Proc. Natl. Acad. Sc. USA Vol. 84, pp. 7832-7836, November 1987 Bochemstry Mechansm of epdermal growth factor receptor autophosphorylaton and hgh-affnty bndng (receptor-tyrosne knases/lgand-nduced receptor dmerzaton) MARIANNE BONI-SCHNETZLER AND PAUL F. PILCH Boston Unversty School of Medcne, Department of Bochemstry, 80 East Concord Street, Boston, MA 02118 Communcated by Stanley Cohen, July 16, 1987 ABSTRACT Epdermal growth factor (EGF) receptor monomers and noncovalently assocated dmers were solated by sucrose densty gradent centrfugaton, and ther respectve bndng and autophosphorylaton actvtes were determned. We fnd that monomers are low-affnty receptors and dmers are hgh-affnt receptors. In the absence of EGF, dmers exhbt a 4-fold hgher autophosphorylaton actvty than do monomers. Addton of EGF ncreases autophosphorylaton on monomers an average of 4.8-fold but has a mnmal effect on autophosphorylaton of dmers. Furthermore, EGF bndng shfts the receptor monomer-dmer equlbrum to the dmer form. We conclude that EGF stmulates n vtro receptor autophosphorylaton by nducng knase-nactve receptor monomers to assocate and form receptor dmers, n whch conformaton the autophosphorylaton actvty s enhanced. Epdermal growth factor (EGF) exerts growth-promotng actvtes n a wde range of epthelal and fbroblastc cells. It s wdely accepted that EGF nduces ts bologcal effects through nteractons wth a specfc cell membrane receptor, the EGF receptor, whch s a sngle-chan, membranespannng proten ofmr 170,000 (1). The bochemcal detals of ths nteracton can be summarzed as frst bndng of EGF to the extracellular porton of the receptor and then rapd phosphorylaton of the cytoplasmc doman of the receptor, whch s the result of the receptor's ntrnsc tyrosne knase actvty (1-5). EGF bndng stes appear to be functonally heterogeneous because a small porton of the EGF receptor bnds lgand wth hgh affnty and a large porton bnds lgand wth low affnty (6, 7). The underlyng structural dfference between hgh- and low-affnty bndng stes has not been descrbed. The analyss of the proten sequence deduced from cdna clonng predcts that the extracellular bndng doman and the cytoplasmc knase doman are connected by a sngle membranespannng sequence (8). Ths structural feature mposes apparent mechanstc constrants on transmembrane knase actvaton and on knase actvaton n purfed preparatons of detergent-solublzed receptors. A possble mechansm could nvolve an assocaton of two or more EGF receptor molecules such that proten-proten nteracton could lead to the conformatonal changes requred for knase actvaton. The potental role of assocaton between EGF receptor molecules was suggested prevously: macroaggregaton of EGF receptors was correlated wth stmulaton of mtogeness by EGF (9). Also, ant-receptor antbodes that stmulate EGF receptor knase functon lose ther actvty when ther monovalent Fab' fragments are used (10). Furthermore, the exstence of the EGF receptor dmers n membranes was demonstrated by chemcal crosslnkng (11). The publcaton costs of ths artcle were defrayed n part by page charge payment. Ths artcle must therefore be hereby marked "advertsement" n accordance wth 18 U.S.C. 1734 solely to ndcate ths fact. In the present study, we have solated natve EGF receptor monomers and dmers from A431 cell membranes, and we have examned ther potental to bnd EGF and to undergo autophosphorylaton, the latter beng an assay for the sgnal transfer from the lgand-bndng doman to the knase doman. Furthermore, we examned the effect of EGF on the monomer-dmer equlbrum. The results strongly support a mechansm for n vtro knase actvaton n whch the key regulatory step s the EGF-dependent converson of receptor monomers to dmers. These noncovalently assocated receptor dmers exhbt a hgher autophosphorylaton actvty than monomers and also have a