Glycosylation of the Epidermal Growth Factor Receptor in A-431 Cells

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 19M by The Amerian Soiety of Biologial Chemists, In Vol. 259, No. 20, Issue of Otober 25, pp ,1984 Printed in U. S. A. Glyosylation of the Epidermal Growth Fator Reeptor in A-431 Cells THE CONTRIBUTION OF CARBOHYDRATE TO RECEPTOR FUNCTION* (Reeived for publiation, January 16, 1984, and in revised form, July 6, 1984) Ann Mangelsdorf SoderquistSg and Graham CarpenterSllIl From the $Department of Biohemistry and lldiuision of Dermatology, Vanderbilt University Shool of Mediine, Nashville, Tennessee A-431 ells were treated with inhibitors of either N- linked glyosylation (tuniamyin or gluosamine) or of N-linked oligosaharide proessing (swainsonine or monensin) to examine the glyosylation of epidermal growth fator (EGF) reeptors and to determine the effet of glyosylation modifiation on reeptor funtion. The reeptor was found to be an M, = 130,000 polypeptide to whih a relatively large amount of ar- bohydrate is added o-translationally in the form of N- linked oligosaharides. Proessing of these oligosaharides aounts for the 10,000-dalton differene in eletrophoreti migration between the M, = 160,000 preursor and M, = 170,000 mature forms of the reeptor. No evidene was found for 0-linked oligosaharides on the reeptor. M, = 160,000 reeptors resulting from swainsonine or monensin treatment were present on the ell surfae and retained full funtion, as judged by 1-EGF binding to intat ells and detergent-solubilized extrats and by in vitro phosphoryla- tion in the absene or presene of EGF. On the other hand, when ells were treated with tuniamyin or gluosamine, ligand binding was redued by more than 50% in either intat ells or solubilized ell extrats. The M, = 130,000 reeptors synthesized in the presene of these inhibitors were not found on the ell surfae. In addition, no M, = 130,000 phosphoprotein was deteted in the in vitro phosphorylation of tuniamyin or gluosamine-treated ells. It appears, therefore, that although terminal proessing of N- linked oligosaharides is not neessary for proper transloation or funtion of the EGF reeptor, the addition of N-linked oligosaharides is required. EGF exerts its mitogeni effet on target ells by interating with speifi ell surfae reeptors (1). Sine the omplex sequene of events that take plae between EGF binding to * This work was supported by Researh Grant CA24071 from the National Caner Institute and BC294B from the Amerian Caner Soiety. A portion of this work was presented at the Annual Meeting of the Amerian Soiety for Cell Biology, San Antonio, TX, Nov. 29- De. 3, The osts of publiation of this artile were defrayed in part by the payment of page harges. This artile must therefore he hereby marked advertisement in aordane with 18 U.S.C. Setion 1734 solely to indiate this fat. Supported in part by National Caner Institute Training Grant CA Reipient of an Established Investigator Award from the Amerian Heart Assoiation. The abbreviations used are: EGF, epidermal growth fator; SDS, sodium dodeyl sulfate; Endo H, endo-p-n-aetylgluosaminidase H; DMEM, Dulbeo s modified Eagle s medium; Met- MEM, methionine-free modified Eagle s medium; ConA, onanavalin A; Hepes, 4- (2-hydroxyethyl)-l-piperazineethanesulfoni aid. its reeptor and DNA synthesis has been diffiult to unravel in whole ells, onsiderable researh effort has been onentrated on the biohemial properties of the reeptor. Progress in this area has been greatly failitated by the disovery that A-431 ells, an epidermoid arinoma ell line, have approximately 2 X lo6 reeptors/ell (2). Using these ells, it has been possible to study the funtional aspets of the reeptor in situ and to purify suffiient reeptor to allow biohemial analyses to be performed in vitro. As a result of these studies, an M, = 170,000 protein has been identified that has both lz5i-egf binding and EGF-dependent protein kinase ativities and that ats a as substrate for EGF-stimulable phosphorylation (as reviewed in Refs. 1 and 3). Several lines of indiret evidene have suggested that the EGF reeptor is a glyoprotein. These inlude dereased lz5i- EGF binding to glyosylation defetive ells (4), the inhibition of lz5i-egf binding to fibroblasts by letins (5), and the speifi binding of affinity-purified EGF reeptor to lentilletin Sepharose (6). In the present study, glyosylation of the reeptor from A-431 ells has been demonstrated diretly by inorporation of radiolabeled monosaharides into the immunopreipitable M, = 170,000 reeptor, and by arbohydrate-speifi binding of onanavalin A to immuno-isolated reeptors transferred to nitroellulose. A large number of proteins in euaryoti ells have at least some arbohydrate ovalently attahed to them (7); however, the role of arbohydrate in protein funtion remains obsure. With the availability of inhibitors of various steps in the glyosylation of proteins, it is possible to produe glyosylation modified proteins within ells and to examine th effet of hanges in glyosylation on protein loalization and/or funtion. N-Linked glyoproteins are synthesized by the sequential addition of 2 N-aetylgluosamine, 9 mannose, and 3 gluose residues to a dolihol phosphate intermediate, followed by the o-translational transfer of these high mannose ore oligosa- harides to asparagine residues of nasent polypeptide hains (7). Tuniamyin inhibits the addition of the first N-aetylgluosamine residue to the lipid intermediate, thereby preventing the N-glyosylation of proteins (8). N-Glyosylation is also inhibited by gluosamine, although the mehanism of this inhibition is not known (9). One attahed to proteins, the high mannose oligosaharide ores an undergo extensive proessing. This inludes removal of gluose and several of the mannose residues, and the addition of terminal sugars, e.g. N-aetylgluosamine, galatose, fuose, and siali aid, that are harateristi of omplex oligosaharides (10). Golgi mannosidase I1 is responsible for the removal of mannose residues from one of the branhes of the high mannose ore (11). Swainsonine inhibition of this enzyme prevents proessing of this branh

