Biochimica et B iophysica A cta, 1052 (1990) 453-460
453
Elsevier BBAMCR12706
Membrane vesicles of A431 cells contain one class of epidermal growth factor binding sites J o s A . M . B e r k e r s , P a u l M . P . v a n B e r g e n e n H e n e g o u w e n , A r i e J. V e r k l e i j and Johannes Boonstra Department of Molecular Cell Biology, University of Utrecht, Utrecht (The Netherlands)
(Received 9 March 1989) (Revised manuscript received20 November 1989)
Key words: Membranevesicle; EGF receptor; Scatchardanalysis;(A431 cells)
Epidermoid carcinoma A431 cells exhibit two classes of epidermal growth factor (EGF) receptors as deduced from Scatchard analysis. Steady-state binding of EGF to isolated A431 membranes indicated, however, the presence of only one class of EGF binding sites. The apparent dissociation constant (Kd) of these sites was approx. 0.45 nM which is similar to that of the high-affinity receptor of intact A431 cells. These results suggest that the vesicle receptor population consists only of high-affinity receptors. However, further studies indicated that the binding sites were similar to the low-affinity class, since binding of EGF could be blocked entirely by 2E9, a monocional anti-EGF receptor antibody which is able to inhibit specifically EGF binding to low-affinity receptors in A431 cells. The difference in affinity of the receptors in membrane vesicles as compared to intact cells may be explained by differences in biophysical parameters such as diffusion-limited EGF bintH'ng and receptor distribution. Based upon these considerations, it is concluded that membrane vesicles of A431 cells contain one class of EGF receptors which are apparently identical to the low-affinity receptors of intact cells.
Introduction Binding of epidermal growth factor (EGF) to its receptor, a 170 kDa transmembrane glycoprotein, leads to the initiation of a variety of responses in target cells such as receptor phosphorylation, receptor dimerization, protein phosphorylation and DNA synthesis [1-4]. It has been demonstrated in several different cell types that the population of E G F receptors is represented by two classes of receptors which differ with respect to their affinity for the ligand. This affinity is usually determined by equilibrium binding experiments and Scatchard plot analysis. The validity of the discrimination of two E G F receptor subclasses by Scatchard analysis has been, however, frequently questioned [1,5]. Nevertheless, there are several lines of evidence which
Abbreviations: BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagles medium; EGF, epidermal growth factor; FCS, fetal calf serum; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonylfluoride; PVC, poly(vinylchloride). Correspondence: J.A.M. Berkers, Department of Molecular Cell Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands.
support the existence of two classes of binding sites. Firstly, preincubation of different cell types with the phorbolester phorbol-12-myristate-13-acetate results in a complete reduction of EGF binding to the high-affinity receptor class [5-8]. Secondly the high-affinity receptor class of A431 cells can be visualized by treatment of the cells with the non-ionic detergent Triton X-100 and shown to be associated to the cytoskeleton [9-11]. Finally, it has been recently reported that the two receptor classes can be distinguished by the monoclonal anti-EGF receptor antibody 2E9, which is directed against the extracellular domain of the EGF receptor [9,12,13]. Preincubation of A431 and other cell types with saturating concentrations of 2E9 results in a complete inhibition of E G F binding to the low-affinity receptors (2E9-sensitive sites), but not to high-affinity receptors (2E9-insensitive sites) [4,9,12,13]. In order to study the binding characteristics of the E G F receptor classes in more detail, we decided to use a model system and thus avoid interference from metabolic processes, such as receptor biosynthesis, internalization and degradation. A choice was made for a membrane preparation isolated according to the method of Thorn et al. [14]. In this paper we describe the characterization of
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454 EGF binding to membrane vesicles. It is shown that the membrane vesicles contain only one class of EGF receptors. Based upon the unique properties of the anti EGF receptor antibody 2E9 [12,13], it is concluded that this population of receptors is identical to the low-affinity receptor class of intact A431 cells. Materials and Methods
Materials Bovine serum albumin (BSA), leupeptin, trypsin inhibitor, benzamidine, phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma (St. Louis, MO, U.S.A.). Paraformaldehyde from BDH Chemicals (Poole, U.K.), glutar-aldehyde from Fluka (Buchs, Switserland), Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum (FCS) from Gibco Europe B.V. (Paisley, Scotland), Glass microfiber filters GF-B from Whatman Ltd. (Maidstone, England), Tissue culture plates (245 × 245 x 20) were from Nunc (Roskilde, Denmark). Epidermal growth factor (EGF, receptor grade) was from Collaborative Research (Waltham, MA, U.S.A.). Iodine-125 was from New England Nuclear (Boston, MA, U.S.A.). The 2E9 monoclonal antibody and the 281.7 polyclonal antibody against the EGF receptor were prepared as previously described [12,13]. Cell culture A431 cells were grown at 37 °C in DMEM containing 7.5% FCS in a 7% CO 2 humidified atmosphere. For membrane vesicle preparation the cells were grown in 16 tissue culture plates (245 x 245 x 20) to a final density of 70-90% con fluency. Membrane preparation A431 membrane vesicles were prepared according the method of Thom et al. [14]. Briefly, cells were washed twice in phosphate-buffered saline (PBS) (pH 7.4) and once in harvesting buffer (50 mM boric acid, 150 mM NaC1, 1 mM MgC12, 1 mM CaC12, pH 7.4) without proteinase inhibitors. Subsequently, the cells were scraped with a rubber policeman in harvesting buffer in the presence of proteinase-inhibitors: benzamidine (0.2 mM), leupeptin (1 gg/ml), PMSF (0.1 mM) and trypsin inhibitor (50 ftg/ml). The scraped cells were collected by centrifugation at 450 × g and the pellet was suspended in 10 ml of harvesting buffer with proteinase inhibitors. This solution was added drop by drop over a period of 10 rain to 180 ml of extraction buffer (20 mM boric acid, 200 mM EDTA, pH 10.2) stirring continuously. After 10 min stirring, 10 ml of boric-buffer (0.5 M boric acid, pH 10.2) was added. This suspension was centrifuged at 450 x g for 10 rain, the supernatant was centrifuged at 15000 × g for 30 minutes. This pellet was then put on a 35% sucrose cushion in 20 mM Hepes (with proteinase inhibitors)
and centrifuged for 60 minutes at 15 000 × g. The interface (Thorn vesicles) and the pellet were collected and centrifuged separately for 10 minutes at 100000 × g, suspended in 20 mM Hepes (pH 7.4) and subsequently frozen and stored at - 2 0 o C. The entire procedure was performed on ice or at 4 o C. Proteinase inhibitors were used in all buffers, until sucrose centrifugation was completed. Shedding vesicles of A431 cells were prepared according to the method of Cohen et al. [15]. Briefly, cells were washed three times with PBS and once with Ca 2+ and Mg z+ free hypotonic PBS, followed by a 15 min incubation at room temperature in the same buffer. The hypotonic buffer was discarded and cells were washed with vesiculation buffer (100 mM NaC1, 50 mM Na2HPO4, 5 mM KC1 and 0.5 mM MgSO4, pH 8.5). The cells were incubated once again in vesiculation buffer for 20 min at room temperature and 1 h at 37 o C. During this period, the shedding vesicles were formed. The buffer along with the vesicles was decanted and the vesicles were isolated by means of several centrifugation steps essentially as described by Cohen et al. [15]. Protein determination was performed according the BCA-protein assay (Pierce Chemical Company, Rockford, IL, U.S.A.) with BSA as standard. Freeze fracture For freeze fracture, the membrane preparation was impregnated with 25% glycerol, frozen in liquid N z and transferred to a Balzers BAF 300 freeze-etch machine, fractured and replicated following established procedures [16]. The replicas were stripped off and cleaned with sodium hypochlorite and distilled water as described [16]. EGF binding to isolated membranes Association kinetics. The 125I-EGF was prepared by the chloramine-T method [17]. 