hgher affnty for EGF, and we postulate that a smlar mechansm may also work n cells. MATERIALS AND METHODS Preparaton of Soluble EGF Receptor. Membranes were solated by sheddng vescles from A431 cells, and receptors were solublzed as descrbed (1). The glycoproten fracton was solated by bndng and eluton to wheat germ agarose (WGA; E-Y Laboratores, San Mateo, CA) (12) n 30 mm Hepes/0.1% Trton X-100/0.02% azde, ph 7.6. Glycoprotens (100 fg) were fractonated on sucrose densty gradents as descrbed (13) wth the followng changes. The gradent was supplemented wth 10% (vol/vol) glycerol, the centrfugaton tme was ncreased to 18 hr, and the appled g force was 200,000. Durng all purfcaton steps, the buffers were supplemented wth 1 mm phenylmethylsulfonyl fluorde, 10 AM leupeptn, 1 AuM pepstatn A, 0.05 trypsn nhbtor unts of aprotnn per ml, and 1 mm EDTA. Based on tracer EGF bndng and autophosphorylaton, we recovered 95% of the appled sample from sucrose gradents. EGF Bndn, Scatchard Analyss, and Receptor Autophosphorylaton. 12 I-labeled EGF (New England Nuclear) bndng was performed wth a polyethylene glycol precptaton assay essentally as descrbed for the nsuln receptor (13, 14). Brefly, receptor was ncubated for 2 hr at room temperature (bndng reached equlbrum; data not shown) wth 80 pm I251-labeled EGF. Nonspecfc tracer bndng was determned n the presence of 0.2 AM unlabeled EGF (Calbochem) and was 10-13% of the total bndng. Samples for Scatchard analyss were obtaned by poolng receptor monomers and dmers from three gradents. The fractons of the monomer and dmer peaks were subjected to a competton bndng n trplcate, and the data were analyzed by the LIGAND program (15). For autophosphorylaton, receptor preparatons were ncubated for 30 mn at room temperature n the presence or absence of0.2,um EGF and were chlled on ce; phosphorylatons were ntated by the addton of 15 AC (1 GC = 37 kbq) of [_y-32p]atp {50,uM ATP total; prepared from [32P]orthophosphate (New England Nuclear) wth a y prep kt from Promega Botec, Madson, WI}, 10 mm MgCl2, and 4 mm Abbrevaton: EGF, epdermal growth factor. 7832
Bochemstry: B6n-Schnetzler and Plch MnCl2 (fnal concentratons). After 8 mn of ncubaton on ce, the autophosphorylaton reacton was stopped ether by addng 50 mm EDTA (fnal concentraton) or by addng Laemml sample buffer. Autophosphorylaton reaches a plateau for all receptor speces n less than 8 mn. Polyacrylamde Gel Electrophoress and Immunoblottng. Samples were electrophoresed on 3-10% NaDodSO4/polyacrylamde gels as descrbed by Laemml (16). Gels were transferred to ntrocellulose (Schlecher & Schuell) and mmunoblotted as descrbed (17) except that Carnaton dry mlk was used nstead of fetal calf serum. The monoclonal ant-egf receptor antbody used to blot receptor was obtaned from ICN ImmunoBologcals, and the second antbody, 125I-labeled goat ant-mouse IgG, was purchased from New England Nuclear. Autoradography was performed as descrbed (13). Quanttaton of EGF receptor by mmunoblottng was acheved by cuttng and assayng bands from blots. A standard curve of EGF receptor was constructed and was lnear over the range of receptor concentratons we used. All experments were performed at least three tmes, and the standard devatons are gven n Results. RESULTS Isolaton of Noncovalent EGF Receptor Dmers from A431 Membranes. We reasoned that noncovalently assocated EGF receptor dmers would be more lkely to form when the receptor concentraton was hgh. Therefore, we partally purfed and concentrated EGF receptors by wheat germ agarose affnty chromatography and subsequently fractonated monomerc and dmerc EGF receptors by sucrose densty gradents under condtons smlar to those used for separatng monomerc and dmerc nsuln receptor speces (13). Because A431 cells are rch n EGF receptors (ca. 2 x 106 receptors per cell), ths