2 and an result in abnormal retention of high mannose oligosaharides on proteins (12). Alternatively, proessing of the other of the mannose branhes an ontinue, resulting in hybrid N-linked oligosaharides (13). The effet of monensin on intraellular transport is well doumented (14), but this ionophore has also been reported to inhibit the addition of terminal sugars to the N-linked oligosaharides of glyoproteins (15). The initial N-aetylgalatosamine of oligosaharides having 0-linkages to either serine or threonine residue is added to proteins post-translationally within the Golgi (16, 17). Further proessing of 0-linked oligosaharides, inluding hain elongation and the addition of terminal sugars, also takes plae in the Golgi (17). Unfortunately, there are no means omparable to those available for N-linked oligosaharides for perturbing 0-linked oligosaharides and subsequently studying the modified proteins. In the present study, N-linked oligosaharides were found to be largely, if not entirely, responsible for the glyosylation of the EGF reeptor in A431 ells. No evidene was found for oligosaharides having 0-linkages. Therefore, inhibitors of N-linked glyosylation were used to study the effet of arbohydrate modifiation on the loalization, ligand binding, and phosphorylation ativities of the reeptor. EXPERIMENTAL PROCEDURES ~ater~~-tuniamyin and monensin were purhased from Calbiohem-Behring, and gluosamine hydrohloride was obtained from Sigma. Swainsonine was a kind gift from Dr. Harry Broquist, Vanderbilt University. ~[~'SIMethionine (600 Ci/mmol), D-[6-3H]gluosamine hydrohloride (20-40 Ci/mmol), and ~-[2-~H]mannose (10-20 Ci/mmol) were from Amersham; "'I-protein A ( pci/pg) and '251-onanavalin A (30-40 pci/fg) were from New England Nulear; and [y-32p]atp(20-30 Cilmmol) was from ICN. EGF was generously supplied by Dr. Stanley Cohen, Vanderbilt University, and "9-EGF was prepared by the hloramine-?' method (18). Rabbit anti-egf reeptor serum (No. 9861, raised against affinity purified reeptor, has been desribed previously (19). Formalin-fixed Staphyloous aurew ells (Immunopreipitin) were obtained from Bethesda Researh Laboratories, and Endo H was purhased from Miles Laboratories. Eletrophoreti hemials and unlabeled moleular weight standards were from Bio-Rad, and I4C-1abeled moleular weight standards were purhased from Bethesda Researh Laboratories. Enlightening was from New England Nulear, and X-Omat film was purhased from Kodak. Cell Culture, Inh~bitor Trea~men~, Meta~~i and Labeling-A431 stoks were maintained in, and all long-term labeling and inhibitor treatments were arried out in, DMEM plus 10% alf serum, supplemented with Garamyin (M. A. Bioproduts) at 50 pg/ml. Cells were plated out into 35-mm ulture dishes, 24-well plates, or 96-well plates as indiated in eah proedure. Inhibitor treatment and/or metaboli labeling was begun not less than 48 h after plating, when the ells were eit.her just onfluent or approximately 75% onfluent, depending on the intended length of the experiment. When ells were labeled in the presene of inhibitors, they were pretreated at least 30 min prior to the addition of label. In the pulse-hase experiments, ell monolayers were washed twie and refed with methionine-free modified Eagle's medium k tuniamyin. Labeling with [35S]methionine was for the time indiated in the figure legend. Cells were either solubilized immediately, or were washed twie and inubated in fresh Dulbeo's medium + 10% alf serum & tuniamyin for various times before solubilization. inhibitor onentrations were as follows: 2 pg/ml tuniamyin, 1 pg/mi swainsonine, 1 pm monensin, and 10 mm gluosamine. Detergent Solubilization of Cells-For all experiments requiring detergent solubilization, ells were washed four times at 5 "C with alium- and magnesium-free phosphate-buffered saline and were solubilized at 2 X lo6 ells/ml in TGP buffer (1% Triton X-100, 10% glyerol, 20 mm Hepes, ph 7.4,l mm phenylmethylsulfonyl fluoride). Inubation was for 20 min at room temperature. The extrats were used without entrifugation. I ~ ~ u ~ ~ gand i ~ u~~~phore~i l Proedures-Immunopreipitation was performed by adding either normal rabbit serum or anti- EGF Reeptor Glyosylution reeptor serum to aliquots of detergent-solubilized ell extrats at a ratio of 1:lO (v/v). After a 15-min inubation at room temperature, a 10% suspension of S. ourew ells at a volume equal to that of the ell extrat was added. Inubation was ontinued for 15 min at room temperature. The S. aurew ells were pelleted by entrifugation and washed four times with 37 "C RIPA buffer (1% Triton X-100, 1% deoxyholate, 0.1% SDS, 50 mm Tris, ph 8.4, 150 mm NaCl, 0.02% sodium azide). The immune omplexes were dissoiated from the S. aurew ells by boiling 2 min in Laemmli sample buffer (ZO), and eletrophoresis was arried out on 7.5% SDS-polyarylamide gels (20). After eletrophoresis, gels ontaining 3H- or 35S-labeled proteins were treated for fluorography, either aording to Bonner and Laskey (21) or with Enligh~ning, aording to the m~ufa~urer's instrutions. Gels ontaining 3ZP-labeled proteins were first stained with Coomassie. In eah ase, the dried gels were exposed to film at -70 "C in the presene of intensifying sreens. For immunodetetion and onanavalin A binding of reeptors, 100 pl of unlabeled ell extrats were preipitated by inubation with 1 ml of 0 "C aetone for 1 h or were immunopreipitated as above. The preipitates were run on polyarylamide gels and the proteins were transferred eletropho~tially to nitroellulose filters. Transfer was at 100 ma for 18 h in 25 mm Tris, 192 mm glyine at ph 8.2. The filters were ut into five strips, eah having idential samples transferred to it. One nitroellulose strip, ontaining unlabeled moleular weight markers in addition to samples, was stained briefly with Amido Blak. The other four filter strips were first inubated in a buffer ontaining 3% bovine serum albumin (22) to blok nonspeifi binding to the nitroellulose. They were then inubated either in 5 ml of a 1:2000 dilution of antireeptor serum followed by 2 ml of 12'I-protein A (10' pmlml) or in 2 ml of 'Z'I-onanavalin A (lo6 pmlml). Control strips were inubated either in a 1:2000 dilution of normal