Brown, 1977; Brown et al., 1979, 1983)respectively are examples of these two types of receptors.Of particular importance for the regulation of cell growth

are a series of polypeptide growth factors including epidermalgrowth factor (EGF), platelet-derived growth factor (PDGF),insulin and the insulin-like growth factors (IGFs) which allhave distinct cell surface glycoprotein receptors (see reviews:for EGF, Carpenter, 1981; Adamson and Rees, 1981; Das,1983; for PDGF, Antoniades and Williams, 1983; Wester-mark et al., 1983; for insulin and IGFs, Clemmons and VanWyk, 1981; Czech, 1982; Czech et al., 1983). The growth fac-

tor receptors may function directly as signal transducers forthe early events (which can include ion movements, changesin phosphorylation of the receptors and cytoskeletal alter-ations) or the late events which follow ligand binding and canculminate in stimulation ofDNA synthesis and cell division incertain cells (see above reviews). Internalisation of thesereceptors, probably occurs initially through coated pits bysimilar mechanisms established in studies of the LDL re-ceptor (Anderson et al., 1982; Carpentier et al., 1982; Brownet al., 1983). Although the receptors and their ligands are in-ternalised it is not clear if internalised growth factors mediateany of their effects through intracellular mechanisms.The control of biosynthesis and surface expression of

receptors is clearly important in the mediation of selectiveresponses to growth factors and we have studied details of thebiosynthesis of the EGF receptor as a step towards under-standing control of receptor expression.The steps involved in glycoprotein biosynthesis were in-

itially worked out using virus model systems (see review,Klenk and Rott, 1980) but recent studies of cellular glycopro-teins such as the histocompatability antigens (Owen et al.,1980), the transferrin receptor (Omary and Trowbridge,1981; Schneider et al., 1982), insulin receptor (Jacobs et al.,1983), and the LDL receptor (Tolleshaug et al., 1982) showthat similar mechanisms are involved. Addition of N-linkedoligosaccharide side chains occurs co-translationally by trans-fer from a lipid carrier, a process blocked by the antibiotictunicamycin (Struck and Lennarz, 1977). Subsequently pro-cessing and modification of these oligosaccharide chains mayoccur as the glycoproteins are transported through the Golgicomplex, where complexing of oligosaccharide chains canoccur prior to insertion of the mature protein into the surfacemembrane. This process is inhibited by the ionophore mon-ensin (Tartakoff, 1983).We have investigated the biosynthesis and addition of

oligosaccharide chains to the EGF receptor using radio-labelled precursors, the inhibitors tunicamycin and monensin,specific glycosidases and by peptide mapping. Biosynthesis ofthe receptor has been followed using a monoclonal antibodyRI (Waterfield et al., 1982) specific for the human receptor,and the human cell line A431 has been used because it con-tains a 50-fold higher level of receptors than fibroblasts andmost other cells (Fabricant et al., 1977; Wrann and Fox,1979).

ResultsBiosynthesis ofEGF receptor-related peptidesThe biosynthesis of the EGF receptor was studied by labellingA431 cells with [35S]methionine for various times. Cells werelysed in Nonidet P-40 (NP-40), immunoprecipitated with themonoclonal antibody RI, subjected to SDS-gel electro-phoresis and polypeptides revealed by autoradiography of thedried gel. Immunoprecipitates of cells labelled for 20 minshow two polypeptides on SDS-polyacrylamide gels of ap-

© IRL Press Limited, Oxford, England. 531

E.L.V.Mayes and M.D.Waterfild

B

-200 175 _160-145-

- 116

-92'5 95 -_

80-0- 66

1 2 3 4 5 ! 2 3 4 5 6

Fig. 1. Biosynthesis of EGF receptor-related polypeptides in A431 cells labelled with [35S]methionine, [3H]mannose or [3H]fucose. Lysate from labelled A431cells was immunoprecipitated with monoclonal RI and analyzed on a 7% SDS polyacrylamide gel which was then autoradiographed. (A) Cells were labelledwith [35S]methionine as follows: lane 1, 20 min 1 mCi/ml; lane 2, 1 h 500 1tCi/ml; lane 3, 2 h 500 ltCi/ml; lane 4, 4 h 200 /tCi/ml; lane 5, 6 h 200 jtCi/ml;lane 6, 18 h 100 uCi/ml; lane 7, 4 h 200 ICi/ml, non-specific immunoprecipitation (no R1). (B) Cells were labelled with 2-[3H]mannose (lanes 1-3) or6-[3H]fucose (lanes 4-6) as follows: lanes 1 and 4, 20 min 250 ACi/ml; lanes 2 and 5, 4 h 50 ACi/ml; lanes 3 and 6, 18 h 25 yCi/mn. Mol. wts. (x 10-) ofprotein standards and EGF receptor-related polypeptides are indicated.