proten s the major component seen n slver-staned NaDodSO4 gels of gradent fractons. When tracer 125I-labeled EGF bndng to sucrose densty gradent fractons was measured, two peaks of bndng actvty were observed (Fg. 1 Lower), correspondng to EGF receptor dmers (fractons 17-19) and to EGF receptor monomers (fractons 22-26). The gradents were calbrated wth natve nsuln receptor a2,32 (360 kda), wth the reduced nsuln receptor a/3 half (180 kda), and wth 14C-labeled marker protens wth known S values (data not shown; see ref. 13). In contrast to tracer bndng, lttle, f any, receptor dmer was detected by mmunoblottng at the exposure used (Fg. 1 Upper). In sx dfferent receptor preparatons analyzed by sucrose gradents and mmunoblottng, 24 ± 8% of the receptor sedments as dmers (see also Fg. 5). In all of these experments, we noted that the relatve amount of EGF receptor dmer detected by tracer EGF bndng exceeded the relatve amount of receptor dmer detected by mmunoblottng. These data suggest that EGF receptor dmers mght have a hgher affnty for EGF than receptor monomers and, hence mght bnd more tracer EGF. Therefore, we determned the affnty of lgand for the two EGF receptor speces. EGF Receptor Dmers Are Hgh-Affnty Bndng Stes and Monomers Are Low-Affnty Bndng Stes. We added up to 25% sucrose and observed no effect on tracer lgand bndng (data not shown), thus rulng out the possblty that the hgher sucrose concentraton present n the dmer fractons s responsble for the hgher 1251-labeled EGF bndng to ths speces. We then performed a bndng sotherm to EGF receptor monomers and dmers and subsequently determned ther affntes for lgand by Scatchard analyses. A representatve Scatchard plot s shown n Fg. 2, where the dmer shows a hgher affnty for EGF than does the monomer. In three separate experments, the Kd for monomers averaged 1.9 ± 0.4 x 10-8 M, and the average Kd was 4.9 ± 0.4 x 10-9 M for the dmers. Ths result also rules out the possblty that 15- m 10-0CE Proc. Natl. Acad. Sc. USA 84 (1987) 7833 5. 7I -W.,I /.xi \ - 10 20 f racton number 30 FIG. 1. EGF receptor monomers and dmers can be separated on sucrose densty gradents. Soluble EGF receptor was fractonated on sucrose densty gradents as descrbed. Gradent fractons were subjected to EGF bndng at tracer lgand concentraton (80 pm), and the specfc bndng was plotted aganst the gradent fracton number (Lower). The smaller numbers correspond to the bottom of the gradent and the bgger numbers to the top. The autoradogram (Upper) shows a 2-hr exposure of an mmunoblot of gradent fractons wth a monoclonal ant-egf receptor antbody. the polyethylene glycol assay preferentally precptates dmers (thus, enhancng tracer bndng) because ths possblty would affect the measurable number of bndng stes but not the Kd. Note that we made no attempt to equalze the amount of monomer and dmer n these Scatchard plots; thus, these results only apply to the dfferences n affnty of monomers and dmers for EGF. Autophosphorylaton of Dmers and Monomers. We separated EGF receptor monomers and dmers on sucrose gradents and subjected the peak fractons to autophosphorylaton n the absence or the presence of saturatng EGF concentratons. Agan, control experments showed that sucrose does not affect the autophosphorylaton reacton. 0.1 0.05 1 2 37 4 7 6 8 o 11010 BOUND FIG. 2. EGF receptor dmers are hgh-affnty bndng receptors and monomers are low-affnty receptors. EGF receptor monomers (*) and dmers (o) solated by preparatve sucrose densty gradent centrfugaton were subjected to a competton bndng assay wth 121I-labeled EGF at a fnal concentraton of 80 pm as a tracer and wth addton of ncreasng concentratons of unlabeled EGF. The resultng competton bndng curves were transformed to Scatchard plots, and the affntes for EGF were determned wth the ad of the LIGAND analyss (15).