12SI-EGF has been shown to be biological active [18]. Four concentrations of EGF, 0.05, 0.5, 2.5 and 5.5 ng/ml, respectively, consisting of 0.05 or 0.5 ng/ml 125I-EGF (spec. act. varying between 200000 and 700000 cpm/ng) and 2 and 5 n g / m l unlabeled EGF, were incubated with 10 gg of the membrane preparation in 1 ml incubation buffer on ice, (DMEM supplemented with 0.1% BSA and buffered with 25 mM Hepes, pH 7.4) for various periods of time up to 4 h in case of the lowest EGF concentration and 30 min for the higher EGF concentrations, as indicated. Binding was determined by separation of bound and free ligand by filtration (Milh'pore filtration-unit, Milhpore Corp., Bedfort MA, U.S.A.) through a giassfiber filter (Whatman, GF-B). The filter was washed twice with 5 ml of PBS supplemented with 0.1% BSA. Radioactivity remaining on the filters was determined by counting in a gammacounter (Crystal 5410 Multi Detector Ria system, United Technologies Packard, IL,
455 U.S.A.). Nonspecific binding was determined by adding a 1000-fold excess unlabeled E G F to the samples and was less then 1.2% of total binding. Equilibrium binding. Filtration assay: a25I-EGF (0.5 n g / m l ) and unlabeled E G F were mixed to final E G F concentrations varying from 0.025 to 100 n g / m l unless otherwise specified. E G F concentrations smaller than 0.5 n g / m l were obtained by dilution of the 0.5 n g / m l ]25I-EGF solution. Nonspecific binding of these samples was determined separately by addition of a 1000times excess of unlabeled EGF. The protein concentration of the membrane preparation was 10 # g / m l in an incubation volume of 1 ml or 500 #1 (as indicated). Equilibrium binding was measured after 22 h or 30 min (as indicated) by separation of bound and free ligand by filtration as described above. The sensitivity of the assay is approx. 0.03-0.1 fmol 125I-EGF b o u n d / m l . Scatchard analysis was performed using the L I G A N D program as described previously [5,19]. When the antibody 2E9 was used, the membranes were preincubated for 3 h at room temperature before performing the binding assay as described above. Centrifugation assay: equilibrium' E G F binding to 3.75 #g vesicle protein was performed in 150 #1 incubation buffer in centrifugation tubes. At equilibrium the membrane vesicles were collected by centrifugation for 5 min at 100000 × g in an airfuge (Beckman) and the pellet was washed once with PBS. PVC-ELISA plate assay: this experiment was performed as described by Kimball and Warren [20]. In short, the membranes were dispensed into a 96-well poly(vinylchloride) (PVC) plate at 2.5 # g / w e l l in 100 #1 PBS. The plates were dried overnight at 37 o C, washed four times with 200 #1 of binding-buffer, pre-incubated 30 min at room temperature with 150 #1 of binding buffer and incubated with various concentrations of E G F for 1 h at room temperature in 100 #1 binding buffer. After washing four times with 200/~1 cold binding buffer, the wells were cut from the plate with a pair of scissors and the radioactivity was determined. The binding buffer consisted of D M E M supplemented with 0.1% BSA and buffered with 50 m M 1,4-piperazinediethanesulphonic acid (Pipes, p H 6.8).
amined the specific E G F binding kinetics of isolated membranes as shown in Fig. 1. The time necessary to reach steady state is dependent upon the E G F concentration and ranged from approximately 4 h for the lowest concentration used (Fig. lb) to less then 30 min for the higher concentrations (Fig. la). Equilibrium binding studies demonstrate that saturation of E G F binding to vesicles occurs at E G F concentrations above 20 n g / m l at 0 ° C (Fig. 2a). Plotting the data of Fig. 2a in a Scatchard graph, results in a linear relationship (Fig. 2b), suggesting the presence of only one class of E G F binding sites. Analysis of the binding data with the L I G A N D program [19] revealed an E G F receptor class with a K d of 0.63 nM. The A
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Characteristics of EGF binding to isolated plasma membranes One of the most widely used methods to determine the characteristics of binding of a ligand to its receptor is the Scatchard plot analysis. In essence, this method determines the relationship between the ligand concentration and the specific binding at equilibrium conditions, yielding the apparent dissociation constant, Kd, and the maximal number of binding sites. In order to reveal the time required to reach equilibrium, we ex-
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Fig. 1. (a) Association kinetics of EGF binding to membrane vesicles at 0 °C using four different concentrations of EGF, 0.05 ng/ml, 0.5 ng/ml, 2.5 ng/ml and 5.5 ng/ml as described under Materials and Methods. The data points are taken from one representative experiment formed in triplicate, o 0.05 ng/ml EGF; • 0.5 ng/ml EGF; /, 2.5 ng/ml EGF; • 5.5 ng/ml EGF. 0a) Magnificationof the association curve of the lowest EGF-concentration (0.05 ng/ml) shown in la.
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tion is observed in the K o of the various m e m b r a n e preparations. However, the maximal binding differs considerably in the various m e m b r a n e preparations, ranging between 8.7 f m o l / ~ g and 42.0 f m o l / / t g protein. This large variation in maximal binding m a y be due to: (a) differences in cell density-dependent receptor density in the A431 cells [22] or (b) to differences in the m e m b r a n e vesicle ultrastructure. The membranes m a y exhibit a mixed orientation (both inside-out and right-side-out) or m a y appear as multilamellar vesicles, b o t h cases leading to a significant under-estimation of the n u m b e r of receptors per ~g m e m b r a n e protein. Therefore, the ultrastructural characteristics of the isolated m e m b r a n e s was studied b y freeze-fracture electron microscopy.
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Freeze fracture and subsequent replication of the isolated m e m b r a n e s demonstrates that the m e m b r a n e preparation consists mainly of single vesicles, varying in size between 5 0 - 5 0 0 n m diameter (Fig. 4a). Most of the m e m b r a n e s a p p e a r as uni-lamellar vesicles as shown by transversely b r o k e n vesicles (Fig. 4b). Sometimes a few small vesicles are present within larger ones. T h e i n t r a m e m b r a n o u s particles (IMP) are aggregated, m o s t likely due to the conditions under which the vesicles were prepared and the rate of freezing [23]. Freeze-fracture studies of intact cells have demonstrated that the density and appearance of i n t r a m e m b r a n o u s particles differ significantly on the protoplasmic fracture face ( P F F ) as c o m p a r e d to that of the exoplasmic
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Bound (fmol/p.g prot) Fig. 2. (a) Equilibrium binding of EGF to membrane vesicles at 0 ° C using a protein concentration of 10/~g/ml in an incubation volume of 1 nil. Bound EGF was measured after 22 h. Maximal binding was achieved with EGF concentrations above 20 ng/ml. (b) Scatchard analysis of the results of 2a. The data are analyzed using the LIGAND program [15]. The Scatchard graph revealed one class of EGF receptors with an apparent dissociation constant of 0.63 nM. Each point is the average of triplicate determinations from a representative experiment. The inset represents a 'Klotz plot' of these data, showing that the correct EGF concentration range has been used for the construction of the Scatchard graph.