parent mol. wts. 160 000 and 95 000 (lanes 1 and 2, FigureIA). After cells were labelled for 18 h in the presence of[35S]methionine, the predominantly labelled polypeptide im-munoprecipitated had an apparent mol. wt. of 175 000 (lane6, Figure IA). At intermediate times all three polypeptideswere labelled. The 175 000 mol. wt. polypeptide has an iden-tical mol. wt. to that of the mature receptor characterised pre-viously by Waterfield et al. (1982) using monoclonal RI andsimilar to that observed by others in studies on the phos-phorylated and EGF cross-linked receptor (Baker et al., 1979;Linsley et al., 1979; Cohen et al., 1982a, 1982b; Comens etal., 1982). The mol. wts. of all three polypeptides did not alterwhen gels were run under non-reducing conditions.Limited proteolysis and peptide mappingThe EGF receptor is susceptible to limited proteolysis by tryp-sin (Linsley and Fox, 1980) and by a calcium-dependent pro-tease (Cassel and Glaser, 1982; Yeaton et al., 1983), generat-ing fragments of - 115 000 and 155 000 mol. wt., respective-ly, from the mature receptor of 175 000 mol. wt. The effectsof these proteases on the labelled receptor-related polypep-tides of 160 000 and 95 000 mol. wt. were studied to probestructural similarities between them. A431 cells were labelled,prior to immunoprecipitation, with [35S]methionine either for18 h so that the mature receptor was the major labelledpolypeptide, or for 20 min or 4 h to label the 95 000 and160 000 mol. wt. polypeptides (Figure 2).

Trypsin treatment of the lysed cells resulted in the forma-tion of a 115 000 mol. wt. resistant domain from the maturereceptor, by loss of fragments with a total mol. wt. of 60 000(lane 9). Digestion of the 95 000 and 160 000 mol. wt. poly-peptides generated fragments of apparent mol. wts. 98 000,41 000, 39 000 and 37 000 (lanes 7 and 8). Removal of a60 000 mol. wt. fragment from the 95 000 and 160 000 mol.wt. polypeptides would theoretically yield polypeptides of35 000 and 100 000 respectively, similar to those actually ob-served. However, from this experiment, we cannot excludethe possibility that the 37 000-41 000 mol. wt. peptides are

generated from the 160 000 mol. wt. polypeptide, andfragments too small to be observed on the SDS gel are gener-

ated from the 95 000 mol. wt. polypeptide.To probe the effects of the calcium-dependent protease on

532

-175 _160 -

w. - 1 158-

-'-95; _ 4

-200

- 116

.92 5

-66

45

39

4*~

Fig. 2. The effect of limited digestion with trypsin or calcium-dependent

protease on EGF receptor-related polypeptides. A431I cells were labelled

with [35S]methionine: lanes 1, 4 and 7, 200 ACilml for 20 mni; lanes 2, 5

and 8, 50 tiCi/ml for 4 h; lanes 3, 6 and 9, 25 itCi/ml for 18 h. After

labelling cells were treated as described below, prior to immuno-

precipitation with RI and analysis on a 5-10% polyacrylamide gradient

SDS gel: lanes 1 3, lysed immediately; lanes 4-6, incubated in mM

Tris HCI pH 7.4, 9 mM CaC12, scraped after min with a rubber

policeman, and left for 5 min prior to addition of an equal volume of

lysis buffer; lanes 7-9, incubated with trypsin in lysis buffer for 5 minprior to addition of phenyl methyl sulphonyl fluoride and trypsin inhibitor

(final concentrations of 0.5 mM and mg/ml, respectively). Mol. wts.

(X 10-3) of protein standards and EGF receptor-related proteins are in-

dicated.

the receptor-related polypeptides, A43 1 cells were lysed in the

presence of calcium in hypotonic buffer conditions which

cause cleavage of the mature receptor and a reduction in

its mol. wt. by 20 000 (lane 6). As shown in Figure 2 (anes 4

and 5) the 160 000 mol. wt. receptor-related polypeptide is

A

175 -..

160-

95-..-

*5:-200

-116-92*5

-66

owl .: ''A

Biosynthesis of the EGF receptor in A431 cells

0 50 100

FRACTION No.

Fig. 3. Comparative peptide maps of EGF receptor-related polypeptides. The 95 000, 160 000 and 175 000 mol. wt. receptor-related polypeptides, labelledwith [35S]methionine and [14C]carboxymethylated cysteine were prepared by preparative gel electrophoresis and digested with TPCK-trypsin as described inMaterials and methods. The resultant peptide mixtures were analyzed by reverse phase h.p.l.c. in 0.1 trifluoroacetic acid. Peptides were eluted with a

0-60010 acetonitrile gradient over 60 min. 0.5 min fractions were collected, and, after addition of 10 ml scintillation fluid, each counted for 10 min in a

Beckman LS700 with an open channel.

also cleaved by this protease with the loss of a 20 000 mol.wt. fragment whilst the 95 000 mol. wt. polypeptide is notcleaved.