rabbit serum followed by '251-protein A or in 1*51-onanavalin Apreinubated with 0.1 M or-methylmannoside. All antiserum, protein A, and onanavalin A inubations were arried out in SDS-Triton washing buffer (50 mm Tris-HCI, ph 7.4, 5 mm EDTA, 150 mm NaCl, 0.25% gelatin, 0.5% Triton X-100, 0.1% sodium dodeyl sulfate), and eah inubation was for 1 h at room temperature. The filters were washed extensively in several hanges of buffer before being dried and exposed to film at -70 "C with intensifying sreens. antit tat ion of the immun~e~ted reeptor forms was by densitometer sanning of the autoradio~aphs. Endoglyosidase Treatment-For endoglyosidase H digestion, ells grown in 35-mm dishes were labeled with [35S]methionine for either 1 h in the absene of inhibitors or for 24 h in the presene or absene of tuniamyin or swainsonine. Reeptors were immunopreipitated from tripliate aliquots of eah ell extrat. Endo H digestion of reeptor forms from 24-h labeling rt inhibitors was done diretly on washed S. uureus pellets. The immunopreipitates from 1-h labeled ells were first eletrophore~d on SDS gels, then the M, = 160,~O bands were ut from the gel, the protein was eluted into alium- and magnesium-free phosphate-buffered saline overnight at 4 "C, and the eluted protein was preipitated with old aetone. For eah set of S. aurew pellets or aetone preipitates, one was resuspended in Laemmli sample buffer and stored at -20 "C, a seond was inubated for 18 h at 37 "C in 50 pl of buffer (0.1 M sodium itrate, ph 6.0,0.1% SDS), and the third was inubated in buffer plus 5.0 munits of Endo H. The inubated samples were mixed with 50 pl of double-stren~h Laemmli sample buffer and were boiled 2 min along with the stored, uninubated samples. Samples were analyzed by SDS-polyarylamide gel eletrophoresis. Inubation in buffer alone had no effet on the eletrophoreti migration of reeptor protein. lz5i-egfbinding to Whole Cells and Cell Extra~ts-~~~I-EGF binding to whole A-431 ells was performed as in Haigler et al. (23). Cells were grown in 24-well plates in the absene or presene of inhibitors. For eah treatment, 25 ng of '*'I-EGF was added to tripliate wells ontaining 250 pi of binding medium; for Sathard analysis (24, between 0.25 and 50 ng '"1-EGF were added per well. Inubation was for 1.5 h at 4 "C. Nonspeifi binding of labeled ligand was measured by adding at least a 40-fold exess of unlabeled EGF to one of the three wells for eah treatment. Y-EGF binding to solubilized extrats of unlabeled, inhibitortreated ells followed the proedure of Carpenter (25). Briefly, 20-pl aliquots of TGP extrats (representing 40,000 ells) were inubated 15 min at room temperature with 100 ng of T-EGF in the absene or presene of a 250-fold exess of unlabeled EGF. The ligandreeptor omplexes were then preipitated with 8.5% polyethylene glyol, the preipitates olleted on ellulose aetate filters, and the

3 12588 Glyosylution EGF Reeptor amount of radioativity present was determined in a y ounter. A. B. Identifiation of Modified Reeptor Forms on the Cell SurfaeCells were grown in 96-well plates in DMEM + 10% alf serum and l ad b a b d e f were labeled for 24 h with 5 pci of [%S]methionine in the absene or presene of inhibitors. Wells, eah representingells from apartiular treatment, were then washed twie to remove the alf serum andwere refed with100 pl of serum-free DMEM a t room temperature. T o isolate EGF reeptors present on the ell surfae, a saturating amount of antireeptor serum was added to eahwell and inubated a t room temperature for 30 min. The ells werethen washed 4 times withold alium- and magnesium-free phosphate-buffered saline to remove unbound antiserum andwere solubilized 20 min a t room temperature in 100 pl of TGP. The solubilized extrats were transferred to tubes omplexes, and S. aureus ells were added to preipitate the immune whih ontained labeled EGF reeptors that were present on the surfae of the intat ells. The reeptors remaining in the supernatants from these "surfae" immunopreipitates were isolated by the addition of antireeptor serum ands. aureus ells to the supernatants and olletion of the preipitates by entrifugation. All S. aureus pellets were then washed as usual and analyzed on Laemmligels. T o show the speifiity of thesurfaeimmunopreipitation,normal rabbit serum was added to a seond set of untreated and inhibitor-45 treated ells and the immunopreipitateswere proessed as above. I n Vitro Phosphorylation and Irnrnun~preipitation-[y-~'P]ATP phosphorylation of unlabeled, TGP-solubilized ells was performed FIG. 1. Carbohydrate inorporation into the EGF reeptor. as in Cohen etal. (26). The 60-pl reation mixture ontained 20 pl of A, A-431 ells were labeled for 24 h with either [%]methionine (lanes ell extrat (representing 40,000 ells) from ontrol and 48-h inhibi- a and d), [3H]gluosamine (lanes b and e ), or [3H]mannose (lanes tor-treated ells, 20 mm Hepes, ph 7.4, 2 mmmnc12, 20 pm ortho- and f 1. Immunopreipitation and SDS-gel eletrophoresis were pervanadate, and 15 p~ [y-32p]atp (2 pci). For EGF stimulation of formed as desribed under "Experimental Proedures." Immunoprephosphorylation, the extratswere preinubated with 100 ngof EGF ipitations were either with nonimmune (lanes a-) or antireeptor for 15 min a t 25 "C. The mixture was then hilled to 0 "C before ATP (lanes d-f) serum. B, aliquots of unlabeled extrats from untreated was added. Inubation of the phosphorylation was for 15 mina t 0 "C A-431 ells were either aetone-preipit.ated (lanes a and ) or imand was stopped by the addition of 200 pl of RIPA buffer ontaining munopreipitated (lanesb and d),eletrophoresed on SDS-polyaryl10 mm pyrophosphate. The phosphorylated reeptors were immuno- amide gels, and transferred eletrophoretially to nitroellulose filpreipitated asabove, run on gels, and visualized by autoradiography. ters. EGF reeptors were deteted with antireeptor serum followed To determine the effet of phosphate inorporation on the eletro- by lz51-proteina (lanes a and b) or with 12sI-onanavalin A (lanes phoretimigration of reeptors, 20 pl of ['lss]methionine-labeled and d). The moleular weight standards are myosin ( M, = 200,000), extrats from ontrol