7834 Bochemstry: Bon-Schnetzler and Plch Furthermore, no phosphatase actvty was observed n the monomer and dmer fractons (data not shown). One band was found to be phosphorylated, correspondng to the ntact 170-kDa form of the EGF receptor (Fg. 3 Upper). Note that the monomer fracton was more responsve than the dmer fracton to EGF. We examned ths effect n more detal n another experment (Fg. 3 Lower) by subjectng every fracton of the gradent to autophosphorylaton and then quanttatng the extent of phosphate ncorporaton. EGF sgnfcantly ncreased the autophosphorylaton of the monomer fracton (fractons 22-27) but dd not apprecably stmulate the autophosphorylaton of the dmers n ths preparaton (fractons 17-21). In four dfferent experments, we found that the stmulaton of autophosphorylaton by EGF was an average of 1.4-fold n the dmer fracton and 4.8-fold n the monomer fracton (see also Fg. 4). In the case of autophosphorylaton n the absence of EGF, monomers and dmers showed the same level of autophosphorylaton (Fg. 3 Lower), whereas n mmunoblottng experments 76% of the receptors sedmented as monomers and 24% as dmers. These data suggest that the autophosphorylaton capacty of receptor dmers s hgher than that of monomers n the absence of EGF. To test ths possblty, we normalzed the extent of autophosphorylaton for the amount of ntact 170-kDa spe- 7 17 1 8 24 25 6 E 90.4 3 /1 2 ~ / 2'0 fracton number FIG. 3. Autophosphorylaton of monomers and dmers n the absence or presence of EGF. (Upper) Monomer (lanes 24/25) and dmer fractons (lanes 17/18) wth the hghest bndng actvty were subjected to autophosphorylaton n the presence (lanes +) or absence (lanes -) of EGF as descrbed. The samples were separated n a 3-10o NaDodSO4/polyacrylamde gel, and the bands were vsualzed by autoradography. (Lower) In a separate experment, each fracton of the gradent was subjected to autophosphorylaton n the presence (-) or absence (----) of EGF, and separated n NaDodSO4/polyacrylamde gels as descrbed n A. The 32p ncorporaton nto receptor bands was quanttated by excsng the bands and assayng the Cerenkov radaton. 0 Proc. Natl. Acad Sc. USA 84 (1987) 4-Q 14 12 14 16 18 20 22 24 26 28 30 fracton number FIG. 4. Normalzaton of autophosphorylaton for receptor amount. EGF receptor monomers and dmers were separated by sucrose densty gradents and fractonated nto 100-,ul fractons. Of each fracton, 20 ul was subjected to autophosphorylaton n the absence (-) or the presence (---) of EGF. The amount of 32p ncorporaton was determned as descrbed n Fg. 3. Of the fractons, 50,ul was subjected to mmunoblottng as descrbed, and the relatve amounts of the ntact 170-kDa receptor form was determned by usng a standard curve. The autophosphorylaton was then normalzed for receptor amount and expressed n arbtrary unts. Fractons 14-18 correspond to EGF receptor dmers, and 22-28 correspond to EGF receptor monomers. ces n each gradent fracton as determned by mmunoblottng. Ths method s advantageous n comparson to other assays because t allows us to dstngush between the knase-actve 170-kDa form and the proteolytcally derved lower molecular weght receptor forms that showed no autophosphorylaton actvty. Also, the dfferent affntes of receptor monomers and dmers dd not allow the use of tracer EGF bndng to normalze the receptor amount. The normalzed autophosphorylaton data n each fracton were expressed as arbtrary unts (Fg. 4). A comparson of the phosphorylaton n the absence of EGF between monomers and dmers revealed that the dmers have a much hgher autophosphorylaton actvty than the monomers. The average dfference from three preparatons was 4.1-fold. The EGF-stmulated autophosphorylaton of the monomers corresponded to that obtaned wth the dmers n the absence of EGF. In some experments, the dmers could not be further stmulated by the