These results suggest that the 160 000 and 95 000 mol. wt.polypeptides contain similar trypsin-resistant domains to thatof the mature receptor, however only the 160 000 mol. wt.polypeptide contains the cleavage site which is susceptible tothe calcium dependent protease.More detailed analysis of the structural relationship be-

tween the 175 000, 160 000 and 95 000 mol. wt. polypeptideswas carried out by peptide mapping using reverse phaseh.p.l.c. Immunoprecipitates from [35S]methionine-labelledcells were purified by preparative SDS-gel electrophoresis anddigested with trypsin as described in Materials and methods.Peptides were fractionated by reverse phase h.p.l.c. andradioactivity in eluted fractions measured by scintillationcounting. Results illustrated in Figure 3 show that the trypticdigests of the 175 000 and 160 000 mol. wt. polypeptides bothcontain labelled peptides which elute from the reverse phasecolumn at similar concentrations of acetonitrile. In addition,the digests of these two polypeptides each have unique pep-tides. The 95 000 mol. wt. polypeptide digest contained pep-tides which were eluted at similar concentrations of aceto-nitrile from the reverse phase column as other peptides ob-served in the digests of the 160 000 and 175 000 mol. wt.polypeptides. However, as expected, the peptide maps of the160 000 and 175 000 mol. wt. polypeptide digests containpeptides not recovered from digests of the 95 000 mol. wt.polypeptide.

Addition of oligosaccharide side chains to the EGF receptorand its related polypeptidesTo study the oligosaccharide side chains which are added to

the polypeptides immunoprecipitated by monoclonal anti-body RI, A431 cells were labelled with [3H]mannose or[3H]fucose. Analysis of immunoprecipitates of cells labelledfor 20 min, 4 h and 18 h are shown in Figure lB. After20 min of labelling of cells with [3H]mannose, polypeptidesof 95 000 and 160 000 apparent mol. wt. were detectedtogether with two polypeptides not observed in labellingstudies with [35S]methionine, which have apparent mol. wts.of 80 000 and 145 000. A labelled polypeptide of apparentmol. wt. 170 000-175 000 can also be seen after both the20 min and 4 h labelling periods. After 18 h the mature re-ceptor is the only significantly labelled polypeptide. In con-trast to the studies with [3H]mannose, when [3H]fucose wasused to label cells the mature receptor was the only labelledpolypeptide observed either by continuous labelling for 18 hQane 6, Figure IB) or by pulse-labelling for 2 h followed by a

chase with unlabelled fucose for 4 h or more (results notshown). These results suggest that only the mature receptorhas fucose containing complexed N-linked oligosaccharidechains while the other receptor-related polypeptides probablycontain non-complexed N-linked oligosaccharide chains.The RI immunoprecipitates were then digested either with

neuraminidase to remove sialic acid residues or with endogly-cosidase H to remove N-linked oligosaccharide chains of thehigh-mannose type. Results obtained are shown in Figure 4.Neuraminidase digestion of immunoprecipitated [35S]methio-nine labelled mature receptor resulted in a reduction in ap-parent mol. wt. from 175 000 to 165 000 (see Figure 4A)while digestion of immunoprecipitates with endoglycosidaseH caused a reduction in apparent mol. wt. from 175 000 to167 000 (see Figure 4C). Therefore, the mature receptor mustcontain both high-mannose and complex N-linked oligo-saccharide chains.

533

15000

CPM

5000

0

B '

Z160-~ ;090:0*00*0w;168-138

95-#m

68-p-66

4-

1 2 3 4 5 1 2 3 456 7 89 1 2 3 4 5 6 7 8

Fig. 4. The effect of neuraminidase and endoglycosidase digestions on EGF receptor-related polypeptides. RI immunoprecipitates from A431 cells labelledwith [35S]methionine were treated with neuraminidase or endoglycosidase H as described in Materials and methods, and then analyzed on 7% polyacrylamideSDS gels followed by autoradiography. (A) Cells labelled for 18 h with 50 /Ci/m1 [35S]methionine. Immunoprecipitates were treated with the followingamounts of neuraminidase: lane 1, 0 m units; lane 2, 10 m units; lane 3, 100 m units; lane 4, 200 m units; lane 5, cells labelled for 1 h with 200 ACi/ml[35S]methionine prior to immunoprecipitation and not treated with neuraminidase. (B) Cells labelled for 1 h with 500 MCi/ml [35S]methionine. Immuno-precipitates were treated with the following amounts of endoglycosidase H: lane 1, 0 m units; lane 2, 1 m unit; lane 3, 5 m units; lane 4, 10 m units; lane 5,50 m units; lane 6, 100 m units; lane 7, 300 m units; lane 8, 200 m units; lane 9, 1000 m units. (C) Cells labelled for 18 h with 50 MCi/ml [35S]methionine.Immunoprecipitates were treated with the following amounts of endoglycosidase H: lane 1, 0 m units; lane 2, 5 m units; lane 3, 10 m units; lane 4, not rele-vant; lane 5, 100 m units; lane 6, 200 m units; lane 7, 500 m units; lane 8, 1000 m units. Mol. wts. (x 10-) of protein standards and EGF receptor-relatedproteins are indicated.