and 24-h tuniamyin-treated ells were sub- phosphorylase b ( M, = 92,500), bovine serum albumin ( M, = 68,000), = 45,000). jeted to the samevitro in phosphorylation onditions as above, exept and ovalbumin (M, in the presene of 15 P M unlabeled ATP. Immunopreipitation and TABLE I eletrophresis were as above. For omparison, the [%]methioninelabeled, phosphorylated reeptors from eah extrat were run alongeffet of glyosylatwn inhibitors on the inorporation of radiolabeled sideimmunopreipitates of unphosphorylated,[35s]methionine-lamethionine or monosaharides into trihloroaeti aid-insoluble material in A-431 ells beled extrats. Cells were grown in 24-well plates in the absene or presene of RESULTS inhibitors and were labeled for 48 h with 1 pci/ml [%S]methionine, 5 pci/ml [3H]gluosamine, or 5 pci/ml of [3H]mannose. Dupliate Glyosylation of the EGFReeptor-To demonstrate diretly wells were solubilized in 1.0 ml of N NaOH, and the labeled proteins arbohydrate inorporation into the EGF reeptor, A-431 ells were trihloroaeti aid preipitated, olleted on glass fiber filters, were metabolially labeled with [3HJgluosamineor ["]manand ounted in a sintillation ounter. The amount of label inorpoof inhibitors was normalized to ounts/min/ell nose for 24 h. Fig. l. 4 shows the inorporationof both arbo- rated in the presene hydrate labels, as well as [3sSS]methionineinto the immuno- and was ompared to the inorporation into untreatedontrols. The values represent the means of a t least three experiments with the preipitated M, = 170,000 reeptor. The glyoprotein nature range from these experimentsgiven in parentheses. 1 of the reeptor was also demonstrated by 1251-onanava1inA binding to isolated reeptor transferred from SDS-polyarylamide gels to nitroellulose (Fig. 1B). A single M, = 170,000 band was visualized by immunodetetion with antireeptor serum followed by "'I-protein A in both thewhole ell extrat (lane a) and the immunopreipitation of ell extrat (lane b). This M, = 170,000 reeptor band ould also be visualized with "'1-ConA. The ConA bound to several proteins in the whole ell extrat (lane ), but only to the M, = 170,000 and two other proteins ( M, = 160,000 and 90,000) in the transferred immunopreipitate (lane d). No l'sii-labeled bands were deteted when dupliate strips were inubated in either normal rabbit serum prior to '251-protein A or in "'I-ConA preinubated with a-methylmannoside. Neither the M, = 160,000 nor the M, = 90,000 protein is immunodeteted in A-431 extrat transferred to nitroellulose, although both proteins are struturally related to the mature M, = 170,000 reeptor (data not shown). Low levels Inhibitor Tuniamyin Swainsonine Monensin Gluosamine % of inorporation into ontrol ells I%IMethionine 82 (70-99) 94 (78-107) 99 (84-107) 80 (57-105) I3H1Gluossmine PHlMannose 24 (18-28) 98 (78-114) 90 (82-106) 3 (2-5) 18 (11-21) 99 (78-126) 109 ( ) 18 (11-30) of the M, = 160,000 protein in A-431 ells' may explain its lak of detetion. The M, = 90,000 protein, on the other hand, is present in suffiient quantities to be deteted, but loses antigeniity upon denat~ration.~ Table I shows the effet of N-linked glyosylation inhibitors onthe inorporation of ["SJmethionine, [3HH]gluosamine, and ['HHJmannose into trihloroaeti aid-preipitable ma- 'Stoshek, C., Soderquist, A. M., and Carpenter, G. (1984) Endorinology, in press. 'A. M. Soderquist, personal observation.

4 EGF Reeptor Glyosylation terial in these ells. After 48 h of treatment, none of the a b d e inhibitors signifiantly affeted protein synthesis, as judged by inorporation of ["S]methionine into total ell protein. However, bothtuniamyinand gluosamine dramatially redued (greater than75%) the inorporationof labeled sugars into trihloroaeti aid-preipitablemaromoleules. Swainsonine and monensin, on the other hand, had little effet on the inorporation of mannose or gluosamine into total ellular protein. Immunopreipitation of reeptors from A-431 ells labeled for 24 h with either [3sS]methionine or [3H]gluosamine (or ['Hlmannose, data not shown) revealed the effet of eah inhibitor on eletrophoreti migration of the reeptor and inorporation of arbohydrate into the reeptor(fig. 2). [35S] Methionine labeling of the ells in the presene of either tuniamyin orgluosamine resulted ina immunopreipitable reeptor of approximately M, = 130,000. As expeted, arbohydrate label was not detetable in immunopreipitates from ells treated with either of these inhibitors. However, [3H] gluosamine and[3h]mannoseas well as [''S]methionine-45 labeled reeptor of M, = 160,000 was preipitated from ells treated with either swainsoniue or monensin. Sine high mannosen-linked oligosaharideores are addedo-translationally to proteins, we sought to isolate newly synthesized reeptor protein having only high mannose FIG. 3. Pulse-hase of A-431 ells. Cells were labeled with [%SI hains attahed.fig. 3 shows that both the M, = 160,000 and methionine for 1 h (lane 6 ) or hased in omplete medium for 1 h 90,000 reeptor forms were speifially immunopreipitated (lane ), 2 h (lane d ), or 4 h (lane e ) following the pulse. Lanes 6-e from ells labeled for 1h with [3sS]methionine. These proteins represent reeptors immunopreipitated with antireeptor serum. were also immunopreipitated from ells labeled for as little Lane a represents an immunopreipitation with normal rabbit serum as 5 min with [3sS]methionine. When ells were hased with from ells labeled for 1 h. fresh medium following the 1-h pulse,a shiftinreeptor migration t o M, = 170,000 was seen. Within 4 h both of the SW T M 1 HR lower moleular weight reeptor speies had disappeared and only the mature M, = 170,000 reeptor was present in the ells. As will be disussed later, the M, = 160,000 reeptor is the preursorof the M, = 170,000 mature reeptor. The M, = 90,000 protein has been studied extensively and is the preur- - a b a e f g h i j D D -45 FIG. 2. Effet of glyosylation inhibitors on EGF reeptor migration and arbohydrate inorporation. EGF reeptors were immunopreipitated from ontrol or inhibitor-treated A-431 ells metabolially labeled for 24 h with either ["S]methionine (lanesa-e) or [3H]gluosamine (lane f-j). Cells were either untreated (lanes a and f ) or treated with 2 pg/ml tuniamyin (lanes 6 and g), 1pg/ml swainsonine (lanes and h), 1p~ monensin (lanes d and i) or 10 mm gluosamine (lanes e and j ). Unless otherwise indiated, the arrowheads on this and other figures designate the positions of the M,= 170,000 (top), M, = 160,000 (middle), and M. = 130,000 (bottom) reeptor speies. FIG.4. Endoglyosidase digestion of glyosylation modified reeptors. Cells were labeled with.