' K l o t z plot' (Fig. 2b, inset) shows a typical sigmoidal curve indicating that a correct E G F concentration range has been used [21]. Essentially similar results were obtained in experiments repeated at r o o m temperature using a 30 rain incubation period as shown in Fig. 3. U n d e r these conditions the K d was calculated as 0.45 n M (S.E.M. = 0.052 nM, n = 10) while approx. 1 . 6 . 1 0 4 receptors//~g m e m b r a n e protein were detected. T h e plot shown in Fig. 3 is representative of a n u m b e r of experiments (n = 10) using m e m b r a n e preparations from different isolation procedures. As noted above, little varia-
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Fig. 3. Scatchard analysis of EGF binding to membrane vesicles (e) and residual EGF bindi+n~(o) after 2 h preincubation with 40 ~tg/ml 2E9 in 500 ~tl. Both EGF and 2E9 binding are carried out at room temperature. Scatchard analysis revealed a best fit according to the one site model with apparent dissociation constants (K d = 0,45 nM and 0.30 nM after 2E9 preincubation revealing approx. 10% residual binding.
457
Fig. 4. Freeze-fracture micrograph of membrane vesicles of A431 cells. Freeze fracturing has been performed as described under Materials and Methods. (a) Overview. Bar represents 1 #m. (b) Transversely broken vesicle as indicated by the arrow head. Bar represents 0.2 gm. (c) Concave and convex replicas of the vesicles exhibiting (IMP) distributions of the protoplasmic fracture fact of intact cells. Bar represents 0.2 #m. The arrows denote the direction of shadowing. fracture face ( E F F ) [24]. Since the m e m b r a n e vesicle p r e p a r a t i o n c o n t a i n s b o t h concave a n d convex replicas of vesicles with a n i n t r a m e m b r a n o u s particle distribution characteristic for a p r o t o p l a s m i c fracture face of
i n t a c t cells, it c a n be c o n c l u d e d that the m e m b r a n e vesicle p r e p a r a t i o n exhibits a mixed o r i e n t a t i o n (Fig.
4c). I n addition, the o r i e n t a t i o n of the m e m b r a n e vesicle
458 preparation was studied with an ELISA assay, using antibodies 2E9 and 281-7, directed against the external and cytoplasmic domain of the receptor, respectively. A positive signal is obtained in the presence of both 2E9 and 281-7, again indicating that the vesicle preparation displays both right-side-out and inside-out vesicles (data not shown). These results demonstrate that the membrane preparation consists of a heterogeneous population, both with regard to size and to orientation of the EGF-receptor. This phenomenon can be expected to contribute to the observed differences in maximal number of receptors per #g protein in the various vesicle preparations.
Effect of the experimental conditions on EGF binding characteristics The data presented above indicate the presence of only one class of binding sites in isolated plasma membranes of A431 cells. These data, however, differ to those obtained with intact cells, in which two classes of binding sites have been detected [9,10]. Contradicting results have been reported on EGF binding to isolated plasma membranes. Several reports describe a curvilinear Scatchard plot of EGF binding in isolated plasma membranes from A431 and other celltypes [20,25-27,32], while others report only one class of EGF binding sites [15,28-30]. In order to establish whether these contradictory results are due to differences in experimental binding procedures used, the equilibrium EGF binding to membrane vesicles was performed in different ways with respect to the separation of free and bound ligand. Immobilization of the membrane vesicles on a PVC-ELISA plate [20] yielded a
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Fig. 5. Effect of the 2E9 antibody on EGF binding in A431 cells (o) and membrane vesicles (O). After 3 h preineubation with various concentrations of 2E9 the cells and membranes were incubated with 0.5 ng/md 12SI-EGF for 30 min at room temperature, whereafter the percentage of binding was determined.
linear Scatchard plot, suggesting one class of binding sites with a K d of 0.54 nM (data not shown). It is known that the filtration method used in quantitative radio-receptor assays can disturb the equilibrium between the bound and free fractions [31]. Therefore, EGF binding was also measured by the centrifugation assay. A small decrease of the affinity was observed (K d = 1.28 nM), but still only one class of receptors was detected (data not shown). Another reason for the apparent presence of only one class of binding sites could be the presence of a binding site with a much higher affinity for EGF than 0.45 nM. Therefore, binding was studied using EGF concentrations ranging from 0.025 ng/ml to 50 ng/ml, but again only one class of binding sites was detected (Fig. 2b). A second receptor class could also originate from differences in affinity caused by iodination of EGF. Association experiments using different mixtures of labeled and unlabeled EGF did not influence the kinetic parameters (data not shown), and recently it has been shown that iodination of the EGF molecule using different methods did not affect the results obtained [32]. In addition to the conditions under which binding is performed, the method by which the membranes are prepared may influence EGF binding characteristics. If vesicles are prepared by swelling and shrinking of A431 cells according to the method of Cohen et al.[15], the same results are obtained and only one site with a K d = 0.46 nM is detected (data not shown). In summary, these results demonstrate that the presence of one class of EGF binding sites in A431 membrane vesicles is not dependent upon the experimental conditions used.
The nature of EGF binding site The binding sites in the membrane vesicles appear to be of the high-affinity class, based upon the Kd value of approx. 0.5 nM [9,10]. Recently, the monoclonal antiEGF receptor antibody 2E9 has been described to discriminate between high- and low-affinity receptors in A431 cells [13]. This antibody prevents EGF binding to the low-affinity site, but not to the high-affinity site. Therefore low-affinity receptors in intact cells are 2E9sensitive and the high-affinity receptor sites appear to be 2E9-insensitive. Preincubation of the vesicles with 40 /ag/ml 2E9 for 3 h caused a dramatic decrease of the maximal EGF binding to values of approx. 10% (Fig. 3b). The remaining sites revealed about the same affinity as the control (K d = 0.30 nM 5:0.075 nM). Therefore it can be concluded that the majority of the binding sites in the membrane vesicles respond to 2E9 preincubation as do the 2E9-sensitive receptors in intact A431 cells. To investigate the nature of the residual sites, membrane vesicles were incubated with increasing con-
459 centrations of the antibody 2E9 at a relative low EGF concentration (0.5 ng/ml). Under these conditions the high affinity binding sites of intact cells are occupied by EGF to a larger extent than the low affinity sites [13]. At equilibrium, the EGF binding to the vesicles, after 2E9 preincubation, was measured as a percentage of the control value (Fig. 5). The residual binding reached a minimal value of 3.6% (S.E.M. = 0.28%, n = 11). So an almost complete inhibition of the EGF binding is possible with the antibody 2E9 at concentrations up to 200 #g/ml. The reason that antibody 2E9 is not able to completely reduce the EGF binding is caused by the dissociation of the antibody due to the relative low affinity of the antibody for the EGF receptor [13]. From these data it is concluded that membrane vesicles of A431 ceils contain one class of 2E9-sensitive EGF binding sites identical to the 2E9-sensitive (i.e., low affinity) binding sites in intact ceils. A rather small fraction of binding sites are 2E9-insensitive and may originate from the high-affinity sites in intact cells. Since the K d of the 2E9-sensitive sites is decreased from 7 nM in intact cells to 0.45 nM in membrane vesicles further discrimination on the basis of affinity between the 2E9-sensitive and -insensitive sites in membrane vesicles is not possible. The possible reason for the decrease of the K d will be discussed.