Distinctly different results were obtained in similar studiesof the 160 000 and 95 000 mol. wt. receptor-related polypep-tides showing that these polypeptides have no complexedN-linked oligosaccharide chains. Thus, endoglycosidase Htreatment of the 160 000 and 95 000 mol. wt. receptor-relatedpolypeptides caused shifts in apparent mol. wt. of these poly-peptides to 138 000 and 68 000 when immunoprecipitateswere incubated in the presence of 1 unit of the enzyme (Figure4B). When intermediate amounts of endoglycosidase H(1-300 m units per immunoprecipitate) were used, severaldiscrete intermediate forms of the polypeptides were ob-served. For the 160 000 mol. wt. polypeptide, intermediatesof mol. wts. 158 000, 156 000, 155 000, 149 000, 145 000,142 000 and 138 000 were observed, and for the 95 000 mol.wt. polypeptide intermediates of mol. wts. 93 000, 90 000,88 000, 86 000, 84 000, 81 000, 78 000, 76 000, 73 000,70 000 and 68 000 were found. These discrete shifts in appar-ent mol. wt. were more easily observed for the 95 000 proteinthan for the 160 000 mol. wt. protein, because for the samedecrease in apparent mol. wt. the change in electrophoreticmobility is greater (relative distance of migration is pro-portional to logarithm of mol. wt.). Eleven discrete shiftswere observed for the 95 000 mol. wt. polypeptide, suggestingthat this polypeptide has approximately 11 N-linked high-mannose chains. The average difference in apparent mol. wt.between the discrete bands observed for the 95 000 mol. wt.polypeptide was 2500, which is similar to the predicted mol.wt. for removal of an individual high-mannose oligosac-charide chain. The total decreases in apparent mol. wt. forthe 160 000 and the 95 000 mol. wt. proteins were -22 000and 28 000, respectively. It is possible that these two receptor-related polypeptides have very similar oligosaccharide struc-tures and the difference between the total decrease in ap-

parent mol. wt. observed may be solely due to the difficultyof measuring such a small percentage change in the mol. wt.

175 5160=138-o

95-.

..68.

68 --O

1 2 3 4

-200

-116

- 92'5

-66

5

Fig. 5. The effect of tunicamycin on biosynthesis of EGF receptor-relatedpolypeptides. A431 cells were incubated in the absence or presence oftunicamycin for 2 h prior to labelling for 4 h with 200 ACi/ml[35S]methionine. Lysates were immunoprecipitated in the presence or

absence of RI, and analyzed on a 7% polyacrylamide SDS gel followed byautoradiography. Lane 1, 0 yig/ml tunicamycin, 0 Ag RI; lane 2, 0 Mg/mltunicamycin, 5 Mg RI; lane 3, 5 ,ug/ml tunicamycin, 5 Mg RI; lane 4,10 Mg/ml tunicamycin, 5 Ag R1; lane 5, 10 Ag/ml tunicamycin, 0 Mg Rl.Mol. wts. (x 10-3) of protein standards and EGF receptor-related proteinsare indicated.

of the 160 000 polypeptide.Limit endoglycosidase H digestion indicates that the mol.

wts. of the peptide precursors which lack N-linked oligosac-charides are 138 000 and 68 000 respectively for the 160 000and 95 000 mol. wt. receptor-related polypeptides. WhenA431 cells were pre-treated with 5 -10 ytg/ml of tunicamycinfor 2 h which inhibits addition of all N-linked oligosaccharidechains and then labelled with [35S]methionine for 4 h, twopolypeptides of mol. wts. 138 000 and 68 000 could be im-munoprecipitated by monoclonal RI (Figure 5).

534

E.L.V.Mayes and M.D.Waterfield

A

175 -_160-

C u

95-..

-200

-116

- 92'5. .. _ s"u . . ............. u,,, a., .. B

Biosynthesis of the EGF receptor in A431 cells

A.

175-9.1609-* wwryw

115 -

95~..9**SO*O*

1 2 3 4 5 6 7 8 9

C.

1 2 3 4 5 6 7 8 9

2 3 4 5 6 7 8 9

D.

2 3 4 5 - 7 8 9

Fig. 6. The effect of monensin on biosynthesis of EGF receptor-related polypeptides analyzed by a pulse-chase experiment. 2 x 106 A431 cells in lowmethionine DMEM 57o heat-inactivated, dialyzed FCS were pretreated for 2 h in the presence (C and D) or absence (A and B) of 1 /M monensin prior tolabelling with 250 1Ci/ml [35S]methionine for 5 min. The medium was replaced with DMEM plus or minus monensin and the label chased for various timesprior to lysis. RI immunoprecipitates of the lysates (A and C) or growth medium (B and D) were analyzed on a 707o polyacrylamide SDS gel followed byautoradiography. Chase times were as follows: lane 1, 0 min; lane 2, 15 min; lane 3, 30 min; lane 4, 45 min; lane 5, 60 min; lane 6, 90 min; lane 7, 120 min;lane 8, 165 min; lane 9, 210 min. Mol. wt. (x 10-) of protein standards and EGF receptor-related proteins are indicated.