[%]methionine for 24 h in the absene of inhibitors (C) or the presene of swainsonine (SW) or tuniamyin ( 7 " ) or were labeled for 1 h in the absene of inhibitor (I HI?). Immunopreipitated reeptors were inubated in either the absene (-) or presene (+) of Endo H, as desribed under "Experimental Proedures" prior to eletrophoresis. sor of a n M,= 110,000 sereted form of the reeptor.' Endoglyosidase H speifially hydrolyzes both high mannose (27), and hybrid (13) N-linked hain from glyoproteins. Endo H treatment of [3sS]methionine-labeled reeptors immunopreipitated from ontrol,tuniamyin-, and swainsonine-treated ells, as well as from ells labeled 1 h with [3sS] methionine, was employed to onfirm the ontribution of N linked oligosaharides to thevarious reeptor forms (Fig. 4). The M, = 130,000 reeptorspeiesfromtuniamyintreated ells was unaffeted by Endo H digestion. The M, = 160,000 reeptor forms from swainsonine-treated ells and from ells labeled for1h with [3sS]methioninewere, however, 'A. M.Soderquist, L.A. Faulkner, C. Stoshek, and G. Carpenter, manusript in preparation.

5 12590 Glyosylation EGF Reeptor only highmannose shifted tom,= 130,000. This onfirms that or hybridoligosaharides are bound to the M, = 160,000 reeptor forms. Digestion with Endo H aused only a slight derease in the apparentmoleular weight of the M,= 170,000 reeptor from ontrol ells, onsistent with its having primarily omplex N-linked oligosaharides. This small shift in migration suggests that the mature reeptor has several Nlinked oligosaharides, some of whih are either high mannose or hybrid. A-431 ells were also labeled with ['H]gluosamine in the presene or absene of swainsonine and the immunopreipitated reeptor was treated with endoglyosidase H. No labeled reeptor speies ould be deteted after Endo H treatment of the M,= 160,000 reeptor from swainsonine-treated ells, while a reeptor band at slightly lessthan M, = 170,000 was deteted when the immunopreipitate from ontrol ells was digested with Endo H. The data from inhibitor and Endo H studies indiate that theegf reeptor from A-431 ells ontainssubstantial amounts of N-linkedarbohydrate. The pulse-hase data suggest that it is the o-translational addition of high mannose hains that is responsible for the bulk of this arbohydrate, sine an Endo H-sensitivereeptor preursor of M, = 160,000 is immunopreipitated from ells labeled for short periods with [3sS]methionine. There is evidene to suggest that no 0-linked oligosaharides are present on the reeptor. No [3H]gluosamine-labeled reeptor ould be deteted in immunopreipitates from ells treated with tuniamyin. This is despite the fat that ells were labeled for 24 h with [3H]gluosamine, whih in A-431 ells is onverted within 20 h to N-aetylgalatosamine and siali aid of the same or nearly the same speifi ativity (28). Sineboth of thesesugarsare ommon in 0-linked oligosaharides, some radioativity would be expeted in the reeptor despite theabsene or removal of N-linked oligosaharides if 0-linked oligosaharides were present. Also, the EGF reeptor is synthesized as a M,= 130,000 protein in the presene of tuniamyin and does not undergo any moleular presene of the inhibitor weight shift duringa 4-h hase in the (Fig. 5). In ontrast, there is a 49,000-dalton shift in eletrophoreti migration of the low density lipoprotein reeptor in A-431 ells despite tuniamyin treatment (28). This shift reflets the addition of terminal sugars to the reeptor's linked oligosaharides. In addition to this indiret evidene, arbohydrate analysis by the methods used to study low density lipoprotein reeptor glyosylation has failed to reveal any 0-linked oligosaharides on the EGF reeptor from A-431 ells.s Based on this ombined evidene, it seems unlikely that 0-linked sugars ould ontribute more than minimally to the glyosylation of the EGF reeptor in these ells. The M, = 130,000 reeptorformshould,therefore, represent theaglyoreeptor. '"I-EGF Binding to Inhibitor-treated Whole Cells-To determine the effet of glyosylation-modifiation on ligand binding, ells were treated with the various inhibitors of Nlinked glyosylation for 24 or 48 h before being assayed for 12'I-EGF binding. The results areshown in Table 11. Binding of T - E G F to swainsonine- or monensin-treated ells was not less than binding to untreated ells whether treatment was for 24 or 48 h. On the other hand, at the end of 24 h of treatment with tuniamyin or gluosamine, ligand binding per ell was approximately 70% of the ontrol value. After 48-h of treatment with these inhibitors, binding was dereased to approximately 40% of that to untreatedells. The half-life of the M, = 170,000 reeptor in A-431 ells is R. D.Cummings, A. M. Soderquist, and G. Carpenter, manusript in preparation. a b d e f g h i j -45 FIG. 5. Reeptor synthesis in the presene of tuniamyin. Reeptors were immunopreipitated from ells labeled with ["SI methionine for 15 min in the presene of tuniamyin (lane d ) or were hased in omplete medium ontaining tuniamyin for 15 min (lane e),30 min (lane f 1, 1 h (lane g), 2 h (lane h ), or 4 h (lane i ). Lane represents reeptors from ells labeled for 15 min in the absene of inhibitor while lane j represents reeptor from ells hased in omplete medium for 4 h. Lanes a and b represent immunopreipitation using normal rabbit serum from ells labeled for 15 min in the absene or presene of tuniamyin, respetively. TABLEI1 Effet of glyosylation inhibitorson lz51-egfbinding to intat A-431 ells Cells were grownfor 48 h in the absene or presene of glyosylation inhibitors before being assayed foriz5i-egf binding as desribed under "Experimental Proedures."The amount of Y - E G F bound to ells of eah treatment was normalized to ounts/min/ell. The values represent