addton of EGF (see Fg. 3), whereas n other experments, at most a 2-fold stmulaton was observed (Fg. 4). The average stmulaton was 1.4-fold. The ncreased knase actvty of dmers n the absence of added EGF was ntrnsc to the dmers and was not the result of the presence of EGF, whch remaned bound to the receptor durng the receptor solaton. EGF was effcently removed durng membrane solublzaton n Trton X-100 (1.5%) and the subsequent steps of washng wheat germ agarose-bound receptor and of centrfugaton. Labeled EGF, added to receptors pror to purfcaton, was undetectable n sucrose gradent fractons (data not shown). Furthermore, the resultng dmer preparatons were fully capable of bndng tracer concentratons of EGF (see Fg. 2) wth a hgh affnty. We conclude from these data (t) that the monomerc form s the reactve speces wth regard to EGF-stmulated autophosphorylaton and (h) that the dmerc form represents an already actvated receptor form. EGF Shfts the Receptor Monomer-Dmer Equlbrum to the Dmer Form. The actvated state of natve dmers could possbly be the result of a receptor modfcaton that occurred by some cellular factors n vvo. Alternatvely, snce we
observe n vtro that the monomerc form s most susceptble to knase actvaton by EGF, t s also possble that EGF allows enhanced autophosphorylaton to occur by nducng an assocaton of receptor monomers to the dmerc speces. To examne whether EGF ndeed nfluences the monomerdmer equlbrum, we treated receptor samples pror to centrfugaton wth near-saturatng concentratons of EGF (0.2,tM) for 30 mn at room temperature or wth buffer alone. Subsequently, the monomers were separated from dmers by sucrose densty gradent centrfugaton. The gradent fractons were subjected to mmunoblottng (Fg. 5 Upper), and the 1251I-labeled receptor bands were assayed for radoactvty (Fg. 5 Lower). The curve wth the sold lne shows the mgraton profle of the untreated receptor. As observed prevously, most of the receptors sedmented n fractons 22-25, correspondng to the monomers. Upon addton of EGF, however, most of the receptor was found n fractons 17-19, correspondng to the EGF receptor dmers (dashed lne). These data show that EGF bndng nduces a specfc receptor dmerzaton. EGF-nduced receptor assocaton also could be vsualzed by chemcal crosslnkng of receptor dmers (Fg. 6). In ths experment receptor samples ncubated n the presence or a b 7 6 Bochemstry: B6n-Schnetzler and Plch 15 16 17 18 19 20 21 22 23 24 25 26 27 42; -.. t 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 fracton number FIG. 5. EGF shfts the monomer-dmer equlbrum to the dmer form. (Upper) Soluble receptor that had been bound to and eluted from wheat germ agarose was treated for 30 mn at room temperature wth 0.2,uM EGF (Upper b) or wth buffer as control (Upper a). The samples were separated on sucrose densty gradents; for the EGF-treated sample, EGF was ncluded throughout the gradent. The gradent fractons were then subjected to mmunoblottng. Note that only half offracton 17 was loaded. (Lower) The mmunoblot was quanttated by excsng the bands correspondng to the ntact and proteolytcally derved receptor forms and by countng the bound 1251 wth a y counter (y-axs). The label was plotted aganst the fracton number. -----, Mgraton profle of samples treated wth EGF; untreated control sample. Proc. Nat. Acad. Sc. USA 84 (1987) 7835 Mo. S 1 2 3 4-214 111-68 -45 FIG. 6. Chemcal crosslnkng of EGF receptor dmers. The receptor-contanng glycoproten fracton (20,ug) was treated for 30 mn at room temperature wth 0.2 AM EGF (lanes 1 and 3) or wth buffer (lanes 2 and 4). Subsequently the samples (lanes 3 and 4) were crosslnked on ce wth 2 mm dsuccnmdyl suberate accordng to prevously publshed procedures (18). Lanes 1 and 2 were not crosslnked. Receptor bands were vsualzed by mmunoblottng. D, EGF receptor dmer; M, EGF