The effects of monensin on biosynthesis of EGF receptor-related polypeptidesMonensin inhibits glycoprotein transport in the Golgi mem-branes and thus interferes with the complexing of N-linkedoligosaccharide chains. Experiments were carried out in thepresence or absence of monensin using A431 cells pulse-labelled with [35S]methionine for 5 min and then chased forvarious times up to 210 min. Immunoprecipitates of A431cell lysates and growth medium made with monoclonal RIwere analysed by SDS-polyacrylamide gel electrophoresisfollowed by autoradiography of the gel (Figure 6); the radio-activity present in each polypeptide was estimated by densi-tometric scanning of the autoradiograph. After the 5 minpulse-label both the 95 000 and 160 000 mol. wt. polypeptideswere labelled. As the chase time was increased, the label inthese polypeptides decreased and label appeared in themature receptor (175 000 mol. wt.) and 210 min after thestart of the chase period this was the major labelled polypep-tide immunoprecipitated (Figure 6A). The ratio of radio-activity in the 160 000 mol. wt. polypeptide to that in the

95 000 mol. wt. polypeptide was constant throughout thechase period while the total label in these two polypeptidesdecreased as label accumulated in the mature receptor. In thepresence of monensin the mature receptor was not formedand increased amounts of radioactivity were found in the160 000 mol. wt. polypeptide compared with the 95 000 mol.wt. polypeptide (Figure 6C). This suggests that the 160 000mol. wt. polypeptide, which accumulates in the Golgi com-plex in the presence of monensin, is a precursor of the175 000 mol. wt. mature receptor. No clear precursor-product relationship to the 160 000 mol. wt. polypeptide canhowever be shown for the 95 000 polypeptide.

Immunoprecipitates of the cell growth medium showed thepresence of a 115 000 mol. wt. polypeptide at late chase times(>90 min) (Figure 6B). This polypeptide could be either a

mature form of the 95 000 mol. wt. polypeptide which issecreted after addition of terminal sugars, or a proteolyticdegradation product of the mature cell-surface expressedreceptor. Consistent with either origin, the mol. wt. of thispolypeptide in the presence of monensin is 95 000 (Figure

535

B.

-200

_165155

-116

- 92'5

160-O- *

95-..

0 Jo

- 200

- 165155

-116

-92 5

E.L.V.Mayes and M.D.Waterfield

6D); that is, either the non-complexed 95 000 mol. wt. poly-peptide, or a degradation product of the non-complexed160 000 mol. wt. receptor precursor. The amount of the95 000 mol. wt. polypeptide in the medium was considerablyless than the amount of 115 000 mol. wt. polypeptide ob-served in the absence of monensin.

DiscussionThe 175 000 mol. wt. EGF receptor is the predominant label-led protein immunoprecipitated by monoclonal RI (Water-field et al., 1982) from lysates of A431 cells which have beenlabelled for 18 h with [35S]methionine. When cells were label-led for times as short as 5 min, two polypeptides of apparentmol. wts. 95 000 and 160 000 could be immunoprecipitated.To establish whether both of these polypeptides were

related to the EGF receptor a number of different approacheswere taken, including protease and glycosidase digestion andthe use of inhibitors of glycoprotein biosynthesis. Both lim-ited proteolysis with trypsin and tryptic peptide mapping byreverse-phase h.p.l.c. indicate that these polypeptides are in-deed related to the EGF receptor. In addition, both containcomparable numbers of N-linked high-mannose chains, anddiffer from the mature EGF receptor which contains pre-dominantly complexed N-linked chains. In contrast to the160 000 mol. wt. polypeptide and the mature receptor, the95 000 mol. wt. polypeptide appears to lack the cleavage sitefor the calcium-dependent protease as well as at least onetryptic peptide observed in the h.p.l.c. peptide map. Pulse-chase studies indicate that the 160 000 mol. wt. polypeptide,and not the 95 000 mol. wt., is the immediate precursor of the175 000 mol. wt. EGF receptor.The experiments reported here do not show whether any

0-linked oligosaccharides are added to the receptor-relatedpolypeptides. However, preliminary experiments using bothneuraminidase and N-acetyl galactosamine oligosaccharidase(which would be expected to remove 0-linked oligo-saccharides) show that neither enzyme alters the apparentmol. wt. of the precursor polypeptides observed in tunica-mycin treated cells (138 000 and 68 000 mol. wt.). This sug-gests that 0-linked oligosaccharides do not make a significantcontribution to the apparent mol. wt. of the EGF receptor.