the means of at least three experiments (exept as noted) with the range from these exderiments given in Darentheses. Inhibitor 96 of binding to ontrol ells 24-h treatment 48-h treatment 59 (45-67) 35 (20-40) 90 (90)" 108 (99-128) 102 (83-120)" 128 ( ) 63 (50-73) 45 (39-52) The means and ranges are from two experiments. Tuniamyin Swainsonine Monensin Gluosamine approximately 20 h (29) and was found to be unaffeted by any of the glyosylation inhibitors used in this study (data not shown).based on this half-life, approximately 45% of the fully glyosylated M,= 170,000 reeptors synthesized prior to treatment would be present after 24 h of inhibitor treatment and about20% would be present after48 h. Sine EGFbinding to whole ells was not diminished after treatmentwith swainsonine or monensin for either 24 or 48 h, the reeptor forms synthesized in thepresene of these inhibitors must be present on the ell surfae and apable of ligand binding. The progressive derease in binding tuniamyinto and gluosaminetreated ells, ontheotherhand, reflets theturnover of funtional reeptors on theell surfae during inhibitor treatment. While the binding affinities and numbersof reeptors/ ell were similar for ontrol, swainsonine-, and monensintreated ells, Sathard analysis (Fig. 6) revealed drastially redued numbers of funtional reeptors on ells treated with tuniamyin or gluosamine. Asmallpopulation of M, = 170,000 reeptors stillpresent on the ells surfae would aount for the low level of '*'I-EGF binding to these ells,

6 EGF Reeptor Glyosylation '1.0" h '251-EGF Bound (ng/io6mls) ells. Nevertheless, a substantial proportion of the fully glyosylated reeptor from untreated ells is loalized to the ell surfae by this tehnique. The absene of M, = 130,000 reeptor in the surfae immunopreipitation from tuniamyin- and gluosamine-treated ells suggests, therefore, that this nonglyosylated form of the reeptor does not reah the ell surfae. It should be noted that both the M, = 160,000 preursor form of the reeptor and the M, = 90,000 reeptor-related protein are apparent in lane b. The prominent M, = 74,000 protein seen in lanes -f is found in both immune and nonimmunepreipitates from tuniamyin- and gluosaminetreated ells. Although it is present only when N-linked glyosylation is inhibited, it is immunopreipitated nonspeifially. The M, = 68,000 protein seen in lane d, as well as in Figs. 2,4, and5, is speifi and represents thenonglyosylated form of the M, = 90,000 protein that is synthesized in the presene of tuniamyin. FIG.6. Sathard analysisof 1261-ECFbinding to inhibitortreated ells. A-431 ells weregrownfor 48 h in the absene of inhibitors (0) or the presene ofswainsonine (A), monensin (e), lzsi-egfbinding to Extrats of Inhibitor-treated Cellstuniamyin (O), or gluosamine (W). Inubation of ells with 1 Despite theirapparent lak of insertionintothe plasma ng/ml of Y - E G F for 1.5 h at 4 "Cwas done in tripliate. Nonspeifi binding was determined in the presene of at least a 40-fold exess of membrane, the intraellular M, = 130,000 reeptors from unlabeled EGF. tuniamyin- and gluosamine-treated ells ould still be a- pable of ligand binding. To study this possibility, aliquots of detergent-solubilized extrats (eahrepresenting thesame number of ells) from ontrol and48-h inhibitor-treated ells were assayed for '*'I-EGF binding, as desribed under "ExP perimental Proedures." In three experiments, the '*'I-EGF binding ativity in extrats from swainsonine- and monensind treated ells averaged 105 and 8096, respetively, of the ontrol w binding ativity. The '"I-EGF bindingativity present in extrats prepared from tuniamyin- andgluosamine-treated ells, however, averaged only 26 and 13%,respetively, of the ontrol value. Sine the low level of binding ativity present in these extrats an be attributed to native M, = 170,000 reeptors remaining after the 48-h inhibitor treatment, the data suggest that intraellular M, = 130,000 reeptors are not apable of '"I-EGF binding. FIG. 7. Identifiation of surfae reeptors. Cells labeled for Rather than being a true refletion of binding ativity, the 24 h with ["SS]methionine in the absene of inhibitors (C) or the low level of bound '2sII-EGFreovered from tuniamyin and presene of tuniamyin ( 7 " ) or gluosamine ( G N ) were subjeted to surfae (lanes a,, and e ) immunopreipitation with antireeptor gluosamine extrats ould reflet different preipitation efserum, aording to the proedures desribed under "Experimental fiienies of polyethylene glyol for the glyosylated and unproedures."lanes b, d, andf, show the immunopreipitable reeptors glyosylated reeptors. However, in ontrol experimentsusing present in the supernatants from these surfae immunopreipitations. [35S]methionine-labeledell extrats, a greater proportion of The arrowheads designate the positions of the M, = 170,000 and M, = 130,000 reeptor from tuniamyin extrats was preip130,000 reeptors. itated by polyethylene glyol than M, = 170,000 reeptor from ontrol extrats. Theabsene of ligand binding to the unglysuggesting that them, = 130,000 reeptors were either absent osylated reeptor was also indiated by the failure of [?3] from the ell surfae or present butnonfuntional. methionine-labeled M, = 130,000 reeptor to bind to EGFImmunopreipitation of Cell Surfae Reeptors-To deter- Affi-Gel, the matrix used to affinity purify EGF reeptor. mine whether the M, = 130,000 reeptors from tuniamyin Phosphorylation of Glyosylation Modified Reeptors-Auand gluosamine treatment were present on the ell surfae, tophosphorylation and phosphorylation of suitable substrates intat ells were inubated with antireeptor serum and the by the EGF reeptor/kinase is enhaned by EGF binding to immune omplexes were preipitated with S. aurew after the M, = 170,000 reeptor(26). Unlabeled extrats from detergent solubilization of the ells (Fig. 7). ontrol and 48-h inhibitor-treated ells were subjeted to the M, = 170,000 reeptors were identified on the surfae of standard in vitro phosphorylation assay followed by immuuntreated ells (lane a), as were M, = 160,000 reeptors on nopreipitation of the phosphorylated reeptor forms (Fig. the surfae of swainsonine- or monensin-treated ells (data 