Discussion The data presented in this paper demonstrate, that membrane vesicles isolated from A431 cells contain a single class of EGF binding sites with an apparent dissociation constant K d of 0.45 nM. In contrast, intact A431 cells have been demonstrated to exhibit two classes of binding sites [9,10,12,13]. The apparent disappearance of one class of binding sites in isolated plasma membranes was shown not to be caused by the experimental procedures. The EGF binding sites present in membrane vesicles exhibit a relative low K d value of approximately 0.5 nM. This value is similar to the K d of the high-affinity binding site of intact cells. Therefore, these results suggest that the EGF receptor present in isolated membranes represent the high affinity receptors of intact cells. It has been previously demonstrated that the monoclonal anti-EGF receptor antibody 2E9 exclusively inhibits binding to the low-affinity binding sites of intact cells [12,13]. In this paper, it is demonstrated that 2E9 almost completely inhibit the binding of EGF to the binding sites present in membrane vesicles. The residual amount of approx. 3% of apparent 2E9-insensitive sites can be expected if only one class of binding sites is present in the membrane vesicles, this fraction being the result of simple equilibrium binding conditions involving a ligand with a relative low affinity [13]. Therefore the most simple explanation of the data is in support of the presence of one class of EGF binding
sites, as based upon the affinity and 2E9 sensitivity. An interesting phenomenon concerns the observation that the 2E9-sensitive receptors display a different apparent affinity when expressed in intact cells (7-8 nM) as compared to membrane vesicles (0.5-0.6 nM). Such a decrease of K d can be the result of a modification of the receptor molecule during the membrane isolation procedure. This, however, seems unlikely, since the properties of the EGF receptor molecule, i.e., receptor dimerization and receptor kinase activity, are not changed in membrane vesicles as compared to intact cells [33-37]. Alternatively, it is of interest that EGF binding to intact cells has been demonstrated to proceed in a diffusion limited manner due to the high receptor density in these cells [38]. Moreover, recently it has been shown that the spatial distribution of the receptors (in solution or in monolayer) is an important factor in regulating the ligand-receptor binding as result of limited diffusion processes [39-42]. These observations demonstrate that the K d values measured in intact cells and isolated membrane vesicles are not simply comparable. In summary, based upon these considerations we conclude that the EGF-receptors present in membrane vesicles of A431 cells represent the low-affinity receptor class of intact cells. It is of interest to speculate how the membrane isolation procedure results in a loss of high-affinity receptors. Recently it has been demonstrated that highaffinity binding sites of A431 cells are associated to the cytoskeleton [9,10]. Since the membrane isolation procedures employed are based upon hypotonic swelling and subsequent vesiculation, these procedures may result in a separation of high and low affinity because high-affinity receptors remain attached to the cytoskeleton. On the other hand, disruption of the association between EGF receptors and the cytoskeleton during the membrane isolation procedure could induce a shift from high to low affinity of the receptors. The conclusion that isolated membrane vesicles of A431 cells contain only low-affinity receptors allows a number of interesting conclusions. It has been well established that EGF causes dimerization of EGF receptors in isolated membrane vesicles [34,35] and therefore we can conclude that low-affinity receptors are able to form receptor dimers. Furthermore, EGF has also been demonstrated to activate the tyrosine-kinase activity of the receptor, resulting in receptor self-phosphorylation [35]. These data, combined with the data presented above, demonstrate that EGF is able to activate low affinity receptors, although in intact cells EGF exerts its effect primarily via the high-affinity site [13].
Acknowledgements The authors would like to thank Ms. Jos~ Leunissen-Bijvelt for the freeze-fracture preparations,
460 D r . L . H . K . D e f i z e f o r t h e g e n e r o u s s u p p l y of t h e m o n o c l o n a l a n t i b o d i e s 2E9 a n d 281-7, D r . P. T h o m a s for c r i t i c a l r e a d i n g o f t h e m a n u s c r i p t a n d J. d e n H a r t i g h f o r his t e c h n i c a l assistance.
References 1 Carpenter, G. (1987) Ann. Rev. Biochem. 56, 881-914. 2 Gill, G.N., Bertics, P.J. and Santon, J.B, (1987) Mol. Cell. Endocrinol. 51,169-186. 3 Schlessinger, J. (1986) J. Cell Biol. 103, 2067-2072. 4 Boonstra, J., Defize, L.H.K., Van Bergen en Henegouwen, P.M.P., De Laat, S.W. and Verldeij, A.J. (1989) in Receptors, Membrane Transport and Signal transduction (Evangelopoulos, A.E., Changeux, J.P., Packer, L., Sotiroudis, T.G. and Wirtz, K.W.A., eds.), NATO ASI Series vol. 29, 162-185, Springer-Verlag, Berlin. 5 Boonstra, J., Mummery, C.L., Van der Saag, P.T. and De Laat, S.W. (1985) J. Cell. Physiol. 123, 347-352. 6 Shoyab, M., De Larco, J.E. and Todaro, G.J. (1979) Nature 279, 387-391. 7 Magun, B.E., Martusian, L.M. and Bowden, G.T. (1980) J. Biol. Chem. 255, 6373-6381. 8 Lockyer, J.M., Bowden, G.T. and Magun, B.E. (1983) Carcinogen 4, 653-658. 9 Van Bergen en Henegouwen, P.M.P., De Kroon, J., Van Damme, H., Verkleij, A.J. and Boonstra, J. (1989) J. Cell. Biochem. 39, 455-465. 10 Wiegant, F.A.C., Blok, F.J., Defize, L.H.K., Linnemans, W.A.M., Verkleij, A.J. and Boonstra, J. (1986) J. Cell. Biol. 103, 87-94. 11 Landreth, G.A., Williams, L.K. and Rieser, G.D. (1985) J. Cell Biol. 101, 1341-1350. 12 Defize, L.H.K., Arndt-Jovin, T.M., Boonstra, J., Meisenhelder, J., Hunter, T., De Hey, H.T. and De Laat, S.W. (1988) J. Cell Biol. 107, 939-949. 13 Defize, L.H.K., Boonstra, J., Meisenhelder, J., Kruyer, W., Tertoolen, LG.J., Tilly, B.C., Hunter, T., Van Bergen eta Henegouwen, P.M.P., Moolenaar, W.H. and De Laat, S.W. (1989) J. Cell Biol. 109, 2495-2507. 14 Thorn, D., Powell, A.J., Lloyd, C.W. and Rees, D.A. (1977) Biochem. J. 61,187-194. 15 Cohen, S., Ushiro, H., Stoscheck, C. and Chinkers, M. (1982) J. Biol. Chem. 257, 1523-1531. 16 Burger, K.N.J., Knoll, G. and Verkleij, A. (1988) Biochim. Biophys. Acta 939, 89-101. 17 Comens, P.C., Simmer, R.L. and Baker, J.B. (1982) J. Biol. Chem. 257, 42-45.