These results together show that in A431 cells biosynthesisof the EGF receptor involves co-translational addition ofseven or more N-linked high-mannose oligosaccharide chainsonto a polypeptide of apparent mol. wt. 138 000 to form a160 000 mol. wt. intermediate. The majority of these oligo-saccharide chains are subsequently modified by addition ofterminal sugars (including fucose and sialic acid) to give themature 175 000 mol. wt. form of the receptor. Recent studieson the insulin receptor (Kasuga et al., 1982; Deutsch et al.,1983; Jacobs et al., 1983; Ronnett et al., 1983) suggest thatthe a and,B subunits are synthesized as a single polypeptidechain (180 000- 190 000 mol. wt.) which undergoes similaroligosaccharide processing to that reported here, resulting inaddition of 30 000 apparent mol. wt. of N-linked oligosac-charides. In contrast, the LDL receptor (120 000 mol. wt.)contains only 8000 apparent mol. wt. of N-linked oligosac-charide chains (Tolleshaug et al., 1982).The 95 000 mol. wt. receptor-related polypeptide has a

polypeptide core of 68 000 mol. wt. with up to 11 N-linkedhigh-mannose oligosaccharide chains. Several mechanisms.for the origin of this polypeptide and the II15 000 mol. wt.polypeptide observed in the growth medium can be proposed,

536

all of them consistent with the experimental observations. (i)The 95 000 mol. wt. polypeptide is a proteolytic degradationproduct of the 160 000 mol. wt. precursor. The 115 000 mol.wt. is the corresponding polypeptide from the mature 175 000receptor. (ii) The 95 000 polypeptide is a precursor of the160 000 polypeptide, the conversion perhaps being due torapid addition of 65 000 mol. wt. of polypeptide. The associ-ation of the two polypeptides must involve a linkage resistantto SDS and reducing agent, perhaps covalent as occurs inbacteriophage X head assembly (Hendrix and Casjens, 1974).Proteolysis of the mature receptor gives rise to the 115 000polypeptide observed in the growth medium. (iii) The 95 000polypeptide is translated from a truncated form of themRNA coding for the 160 000 polypeptide which lacks theregion coding for a domain that includes the transmembraneregion. Both the 95 000 and the 160 000 mol. wt. polypep-tides undergo addition of terminal sugars to some of theN-linked oligosaccharide chains producing respectively the115 000 and 175 000 mol. wt. polypeptides. The 115 000 mol.wt. polypeptide is subsequently secreted into the growthmedium whilst the mature 175 000 mol. wt. receptor is trans-ported to the cell surface. The gene coding for the EGF re-ceptor is located on chromosome 7 (Davies et al., 1980;Waterfield et al., 1982). Since A431 cells are known to haveboth normal and translocated chromosome 7s (Shimizu andKondo, 1982; Gill et al., 1983), it is possible that the trunc-ated mRNA may be derived from one or more of additionalcopies of the receptor gene. Alternatively, the two mRNAspecies may arise by differential RNA processing, as observedfor the membrane-bound and secreted forms of the heavy im-munoglobulin chains (Early et al., 1980; Maki et al., 1981;Rogers et al., 1981; Cheng et al., 1982; Tyler et al., 1982;Word et al., 1983).We are now using other human cells in an attempt to deter-

mine whether the 95 000 mol. wt. polypeptide is peculiar toA431 cells. Further clarification of the alternative mechan-isms for the origin of this polypeptide will require structuralanalysis at the nucleic acid level.

Materials and methodsGrowth of cells and monoclonal antibody productionA431 cells (a gift from G. Todaro) were grown in Dulbecco's modified Eagle'smedium (DMEM) containing 10% fetal calf serum (FCS) in an atmosphere of10% C02-90Wo air at 37°C.The monoclonal antibody, RI, was isolated and purified as described in a

previous paper (Waterfield et al., 1982).Biosynthetic labelling of cellular proteinsA431 cells were labelled with [35S]methionine (>800 Ci/mmol, Amersham) inDMEM containing 10% of the normal methionine concentration plus 5%dialyzed FCS(labelling medium) or with L-[6-3H]fucose (20 Ci/mmol, Amer-sham) or D-[2-3H]mannose (5 Ci/mmol, Amersham) in DMEM containing10% of the normal glucose concentration plus 5% FCS. For pulse-chaselabelling experiments the cells were pre-incubated in labelling medium for 2 hprior to the addition of [35S]methionine. After 5 min the labelling mediumwas replaced with DMEM, 10% FCS (dialyzed and heat-inactivated at 56°C).Inhibition of glycosylationTo inhibit N-linked glycosylation, A431 cells were grown in labelling mediumcontaining 5 or 10 itg/ml tunicamycin [diluted from a stock solution dissolvedin dimethyl sulphoxide (DMSO)] and 50 ,M leupeptin (both tunicamycin andleupeptin were purchased from Sigma). To ensure that the pool of lipid-linkedoligosaccharide was depleted, the cells were maintained in the tunicamycin-containing medium for 2 h prior to addition of radioactive label.

Monensin (Calbiochem) was added to labelling medium to a final concen-tration of I itM (diluted from a stock solution dissolved in DMSO), alongwith 50 AM leupeptin. Cells were maintained in this medium for 2 h prior tothe addition of radioactive label.