8A) to determine if the glyosylation modified forms ould not shown). M, = 130,000 reeptors, however, were absent in atasphosphate aeptors. Kinase ativity ould not be the ell surfae immunopreipitations from tuniamyin- or established with ertainty for any of the modified reeptor gluosamine-treated ells. Sine thebasal and lateralsurfaes forms beause immunodetetionrevealed M, = 170,000 reepof ells growing in a monolayer may not be available to tors in all exept the swainsonine extrats (data notshown). antiserum, quantitative preipitation of surfae reeptors is Phosphorylation of the M, = 170,000 reeptor from unnot possible. As is apparent in lane b, more M, = 170,000 treated ells was inreased severalfold in the presene of EGF, reeptor appears in the immunopreipitated supernatant than as antiipated. Thephosphoprotein preipitated from swainin the surfae preipitation, although there is no reason to sonine-treated ells migrated at M, 160,000 and retained its suspet a large intraellular pool of mature reeptors in A- sensitivity to EGF. No M, = 130,000 phosphoprotein was C TM GN a b d e f

7 EGF Reeptor Glyosyhtion ation. Neither basal or EGF-stimulated phosphorylation affeted the migration of the 35S-labeledM, = 170,000or 130,000 C T M SW MN GN C TM reeptor forms. Therefore, phosphate inorporation into M, o-+o-+ = 130,000 reeptors does not explain the appearane of M, = 140,000 phosphoproteins in tuniamyin extrats. In another attempt to determine the nature of the M, = 140,000 phosphoprotein, reeptors were immunopreipitated from unlabeled ontrol and 48-h tuniamyin-treated ells, eletrophoresed on SDS gels, and transferred to nitroellulose (Fig. 9). Immunodetetion with antireeptor serum was used to identify reeptor forms present in eah extrat.only M, = 170,000 reeptor was deteted in untreated ells. The majority of reeptor deteted in tuniamyin-treated ells was M, = 130,000, although M, = 170,000 was also deteted. '251-ConA binding to adupliate nitroellulose strip revealed M, = 170,000, as well as M, = 160,000 and 90,OOO reeptorsin untreated ells. As expeted, unglyosylated M, = 130,000 FIG.8. In vitro phosphorylation and immunopreipitation reeptors were not deteted bycona, but M, = 140,000 of EGF reeptors from inhibitor-treated ells.a, extrats from reeptors were deteted by letin binding to immunopreipiunlabeled ells grown for 48 h in the absene of inhibitor (C), or in tuniamyin ( T M ),swainsonine ( S W ),monensin ( M N ), or gluosa- tates from tuniamyin extrats. These data, along with othwere phosphorylated with[32p]atpin the absene (-) or ers based on [35S]methionine labeling after prolonged tunimine (GN) presene (+) of EGF.Reeptors were then immunopreipitated with amyin treatments (data not shown), suggest that the M, = antireeptorserum. E, [3.5S]methionine-labeledextrats from un- 140,000 protein may represent a partially glyosylated form treated (C) or 24-h tuniamyin-treated( T M ) ells were either im- of the reeptor that is synthesized a t low levels despite tunimunopreipitated (0) or phosphorylated with unlabeled ATP in the amyin treatment. Regardless of the exat nature of the M, absene (-) or presene (+) of EGF before immunopreipitation.the from star indiates the position of the M, = 140,000reeptor speies = 140,000 reeptor form, it is apparent that it is distint the M, = 130,000 reeptor and that it retains ativities that revealed by phosphorylation. are abser?t in the aglyo form of the reeptor. B. A. I I 1 DISCUSSION The experiments reported inthis study have demonstrated o-translational addition of N-linked oligosaharides to the EGF reeptor in A-431 ells and have partially haraterized, C with the use of glyosylation inhibitors, the influene of C arbohydrate on the ellular loalization and biohemial ativities of the reeptor. Sine no evidene ould be found for 0-linked oligosaha rides on the EGF reeptor, the immunopreipitable and imfig. 9. Immunodetetion and '261-ConAbinding to isolated munodetetable M, = 130,000 protein synthesized in the presene of tuniamyin or gluosamine was judged to be the reeptors transferred to nitroellulose. Reeptors wereimmunopreipitated from ontrol (lanes a and ) and 48-h tuniamyin- aglyoreeptor. This onlusion is supported by the absene treated (lanes b and d ) ells, eletrophoresedon SDS-polyarylamide of labeled bandsinimmunopreipitates from [3H]gluosagels, and transferred to nitroellulose. EGF reeptors were deteted mine- or [3H]mannose-labeled ells grown in the presene of with antireeptor serum and '251-protein A (lanes a and b) or with '251-ConA(lanes and d). The arrowheads designate the positions of these inhibitors. After short pulse labeling with [35S]methionine,an M, = the M. = 170,000(top) and M, = 130,000 (bottom) reeptor speies, while the star indiates the position of the M. = 140,000 reeptor 160,000 reeptor having Endo H-sensitive, high mannose Ndeteted with '2SI-ConA. linked hains attahed an be immunopreipitated from A431 ells. Reeptors having the same moleular weights and detetable in immunopreipitates from tuniamyin- or glu- Endo H sensitivity are found in ells treated with either osamine-treated ell extrats, suggesting thatthe M, = swainsonine or monensin. All three M, = 160,000 reeptor forms are shifted to M, = 130,000 byendo H, indiatingthat 130,000 reeptor form does not atas aphosphorylation monensin, as well as swainsonine, prevents proessing of high substrate. An M, = 140,000 phosphoprotein was,however, preipitated from tuniamyin and gluosamine extrats in mannose oligosaharides. Although the 30,000-dalton shift the absene or presene of EGF. Stimulation of the phospho- in eletrophoretimigration resulting from Endo Htreatment rylation of this protein by EGF was not as marked as the may not be a true refletion of the moleular weight of the high mannose hains added to the EGFreeptor, due to the stimulation of phosphorylation of the native reeptor. Sinephosphorylation has been shown to derease the anomalous effets of arbohydrates on protein migration in eletrophoreti migration of proteins on SDS gels (30), the SDS gels (31), it probably indiates that several N-linked possibility that them, = 140,000 protein represents the phos- oligosaharide hainsare presenton eah EGF reeptor phorylated form of the M, = 130,000 reeptor was investi- moleule. The 10,000-dalton differene in eletrophoreti migated. [35S]Methionine-labeled extrats from untreated and gration between the preursor and mature forms of the reeptuniamyin-treated ells were phosphorylated with unlabeled tor ould be due entirely to proessing of high mannose hains. ATP in the presene or absene of EGF, immunopreipitated, The small moleular weight shift seen upon Endo Hdigestion and analyzed by SDS eletrophoresis (Fig. 8B). An aliquot of of the M, = 170,000 reeptor suggests that although most of eah Y3-labeled extrat was also subjeted to immunopreip- the N-linked hains of the mature reeptor are proessed to itation andeletrophoresis without prior in uitro phosphoryl- Endo H resistant, omplex type, one or more remain high