receptor monomer. The numbers denote the poston of prestaned molecular weght markers from Bethesda Research Laboratores shown x 1o-3. absence of 0.2,tM EGF were crosslnked by usng 2 mm dsuccnmdyl suberate and then subjected to NaDodSO4/ polyacrylamde gel electrophoress and mmunoblottng. Upon addton of EGF, receptors could be crosslnked and mgrated as a covalent receptor dmer (lane 3). The dmerc receptor form was absent when no crosslnker was added (lane 1) and was present n low amounts when no EGF was added pror to crosslnkng (lane 4). Apparently because of the poor effcency of chemcal crosslnkng, the amount of dmers detected by ths method was small. However, the method clearly shows that EGF promotes the dmer formaton. Ths fndng s entrely consstent wth the results from the densty gradent analyss, whch allows detecton of all dmers formed. DISCUSSION In the present study we used an n vtro system to examne the potental sgnfcance of EGF receptor assocaton for lgand-nduced autophosphorylaton. Usng sucrose densty gradent centrfugaton, whch allows determnatons of the S value and hence the molecular weght, we found that an average of 24% of solublzed EGF receptors sedment as noncovalent receptor dmers and the rest as monomers. We do not thnk that these ratos correspond to the ratos of receptor monomers and dmers n natve membranes because the receptor concentraton and the envronment s changed upon solublzaton and purfcaton, and, therefore, the dynamc equlbrum of receptor monomers and dmers s expected to be altered as well. However, the fndng of noncovalently assocated protens on sucrose densty gradents s ndcatve for strong and specfc proten-proten nteractons because reactants wth affntes below the nanomolar range typcally dssocate durng centrfugaton. We used ths method to separate receptor monomers from receptor dmers and to subsequently characterze ther functonal propertes. To examne the bndng propertes of monomers and dmers, respectvely, we subjected them to Scatchard analyses and determned that the dmers have an average Kd for EGF bndng of 4.9 x 10-9 M, whereas the monomers have a Kd of 1.9 x 10-8 M. Smlar affnty constants [namely, 1.33
7836 Bochemstry: Bon-Schnetzler and Plch x 10-8 M (1) and 3 x 10-8 M (19)], as we observe for the monomers, have prevously been reported for unfractonated, soluble EGF receptor preparatons. The fndng that EGF causes receptors to dmerze could potentally compromse the determnaton of the bndng affnty to the monomers. However, the receptor concentraton used n the bndng assay s lower by factors of 40-50 than n the EGF-nduced dmerzaton experment, and at ths dluton substantal dmerzaton does not occur. The 4-fold affnty dfference between monomers and dmers observed n vtro s lower than the dfference between the Kd values of hghand low-affnty bndng stes on cells (1, 19). However, the EGF receptor has a much lower apparent affnty for lgand n soluton compared to cells or membranes (1, 19), probably due n part to the presence of detergent and/or absence of phospholpds. Our n vtro fndng that the underlyng structural dfference between hgh- and low-affnty EGF bndng s the receptor assocaton state mght apply also to receptors n membranes. Ths hypothess s consstent wth recent experments of Dunn et al. (20), n whch dfferent vescle pools were solated, wth predomnantly hgh- or low-affnty bndng EGF receptors havng the same molecular weght and the same tryptc dgeston pattern. From the characterzaton of the autophosphorylaton actvty of monomers and dmers, we can draw the followng conclusons. Frst, the monomerc receptor form s most susceptble to stmulaton of autophosphorylaton by EGF, whereas the dmers show a margnal ncrease of autophosphorylaton n the presence of EGF. Second, when the autophosphorylaton actvty was normalzed for the amount of knase-actve