Biosynthesis of the EGF receptor in A431 cells

Immunoprecipitation of EGF receptor with monoclonal antibodyA43 1 cells were lysed and immunoprecipitated with monoclonal antibody RIas described previously (Waterfield et al., 1982), except that 30 Al of a 10%solution of heat-killed formaldehyde fixed Staphylococcus aureus bacteriawere used instead of protein A-Sepharose beads.Enzymatic digestion of immunoprecipitates of EGF receptorFor digestion with endoglycosidase H, immunoprecipitates were boiled in10 mM Tris HCI pH 6.8, I To SDS for 2 min. The supernatants were dilutedwith 9 volumes of 0.15 M sodium citrate pH 5.5. Endoglycosidase H (NewEngland Nuclear) was added and the solution incubated at 37°C overnight.Proteins were precipitated with 10%7o trichloroacetic acid (TCA), and the pre-cipitates, after washing, boiled in gel loading buffer for 2 min (the pH was ad-justed, if necesssary, with 1 M Tris).

Neuraminidase digestion was carried out as follows. Immunoprecipitateswere boiled in 1%o SDS for 2 min, and the supernatant diluted ten fold with50 mM sodium acetate pH 5.5. Neuraminidase (Boehringer Mannheim) andleupeptin (final concentration 50 14M) were added, and the solution incubatedat 37°C overnight. Proteins were precipitated with TCA and treated asdescribed above.Gel electrophoresisOne-dimensional SDS-polyacrylamide gel electrophoresis on slab gels was car-ried out using the buffer system described by Laemmli (1970). Gels were driedunder vacuum after staining with Coomassie brilliant blue and exposed toKodak X-Omat at - 70°C. For visualization of 3H-labelled polypeptides, gelswere pre-treated with 2,5-diphenyloxazole prior to drying as described byBonner and Laskey (1974).H.p.l.c. tryptic peptide maps2 x 107 cells were labelled either with 2.5 mCi [35S]methionine for I h or250 tzCi for 18 h (as described above). Rl immunoprecipitates were dis-sociated in 500 Al of 0.5 M Tris HCI pH 8.5, 6 M guanadine hydrochloride,10 mM dithiothreitol at 37°C for 2 h. 50 ,Ci of iodo-[1-14C]acetamide(>50 mCi/mmol, Amersham International) was added to each supernatant.After 30 min at 4°C, iodoacetamide (recrystallized, Sigma) was added to givea final concentration of 25 mM and the resultant solution incubated for a fur-ther 2 h at 4°C. The samples were dialyzed against 0.1 M ammonium bicar-bonate, and then lyophilized and re-dissolved in gel loading buffer. AfterSDS-gel electrophoresis, the gel was dried without prior staining and auto-radiographed. The labelled bands corresponding to the receptor-related poly-peptides were excised and rehydrated for I h in distilled water. The gel pieceswere extracted 4 times with acetone, and then incubated with 500 yd of 0.1 Mammonium bicarbonate 1 mg/ml TPCK-trypsin (Worthington) at 37°C for18 h. The supernatant was removed and a further 500 pd of 0.1 M ammoniumbicarbonate, I mg/ml TPCK-trypsin added to the gel pieces and incubatedfor 4 h. Peptides were separated by reverse-phase h.p.l.c. on a SynChropakC-18 column (Syn-Chrom, Inc.) with a flow rate of 1 ml/min and a lineargradient of acetonitrile (0.60%o) in 0.1 Wo trifluoroacetic acid over 60 min.Fractions of 500 tl were collected and counted for both 14C and 35S in aBeckman LS700 scintillation counter for 10 min each.

ReferencesAdamson,E.D. and Rees,A.R. (1981) Mol. Cell Biochem., 34, 129-152.Anderson,R.G.W., Brown,M.S., Beisiegel,U. and Goldstein,J.L. (1982) J.

Cell. Biol., 93, 523-531.Anholt,R., Lindstrom,J. and Montal,M. (1984) in Martinosi,E. (ed.), TheEnzymes of Biological Membranes, Plenum Press, NY, 2nd edition, inpress.

Antoniades,H.N. and Williams,L.T. (1983) Fed. Proc., 42, 2630-2634.Baker,J.B., Simmer,R.L., Glenn,K.C. and Cunningham,D.D. (1979) Nature,

278, 743-745.Bonner,W.M. and Laskey,R.A. (1974) Eur. J. Biochem., 46, 83-88.Brown,M.S., Kovanen,P.T. and Goldstein,J.L. (1979) Recent Prog.Hormone Res., 35, 215-257.

Brown,M.S., Anderson,R.G.W. and Goldstein,J.L. (1983) Cell, 32, 663-667.Carpenter,G. (1981) in Baserga,R. (ed.), Handbook of Experimental

Pharmacology, Vol. 57, Springer-Verlag, Berlin, Heidelberg/NY, pp.89-132.

Carpentier,J.L., Gorden,P., Anderson,R.G.W., Goldstein,J.L., Brown,M.S., Cohen,S. and Orci,L. (1982) J. Cell Biol., 95, 73-77.

Cassel,D. and Glaser,L. (1982) J. Biol. Chem., 257, 9845-9848.Cheng,H.-L., Blattner,F.R., Fitzmaurice,L., Mushinski,J.F. and Tucker,P.W. (1982) Nature, 296, 410415.