8 mannose. In addition, [3H]fuose is inorporated into the mature reeptor but not the preursor: demonstrating diretly that the presene of terminal sugars is one of the fators responsible for the dereased eletrophoreti migration of the mature reeptor. There is substantial evidene that the high mannose MI = 160,000 forms of the reeptor resulting from swainsonine or monensin treatment retain full funtion. Transloation of both M, = 160,000 reeptors to the ell surfae has been onfirmed by the surfae immunopreipitation tehnique. Also, 'T-EGF binding to whole ells treated for 48 h with swainsonine or monensin or to solubilized ells from the same treatments is equal to or greater than binding to untreated ells. The M, = 160,000 reeptors from both swainsonine- and monensin-treated ells are also utilized as substrates for EGFdependent phosphorylation. Sine a low level of mature M, = 170,000 reeptor is immunodetetable in extrats from monensin-treated ells, it annot be determined if the M, = 160,000 reeptors from this extrat have kinase ativity or simply serve as substrates for kinase ativity in the mature reeptor. The extrats from swainsonine-treated ells, however, ontain no immunodetetable M, = 170,000 reeptors. It is, therefore, possible that the high mannose form of the reeptor from at least the swainsonine-treated ells retains kinase ativity as well as EGF binding ativity and phosphorylation substrate apaity. In addition, A-431 ells exhibit biologial responses to EGF (ie. ell rounding in alium-free medium (32) and inhibition of ell growth (33)) that are retained by ells treated for 48 h with swainsonine. The presene of M, = 160,000 reeptors on the surfae of monensin-treated ells onfirms that terminal proessing of N-linked oligosaharides of the EGF reeptor, rather than intraellular transport, is what is being affeted by the ionophore. Whether transport or oligosaharide proessing is inhibited by monensin seems to be a property of the glyoprotein under investigation. Monensin inhibits the addition of terminal sugars to both the vestiular stomatitis G protein and the influenza virus hemagglutinin glyoprotein in Madin- Darby anine kidney ells (15). However, the ionophore inhibits the appearane of the vestiular stomatitis G protein, but not the hemagglutinin glyoprotein, on the surfae of these ells. Although inhibition of the terminal proessing of N-linked oligosaharides does not alter the transloation or funtional aspets of the EGF reeptor, the inhibition of N-linked glyosylation does. Although turnover of funtional M, = 170,000 reeptors on the ell surfae explains the loss of lz5i-egf binding to tuniamyin- and gluosamine-treated ells, the nearly parallel derease in Iz5I-EGF binding to detergentsolubilized extrats from the ells suggests that M, = 130,000 reeptors do not bind the ligand. Immunodetetion of M, = 170,000 and 130,000 reeptors in extrats of tuniamyintreated ells reveals nearly the same total amount of reeptor as in untreated ontrols, yet Iz5I-EGF binding is only half of what would be expeted from the amount of Mr = 170,000 present in this extrat. The absene of N-linked oligosaharides has an equally dramati effet on phosphorylation of the reeptor as it does on ligand binding. No phosphorylation of a M, = 130,000 reeptor is detetable in tuniamyin or gluosamine extrats whether or not EGF is added to the assay. In tuniamyin extrats, this is despite the fat that nearly half as muh M, = 130,000 reeptor is present as M, 170,000 reeptor present in untreated extrats. The data suggest that the M, = 140,000 protein that appears in extrats from tuniamyin- and gluosamine-treated ells EGF Reeptor Glyosylation may be a partially glyosylated form of the reeptor synthesized despite glyosylation inhibition. If this is indeed what it represents, then the MI = 140,000 reeptor speies is important beause, unlike the aglyoreeptor, it ats as a phosphate aeptor. Further studies are being arried out to eluidate the nature of this protein. Our results suggest that the presene of N-linked oligosaharides is neessary for EGF reeptor funtion. Although N- linked glyoproteins have been reported to retain funtion despite tuniamyin inhibition of glyosylation (34, 35), the funtional aspets of others seem to be impaired in the absene of N-linked oligosaharides (36, 37). It is oneivable that sugar residues are diretly involved in the ligand binding and phosphorylation ativities of the reeptor. It is more probable, however, that the o-translational addition of N- linked oligosaharides is neessary for establishing a funtional onformation of the EGF reeptor, as has been suggested for the aetylholine reeptor (35) and shown more diretly for the G protein of vesiular stomatitis virus (38). Aknowledgments-We are grateful for the exellent tehnial assistane of Connie Moore and Lynn Shaver. Dr. Christa Stoshek is thanked for helpful suggestions, partiularly for suggesting the proedure for immunopreipitation of surfae-loalized EGF reeptors. Note Added in Proof-Sine submission of this manusript, other reports (39-41) overing various aspets of EGF reeptor biosynthesis have appeared. REFERENCES 1. Carpenter, G. (1983) Mol. Cell. Endorinol. 31, Fabriant, R. N., DeLaro, J. E., and Todaro, G. J. (1977) Pro. Natl. Aad. Si. U. S. A. 74, Soderquist, A. 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