18 Carpenter, G., Lembach, K.J., Morrison, M.M. and Cohen, S. (1975) J. Biol. Chem. 250, 4297-4304. 19 Munson, P.J. and Rodbard, D. (1980) Anal. Biochem. 107, 220239. 20 Kimball, E.S. and Warren, T.C. (1984) Biochim. Biophys. Acta 771, 82-84. 21 Klotz, I.M. (1982) Science 217, 1247-1249. 22 Rizzino, A., Kazakoff, P., Ruff, E., Kuszynski, C. and Nebelsick, J. (1988) Cancer Res. 48, 4266-4271. 23 Knoll, G., Verkleij, A.J. and Plattner (1987) in Cryotechiques in Biological Electron Microscopy, (Steinberg, R.A. and Zierold, K., eds.), pp. 258-268, Springer-Verlag, Berlin. 24 Boonstra, J., Van Belzen, N., Van Maurik, P., Hage, W.J., Blok, F.J., Wiegant, F.A.C. and Verldeij, A.J. (1985) J. Microsc. (Oxford) 140, 119-129. 25 Carpenter, G., King, L., Jr. and Cohen, S. (1979) J. Biol. Chem. 254, 4884-4891. 26 Gullick, W.J., Julian, D., Downward, H., Marsden, J.J. and Waterfield, M.D. (1984) Anal. Biochem. 141,253-261. 27 Murthy, U., Basu, A., Rodeck, U., Herlyn, M., Ross, A.H. and Das, M. (1987) Arch. Biochem. Biophys. 252, 549-560. 28 Hock, .A. and Hollenberg, M.D. (1980) J. Biol. Chem. 255, 1073110736. 29 Lin, P.H., Selinfreund, R., Wakshull, E. and Wharton, W. (1987) Biochemistry 26, 731-736. 30 Payrastre, B., Plantavid, M., Etievan, C., Ribbes, G., Carratero, C., Chap, H. and Douste-Blazy, L. (1988) Biochim. Biophys. Acta 939, 355-365. 31 Ensing, K., Feitsma, K.G., Bloemhof, D., In 't Hout, W.G. and De Zeeuw, R.A. (1986) J. Biochem. Biophys. Meth. 13, 85-96. 32 Marti, U., Burwen, S.J., Barker, M.E.., Huling, S., Feren, A.M. and Jones, A.L. (1989) J. Cell. Biochem. 40, 109-119. 33 Cochet, C., Kashles, O., Chambaz, E.M., Borrello, I., King, C.R. and Schlessinger, J. (1988) J. Biol. Chem. 263, 3290-3295. 34 Fanger, B.O., Stephens, J.E. and Staros, J.V. (1989) FASEB J. 3, 71-75. 35 King, L.E., Carpenter, G. and Cohen, S. (1980) Biochemistry 19, 1524-1528. 36 Hunter, T. and Cooper, J.A. (1981) Cell 24, 741-752. 37 Hunter, T. and Cooper, J.A. (1985) Ann. Rev. Biochem. 54, 897-930. 38 Wiley, H.S. (1988) J. Cell Biol. 107, 801-810. 39 Delisi, C. (1981) Mol. Immunol. 18, 507-511. 40 Delisi, C. and Wiegel, F.W. (1981) Proc. Natl. Acad. SCi. USA 78, 5569-5572. 41 Geurts, B.J. and Wiegel, F.W. (1987) Biophys. Chem. 28, 7-12. 42 Geurts, B.J. (1989) Bull. Math. Bio. 53, 359-379.
453
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Membrane vesicles of A431 cells contain one class of epidermal growth factor binding sites J o s A . M . B e r k e r s , P a u l M . P . v a n B e r g e n e n H e n e g o u w e n , A r i e J. V e r k l e i j and Johannes Boonstra Department of Molecular Cell Biology, University of Utrecht, Utrecht (The Netherlands)
(Received 9 March 1989) (Revised manuscript received20 November 1989)
Key words: Membranevesicle; EGF receptor; Scatchardanalysis;(A431 cells)
Epidermoid carcinoma A431 cells exhibit two classes of epidermal growth factor (EGF) receptors as deduced from Scatchard analysis. Steady-state binding of EGF to isolated A431 membranes indicated, however, the presence of only one class of EGF binding sites. The apparent dissociation constant (Kd) of these sites was approx. 0.45 nM which is similar to that of the high-affinity receptor of intact A431 cells. These results suggest that the vesicle receptor population consists only of high-affinity receptors. However, further studies indicated that the binding sites were similar to the low-affinity class, since binding of EGF could be blocked entirely by 2E9, a monocional anti-EGF receptor antibody which is able to inhibit specifically EGF binding to low-affinity receptors in A431 cells. The difference in affinity of the receptors in membrane vesicles as compared to intact cells may be explained by differences in biophysical parameters such as diffusion-limited EGF bintH'ng and receptor distribution. Based upon these considerations, it is concluded that membrane vesicles of A431 cells contain one class of EGF receptors which are apparently identical to the low-affinity receptors of intact cells.
Introduction Binding of epidermal growth factor (EGF) to its receptor, a 170 kDa transmembrane glycoprotein, leads to the initiation of a variety of responses in target cells such as receptor phosphorylation, receptor dimerization, protein phosphorylation and DNA synthesis [1-4]. It has been demonstrated in several different cell types that the population of E G F receptors is represented by two classes of receptors which differ with respect to their affinity for the ligand. This affinity is usually determined by equilibrium binding experiments and Scatchard plot analysis. The validity of the discrimination of two E G F receptor subclasses by Scatchard analysis has been, however, frequently questioned [1,5]. Nevertheless, there are several lines of evidence which
Abbreviations: BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagles medium; EGF, epidermal growth factor; FCS, fetal calf serum; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonylfluoride; PVC, poly(vinylchloride). Correspondence: J.A.M. Berkers, Department of Molecular Cell Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands.