ntact receptor, we found that, n the absence of EGF, the dmers exhbt on average a 4.1-fold hgher knase actvty than do the monomers. The use of mmunoblottng for normalzaton of the autophosphorylaton actvty allows us to dstngush between the phosphorylatng 170- kda receptor form and the nonphosphorylated 150-kDa receptor form, whch partally cosedments n the monomer fractons. Therefore, we can exclude the possblty that the lower knase actvty of monomers s an artfcal result obtaned f both the amount of knase-actve and knasenactve receptor forms were used for normalzaton of receptor amount. Two lnes of evdence show that EGF nduces a specfc assocaton of receptor monomers to dmers. Frst, f receptor s treated wth saturatng concentratons of EGF, most of the receptors sedment as noncovalent dmers on sucrose densty gradents; second, covalent receptor dmers can be obtaned upon treatment wth EGF and subsequent chemcal crosslnkng. The fndng that EGF nfluences the assocaton state of the receptor together wth the fndng that the dmer represents the actvated receptor form wth regard to autophosphorylaton strongly suggests that EGF nduces autophosphorylaton prncpally by promotng the assocaton of monomerc receptor to the actvated dmer speces. We suggest that ths mechansm s also lkely to be operatve n cells, although ths cannot be drectly tested at present. Our data support the model of EGF receptor actvaton recently proposed by Schlessnger (21) and Yarden and Schlessnger (22, 23). These nvestgatons used nondenaturng electrophoress to assess EGF receptor propertes, whereas we used drect solaton of receptor monomers and dmers by sucrose densty gradents. Nevertheless, our conclusons about the functonal propertes of the EGF receptor are dentcal to those of Schlessnger and Yarden. In contrast, Bswas et al. (24) have used technology smlar to our own to reach conclusons opposte from us and from Yarden and Schlessnger. In the study of Bswas et al., freshly solated receptors were used as a source of monomers Proc. Natl. Acad. Sc. USA 84 (1987) and aged receptors as a source of dmers. Consderng the low stablty of the EGF receptor knase relatve to the hgher stablty of EGF bndng, ths approach may be problematc, partcularly when bndng parameters are used to determne receptor amounts n preparatons of dfferent age and to normalze to the assayed knase actvty (24). We recently have demonstrated the need for a dmerc nsuln receptor structure n lgand-nduced autophosphorylaton (13) and n hgh-affnty lgand bndng (12). The ntact nsuln receptor covalent structure can be consdered analogous to a noncovalent EGF receptor dmer. Isolated nsuln receptor halves (an) are analogous to EGF receptor monomers and are not able to undergo autophosphorylaton f ther nteracton s prevented. Taken together, these data suggest that the mechansm for actvatng autophosphorylaton by lgand-nduced receptor dmerzaton (as observed for the EGF receptor) or by a lgand-nduced conformatonal change n an ntrnscally dmerc structure (as found for the nsuln receptor) mght be a common feature of the broader class of receptor-tyrosne knases. We thank Dr. Marsha Rosner (Massachusetts Insttute of Technology) for provdng the A431 cells, Aram Kalgan for techncal assstance, Tm O'Hare for helpng wth the computer analyses of the bndng data, and Dr. Davd Brautgan (Brown Unversty) for help wth the phosphatase assays. Ths work was supported by Grant DK 36424 from the U.S. Publc Health Servce. P.F.P. s the recpent of a research career development award from the U.S. Publc Health Servce, and M.B.-S. s a postdoctoral fellow of the Juvenle Dabetes Foundaton. 1. Cohen, S., Ushro, H., Stoscheck, Ch. & Chnkers, M. (1982) J. Bol. Chem. 257, 1523-1531. 2. Nexo, E., Hock, R. A. & Hollenberg, M. D. (1979) J. 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