Clemmons,D.R. and Van Wyk,J.J. (1981) in Baserga,R. (ed.), Handbook ofExperimental Pharmacology, Vol. 57, Springer-Verlag, Berlin, Heidelberg/NY, pp. 161-208.

Cohen.S., Ushiro,H., Stoscheck,C. and Chinkers,M. (1982a) J. Biol. Chem.,

257, 1523-1531.Cohen,S., Fava,R.A. and Sawyer,S.T. (1982b) Proc. Natl. Acad. Sci. USA,

79, 6237-6241.Comens,P.G., Simmer,R.L. and Baker,J.B. (1982) J. Biol. Chem., 257,

42-45.Czech,M.P. (1982) Cell, 31, 8-10.Czech,M.P., Oppenheimer,C.L. and Massague,J. (1983) Fed. Proc., 42,

2598-2601.Das,M. (1983) in Kleinzeller,A. and Martin,B.R. (eds.), Current Topics inMembranes and Transport, Vol. 18, Academic Press, NY/London, pp.381-405.

Davies,R.L., Grosse,V.A., Kucherlapati,R. and Bothwell,M. (1980) Proc.NatI. Acad. Sci. USA, 77, 41884192.

Deutsch,P.J., Wan,C.F., Rosen,O.M. and Rubin,C.S. (1983) Proc. Natl.Acad. Sci. USA, 80, 133-136.

Early,P., Rogers,J., Davis,M., Calame,K., Bond,M., Wall,R. and Hood,L.(1980) Cell, 20, 313-319.

Fabricant,R.N., DeLarco,J.E., and Todaro,G.J. (1977) Proc. Natl. Acad.Sci. USA, 74, 565-569.

Gill,G.N., Lazar,C.S., Shimizu,N. and Gamou,S. (1983) Clin. Res., 31,A470.

Goldstein,J.L. and Brown,M.S. (1977) Annu. Rev. Biochem., 46, 897-930.Hendrix,R.W. and Casjens,S.R. (1974) Proc. Natl. Acad. Sci. USA, 71,

1451-1455.Jacobs,S., Kull,F.C., and Cuatrecasas,P. (1983) Proc. Natl. Acad. Sci. USA,

80, 1228-1231.Kasuga,M., Hedo,J.A., Yamada,K.M. and Kahn,C.R. (1982) J. Biol.

Chem., 257, 10392-10399.Klenk,H.-D. and Rott,R. (1980) Curr. Top. Microbiol. Immunol., 90, 19-48.Laemmli,U.K. (1970) Nature, 227, 680-685.Linsley,P.S., Blifeld,C., Wrann,M. and Fox,C.F. (1979) Nature, 278, 745-

748.Linsley,P.S. and Fox,C.F. (1980) J. Supramol. Struct., 14, 461-471.Maki,R., Roeder,W., Traunecker,A., Sidman,C., Wald,M., Raschke,W.

and Tonegawa,S. (1981) Cell, 24, 353-365.Omary,M.B. and Trowbridge,I.S. (1981) J. Biol. Chem., 256, 12888-12892.Owen,M.J., Kissonerghis,A.-M. and Lodish,H.F. (1980) J. Biol. Chem., 255,

9678-9684.Rogers,J., Choi,E., Souza,L., Carter,C., Word,C., Kuehl,M., Eisenberg,D.and Wall,R. (1981) Cell, 26, 19-27.

Ronnett,G.V., Knutson,V.P. and Lane,M.D. (1983) Fed. Proc., 42, 1789.Schneider,C., Sutherland,R., Newman,R. and Greaves,M. (1982) J. Biol.

Chem., 257, 8516-8522.Shimizu,N. and Kondo,I. (1982) Cytogenet. Cell Genet., 32, 316.Struck,D.K. and Lennarz,W.J. (1977) J. Biol. Chem., 252, 1007-1013.Tartakoff,A.M. (1983) Cell, 32, 1026-1028.Tolleshaug,H., Goldstein,J.L., Schneider,W.J. and Brown,M.S. (1982)

Cell, 30, 715-724.Tyler,B.M., Carman,A.F., Gerondakis,S.D., Adams,J.M. and Bernard,O.

(1982) Proc. Natl. Acad. Sci. USA, 79, 2008-2012.Waterfield,M.D., Mayes,E.L.V., Stroobant,P., Bennett,P.L.P., Young,S.,

Goodfellow,P.N., Banting,G.S. and Ozanne,B. (1982) J. Cell, Biochem.,20, 149-161.

Westermark,B., Heldin,C-H., Ek,B., Johnsson,A., Mellstrom,K., Nister,M.and Wasteson,A. (1983) in Guroff,G. (ed.), Growth and MaturationFactors, Vol. 1, Wiley and Sons, pp. 73-115.

Word,C.J., Mushinski,J.F. and Tucker,P.W. (1983) EMBO J., 2, 887-898.Wrann,M.M. and Fox,C.F. (1979) J. Biol. Chem., 254, 8083-8086.Yeaton,R.W., Lipari,M.T. and Fox,C.F. (1983) J. Biol. Chem., 258, 9254-

9261.

Received on 7 November 1983; revised on 16 December 1983

537