support the existence of two classes of binding sites. Firstly, preincubation of different cell types with the phorbolester phorbol-12-myristate-13-acetate results in a complete reduction of EGF binding to the high-affinity receptor class [5-8]. Secondly the high-affinity receptor class of A431 cells can be visualized by treatment of the cells with the non-ionic detergent Triton X-100 and shown to be associated to the cytoskeleton [9-11]. Finally, it has been recently reported that the two receptor classes can be distinguished by the monoclonal anti-EGF receptor antibody 2E9, which is directed against the extracellular domain of the EGF receptor [9,12,13]. Preincubation of A431 and other cell types with saturating concentrations of 2E9 results in a complete inhibition of E G F binding to the low-affinity receptors (2E9-sensitive sites), but not to high-affinity receptors (2E9-insensitive sites) [4,9,12,13]. In order to study the binding characteristics of the E G F receptor classes in more detail, we decided to use a model system and thus avoid interference from metabolic processes, such as receptor biosynthesis, internalization and degradation. A choice was made for a membrane preparation isolated according to the method of Thorn et al. [14]. In this paper we describe the characterization of
0167-4889/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDivision)
454 EGF binding to membrane vesicles. It is shown that the membrane vesicles contain only one class of EGF receptors. Based upon the unique properties of the anti EGF receptor antibody 2E9 [12,13], it is concluded that this population of receptors is identical to the low-affinity receptor class of intact A431 cells. Materials and Methods
Materials Bovine serum albumin (BSA), leupeptin, trypsin inhibitor, benzamidine, phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma (St. Louis, MO, U.S.A.). Paraformaldehyde from BDH Chemicals (Poole, U.K.), glutar-aldehyde from Fluka (Buchs, Switserland), Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum (FCS) from Gibco Europe B.V. (Paisley, Scotland), Glass microfiber filters GF-B from Whatman Ltd. (Maidstone, England), Tissue culture plates (245 × 245 x 20) were from Nunc (Roskilde, Denmark). Epidermal growth factor (EGF, receptor grade) was from Collaborative Research (Waltham, MA, U.S.A.). Iodine-125 was from New England Nuclear (Boston, MA, U.S.A.). The 2E9 monoclonal antibody and the 281.7 polyclonal antibody against the EGF receptor were prepared as previously described [12,13]. Cell culture A431 cells were grown at 37 °C in DMEM containing 7.5% FCS in a 7% CO 2 humidified atmosphere. For membrane vesicle preparation the cells were grown in 16 tissue culture plates (245 x 245 x 20) to a final density of 70-90% con fluency. Membrane preparation A431 membrane vesicles were prepared according the method of Thom et al. [14]. Briefly, cells were washed twice in phosphate-buffered saline (PBS) (pH 7.4) and once in harvesting buffer (50 mM boric acid, 150 mM NaC1, 1 mM MgC12, 1 mM CaC12, pH 7.4) without proteinase inhibitors. Subsequently, the cells were scraped with a rubber policeman in harvesting buffer in the presence of proteinase-inhibitors: benzamidine (0.2 mM), leupeptin (1 gg/ml), PMSF (0.1 mM) and trypsin inhibitor (50 ftg/ml). The scraped cells were collected by centrifugation at 450 × g and the pellet was suspended in 10 ml of harvesting buffer with proteinase inhibitors. This solution was added drop by drop over a period of 10 rain to 180 ml of extraction buffer (20 mM boric acid, 200 mM EDTA, pH 10.2) stirring continuously. After 10 min stirring, 10 ml of boric-buffer (0.5 M boric acid, pH 10.2) was added. This suspension was centrifuged at 450 x g for 10 rain, the supernatant was centrifuged at 15000 × g for 30 minutes. This pellet was then put on a 35% sucrose cushion in 20 mM Hepes (with proteinase inhibitors)
and centrifuged for 60 minutes at 15 000 × g. The interface (Thorn vesicles) and the pellet were collected and centrifuged separately for 10 minutes at 100000 × g, suspended in 20 mM Hepes (pH 7.4) and subsequently frozen and stored at - 2 0 o C. The entire procedure was performed on ice or at 4 o C. Proteinase inhibitors were used in all buffers, until sucrose centrifugation was completed. Shedding vesicles of A431 cells were prepared according to the method of Cohen et al. [15]. Briefly, cells were washed three times with PBS and once with Ca 2+ and Mg z+ free hypotonic PBS, followed by a 15 min incubation at room temperature in the same buffer. The hypotonic buffer was discarded and cells were washed with vesiculation buffer (100 mM NaC1, 50 mM Na2HPO4, 5 mM KC1 and 0.5 mM MgSO4, pH 8.5). The cells were incubated once again in vesiculation buffer for 20 min at room temperature and 1 h at 37 o C. During this period, the shedding vesicles were formed. The buffer along with the vesicles was decanted and the vesicles were isolated by means of several centrifugation steps essentially as described by Cohen et al. [15]. Protein determination was performed according the BCA-protein assay (Pierce Chemical Company, Rockford, IL, U.S.A.) with BSA as standard. Freeze fracture For freeze fracture, the membrane preparation was impregnated with 25% glycerol, frozen in liquid N z and transferred to a Balzers BAF 300 freeze-etch machine, fractured and replicated following established procedures [16]. The replicas were stripped off and cleaned with sodium hypochlorite and distilled water as described [16]. EGF binding to isolated membranes Association kinetics. The 125I-EGF was prepared by the chloramine-T method [17]. 12SI-EGF has been shown to be biological active [18]. Four concentrations of EGF, 0.05, 0.5, 2.5 and 5.5 ng/ml, respectively, consisting of 0.05 or 0.5 ng/ml 125I-EGF (spec. act. varying between 200000 and 700000 cpm/ng) and 2 and 5 n g / m l unlabeled EGF, were incubated with 10 gg of the membrane preparation in 1 ml incubation buffer on ice, (DMEM supplemented with 0.1% BSA and buffered with 25 mM Hepes, pH 7.4) for various periods of time up to 4 h in case of the lowest EGF concentration and 30 min for the higher EGF concentrations, as indicated. Binding was determined by separation of bound and free ligand by filtration (Milh'pore filtration-unit, Milhpore Corp., Bedfort MA, U.S.A.) through a giassfiber filter (Whatman, GF-B). The filter was washed twice with 5 ml of PBS supplemented with 0.1% BSA. Radioactivity remaining on the filters was determined by counting in a gammacounter (Crystal 5410 Multi Detector Ria system, United Technologies Packard, IL,
455 U.S.A.). Nonspecific binding was determined by adding a 1000-fold excess unlabeled E G F to the samples and was less then 1.2% of total binding. Equilibrium binding. Filtration assay: a25I-EGF (0.5 n g / m l ) and unlabeled E G F were mixed to final E G F concentrations varying from 0.025 to 100 n g / m l unless otherwise specified. E G F concentrations smaller than 0.5 n g / m l were obtained by dilution of the 0.5 n g / m l ]25I-EGF solution. Nonspecific binding of these samples was determined separately by addition of a 1000times excess of unlabeled EGF. The protein concentration of the membrane preparation was 10 # g / m l in an incubation volume of 1 ml or 500 #1 (as indicated). Equilibrium binding was measured after 22 h or 30 min (as indicated) by separation of bound and free ligand by filtration as described above. The sensitivity of the assay is approx. 0.03-0.1 fmol 125I-EGF b o u n d / m l . Scatchard analysis was performed using the L I G A N D program as described previously [5,19]. When the antibody 2E9 was used, the membranes were preincubated for 3 h at room temperature before performing the binding assay as described above. Centrifugation assay: equilibrium' E G F binding to 3.75 #g vesicle protein was performed in 150 #1 incubation buffer in centrifugation tubes. At equilibrium the membrane vesicles were collected by centrifugation for 5 min at 100000 × g in an airfuge (Beckman) and the pellet was washed once with PBS. PVC-ELISA plate assay: this experiment was performed as described by Kimball and Warren [20]. In short, the membranes were dispensed into a 96-well poly(vinylchloride) (PVC) plate at 2.5 # g / w e l l in 100 #1 PBS. The plates were dried overnight at 37 o C, washed four times with 200 #1 of binding-buffer, pre-incubated 30 min at room temperature with 150 #1 of binding buffer and incubated with various concentrations of E G F for 1 h at room temperature in 100 #1 binding buffer. After washing four times with 200/~1 cold binding buffer, the wells were cut from the plate with a pair of scissors and the radioactivity was determined. The binding buffer consisted of D M E M supplemented with 0.1% BSA and buffered with 50 m M 1,4-piperazinediethanesulphonic acid (Pipes, p H 6.8).
amined the specific E G F binding kinetics of isolated membranes as shown in Fig. 1. The time necessary to reach steady state is dependent upon the E G F concentration and ranged from approximately 4 h for the lowest concentration used (Fig. lb) to less then 30 min for the higher concentrations (Fig. la). Equilibrium binding studies demonstrate that saturation of E G F binding to vesicles occurs at E G F concentrations above 20 n g / m l at 0 ° C (Fig. 2a). Plotting the data of Fig. 2a in a Scatchard graph, results in a linear relationship (Fig. 2b), suggesting the presence of only one class of E G F binding sites. Analysis of the binding data with the L I G A N D program [19] revealed an E G F receptor class with a K d of 0.63 nM. The A
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Characteristics of EGF binding to isolated plasma membranes One of the most widely used methods to determine the characteristics of binding of a ligand to its receptor is the Scatchard plot analysis. In essence, this method determines the relationship between the ligand concentration and the specific binding at equilibrium conditions, yielding the apparent dissociation constant, Kd, and the maximal number of binding sites. In order to reveal the time required to reach equilibrium, we ex-
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Fig. 1. (a) Association kinetics of EGF binding to membrane vesicles at 0 °C using four different concentrations of EGF, 0.05 ng/ml, 0.5 ng/ml, 2.5 ng/ml and 5.5 ng/ml as described under Materials and Methods. The data points are taken from one representative experiment formed in triplicate, o 0.05 ng/ml EGF; • 0.5 ng/ml EGF; /, 2.5 ng/ml EGF; • 5.5 ng/ml EGF. 0a) Magnificationof the association curve of the lowest EGF-concentration (0.05 ng/ml) shown in la.
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tion is observed in the K o of the various m e m b r a n e preparations. However, the maximal binding differs considerably in the various m e m b r a n e preparations, ranging between 8.7 f m o l / ~ g and 42.0 f m o l / / t g protein. This large variation in maximal binding m a y be due to: (a) differences in cell density-dependent receptor density in the A431 cells [22] or (b) to differences in the m e m b r a n e vesicle ultrastructure. The membranes m a y exhibit a mixed orientation (both inside-out and right-side-out) or m a y appear as multilamellar vesicles, b o t h cases leading to a significant under-estimation of the n u m b e r of receptors per ~g m e m b r a n e protein. Therefore, the ultrastructural characteristics of the isolated m e m b r a n e s was studied b y freeze-fracture electron microscopy.
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Freeze fracture and subsequent replication of the isolated m e m b r a n e s demonstrates that the m e m b r a n e preparation consists mainly of single vesicles, varying in size between 5 0 - 5 0 0 n m diameter (Fig. 4a). Most of the m e m b r a n e s a p p e a r as uni-lamellar vesicles as shown by transversely b r o k e n vesicles (Fig. 4b). Sometimes a few small vesicles are present within larger ones. T h e i n t r a m e m b r a n o u s particles (IMP) are aggregated, m o s t likely due to the conditions under which the vesicles were prepared and the rate of freezing [23]. Freeze-fracture studies of intact cells have demonstrated that the density and appearance of i n t r a m e m b r a n o u s particles differ significantly on the protoplasmic fracture face ( P F F ) as c o m p a r e d to that of the exoplasmic
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Bound (fmol/p.g prot) Fig. 2. (a) Equilibrium binding of EGF to membrane vesicles at 0 ° C using a protein concentration of 10/~g/ml in an incubation volume of 1 nil. Bound EGF was measured after 22 h. Maximal binding was achieved with EGF concentrations above 20 ng/ml. (b) Scatchard analysis of the results of 2a. The data are analyzed using the LIGAND program [15]. The Scatchard graph revealed one class of EGF receptors with an apparent dissociation constant of 0.63 nM. Each point is the average of triplicate determinations from a representative experiment. The inset represents a 'Klotz plot' of these data, showing that the correct EGF concentration range has been used for the construction of the Scatchard graph.
' K l o t z plot' (Fig. 2b, inset) shows a typical sigmoidal curve indicating that a correct E G F concentration range has been used [21]. Essentially similar results were obtained in experiments repeated at r o o m temperature using a 30 rain incubation period as shown in Fig. 3. U n d e r these conditions the K d was calculated as 0.45 n M (S.E.M. = 0.052 nM, n = 10) while approx. 1 . 6 . 1 0 4 receptors//~g m e m b r a n e protein were detected. T h e plot shown in Fig. 3 is representative of a n u m b e r of experiments (n = 10) using m e m b r a n e preparations from different isolation procedures. As noted above, little varia-
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Fig. 3. Scatchard analysis of EGF binding to membrane vesicles (e) and residual EGF bindi+n~(o) after 2 h preincubation with 40 ~tg/ml 2E9 in 500 ~tl. Both EGF and 2E9 binding are carried out at room temperature. Scatchard analysis revealed a best fit according to the one site model with apparent dissociation constants (K d = 0,45 nM and 0.30 nM after 2E9 preincubation revealing approx. 10% residual binding.
457
Fig. 4. Freeze-fracture micrograph of membrane vesicles of A431 cells. Freeze fracturing has been performed as described under Materials and Methods. (a) Overview. Bar represents 1 #m. (b) Transversely broken vesicle as indicated by the arrow head. Bar represents 0.2 gm. (c) Concave and convex replicas of the vesicles exhibiting (IMP) distributions of the protoplasmic fracture fact of intact cells. Bar represents 0.2 #m. The arrows denote the direction of shadowing. fracture face ( E F F ) [24]. Since the m e m b r a n e vesicle p r e p a r a t i o n c o n t a i n s b o t h concave a n d convex replicas of vesicles with a n i n t r a m e m b r a n o u s particle distribution characteristic for a p r o t o p l a s m i c fracture face of
i n t a c t cells, it c a n be c o n c l u d e d that the m e m b r a n e vesicle p r e p a r a t i o n exhibits a mixed o r i e n t a t i o n (Fig.
4c). I n addition, the o r i e n t a t i o n of the m e m b r a n e vesicle
458 preparation was studied with an ELISA assay, using antibodies 2E9 and 281-7, directed against the external and cytoplasmic domain of the receptor, respectively. A positive signal is obtained in the presence of both 2E9 and 281-7, again indicating that the vesicle preparation displays both right-side-out and inside-out vesicles (data not shown). These results demonstrate that the membrane preparation consists of a heterogeneous population, both with regard to size and to orientation of the EGF-receptor. This phenomenon can be expected to contribute to the observed differences in maximal number of receptors per #g protein in the various vesicle preparations.
Effect of the experimental conditions on EGF binding characteristics The data presented above indicate the presence of only one class of binding sites in isolated plasma membranes of A431 cells. These data, however, differ to those obtained with intact cells, in which two classes of binding sites have been detected [9,10]. Contradicting results have been reported on EGF binding to isolated plasma membranes. Several reports describe a curvilinear Scatchard plot of EGF binding in isolated plasma membranes from A431 and other celltypes [20,25-27,32], while others report only one class of EGF binding sites [15,28-30]. In order to establish whether these contradictory results are due to differences in experimental binding procedures used, the equilibrium EGF binding to membrane vesicles was performed in different ways with respect to the separation of free and bound ligand. Immobilization of the membrane vesicles on a PVC-ELISA plate [20] yielded a
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Fig. 5. Effect of the 2E9 antibody on EGF binding in A431 cells (o) and membrane vesicles (O). After 3 h preineubation with various concentrations of 2E9 the cells and membranes were incubated with 0.5 ng/md 12SI-EGF for 30 min at room temperature, whereafter the percentage of binding was determined.
linear Scatchard plot, suggesting one class of binding sites with a K d of 0.54 nM (data not shown). It is known that the filtration method used in quantitative radio-receptor assays can disturb the equilibrium between the bound and free fractions [31]. Therefore, EGF binding was also measured by the centrifugation assay. A small decrease of the affinity was observed (K d = 1.28 nM), but still only one class of receptors was detected (data not shown). Another reason for the apparent presence of only one class of binding sites could be the presence of a binding site with a much higher affinity for EGF than 0.45 nM. Therefore, binding was studied using EGF concentrations ranging from 0.025 ng/ml to 50 ng/ml, but again only one class of binding sites was detected (Fig. 2b). A second receptor class could also originate from differences in affinity caused by iodination of EGF. Association experiments using different mixtures of labeled and unlabeled EGF did not influence the kinetic parameters (data not shown), and recently it has been shown that iodination of the EGF molecule using different methods did not affect the results obtained [32]. In addition to the conditions under which binding is performed, the method by which the membranes are prepared may influence EGF binding characteristics. If vesicles are prepared by swelling and shrinking of A431 cells according to the method of Cohen et al.[15], the same results are obtained and only one site with a K d = 0.46 nM is detected (data not shown). In summary, these results demonstrate that the presence of one class of EGF binding sites in A431 membrane vesicles is not dependent upon the experimental conditions used.
The nature of EGF binding site The binding sites in the membrane vesicles appear to be of the high-affinity class, based upon the Kd value of approx. 0.5 nM [9,10]. Recently, the monoclonal antiEGF receptor antibody 2E9 has been described to discriminate between high- and low-affinity receptors in A431 cells [13]. This antibody prevents EGF binding to the low-affinity site, but not to the high-affinity site. Therefore low-affinity receptors in intact cells are 2E9sensitive and the high-affinity receptor sites appear to be 2E9-insensitive. Preincubation of the vesicles with 40 /ag/ml 2E9 for 3 h caused a dramatic decrease of the maximal EGF binding to values of approx. 10% (Fig. 3b). The remaining sites revealed about the same affinity as the control (K d = 0.30 nM 5:0.075 nM). Therefore it can be concluded that the majority of the binding sites in the membrane vesicles respond to 2E9 preincubation as do the 2E9-sensitive receptors in intact A431 cells. To investigate the nature of the residual sites, membrane vesicles were incubated with increasing con-
459 centrations of the antibody 2E9 at a relative low EGF concentration (0.5 ng/ml). Under these conditions the high affinity binding sites of intact cells are occupied by EGF to a larger extent than the low affinity sites [13]. At equilibrium, the EGF binding to the vesicles, after 2E9 preincubation, was measured as a percentage of the control value (Fig. 5). The residual binding reached a minimal value of 3.6% (S.E.M. = 0.28%, n = 11). So an almost complete inhibition of the EGF binding is possible with the antibody 2E9 at concentrations up to 200 #g/ml. The reason that antibody 2E9 is not able to completely reduce the EGF binding is caused by the dissociation of the antibody due to the relative low affinity of the antibody for the EGF receptor [13]. From these data it is concluded that membrane vesicles of A431 ceils contain one class of 2E9-sensitive EGF binding sites identical to the 2E9-sensitive (i.e., low affinity) binding sites in intact ceils. A rather small fraction of binding sites are 2E9-insensitive and may originate from the high-affinity sites in intact cells. Since the K d of the 2E9-sensitive sites is decreased from 7 nM in intact cells to 0.45 nM in membrane vesicles further discrimination on the basis of affinity between the 2E9-sensitive and -insensitive sites in membrane vesicles is not possible. The possible reason for the decrease of the K d will be discussed.
Discussion The data presented in this paper demonstrate, that membrane vesicles isolated from A431 cells contain a single class of EGF binding sites with an apparent dissociation constant K d of 0.45 nM. In contrast, intact A431 cells have been demonstrated to exhibit two classes of binding sites [9,10,12,13]. The apparent disappearance of one class of binding sites in isolated plasma membranes was shown not to be caused by the experimental procedures. The EGF binding sites present in membrane vesicles exhibit a relative low K d value of approximately 0.5 nM. This value is similar to the K d of the high-affinity binding site of intact cells. Therefore, these results suggest that the EGF receptor present in isolated membranes represent the high affinity receptors of intact cells. It has been previously demonstrated that the monoclonal anti-EGF receptor antibody 2E9 exclusively inhibits binding to the low-affinity binding sites of intact cells [12,13]. In this paper, it is demonstrated that 2E9 almost completely inhibit the binding of EGF to the binding sites present in membrane vesicles. The residual amount of approx. 3% of apparent 2E9-insensitive sites can be expected if only one class of binding sites is present in the membrane vesicles, this fraction being the result of simple equilibrium binding conditions involving a ligand with a relative low affinity [13]. Therefore the most simple explanation of the data is in support of the presence of one class of EGF binding
sites, as based upon the affinity and 2E9 sensitivity. An interesting phenomenon concerns the observation that the 2E9-sensitive receptors display a different apparent affinity when expressed in intact cells (7-8 nM) as compared to membrane vesicles (0.5-0.6 nM). Such a decrease of K d can be the result of a modification of the receptor molecule during the membrane isolation procedure. This, however, seems unlikely, since the properties of the EGF receptor molecule, i.e., receptor dimerization and receptor kinase activity, are not changed in membrane vesicles as compared to intact cells [33-37]. Alternatively, it is of interest that EGF binding to intact cells has been demonstrated to proceed in a diffusion limited manner due to the high receptor density in these cells [38]. Moreover, recently it has been shown that the spatial distribution of the receptors (in solution or in monolayer) is an important factor in regulating the ligand-receptor binding as result of limited diffusion processes [39-42]. These observations demonstrate that the K d values measured in intact cells and isolated membrane vesicles are not simply comparable. In summary, based upon these considerations we conclude that the EGF-receptors present in membrane vesicles of A431 cells represent the low-affinity receptor class of intact cells. It is of interest to speculate how the membrane isolation procedure results in a loss of high-affinity receptors. Recently it has been demonstrated that highaffinity binding sites of A431 cells are associated to the cytoskeleton [9,10]. Since the membrane isolation procedures employed are based upon hypotonic swelling and subsequent vesiculation, these procedures may result in a separation of high and low affinity because high-affinity receptors remain attached to the cytoskeleton. On the other hand, disruption of the association between EGF receptors and the cytoskeleton during the membrane isolation procedure could induce a shift from high to low affinity of the receptors. The conclusion that isolated membrane vesicles of A431 cells contain only low-affinity receptors allows a number of interesting conclusions. It has been well established that EGF causes dimerization of EGF receptors in isolated membrane vesicles [34,35] and therefore we can conclude that low-affinity receptors are able to form receptor dimers. Furthermore, EGF has also been demonstrated to activate the tyrosine-kinase activity of the receptor, resulting in receptor self-phosphorylation [35]. These data, combined with the data presented above, demonstrate that EGF is able to activate low affinity receptors, although in intact cells EGF exerts its effect primarily via the high-affinity site [13].
Acknowledgements The authors would like to thank Ms. Jos~ Leunissen-Bijvelt for the freeze-fracture preparations,
460 D r . L . H . K . D e f i z e f o r t h e g e n e r o u s s u p p l y of t h e m o n o c l o n a l a n t i b o d i e s 2E9 a n d 281-7, D r . P. T h o m a s for c r i t i c a l r e a d i n g o f t h e m a n u s c r i p t a n d J. d e n H a r t i g h f o r his t e c h n i c a l assistance.
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