JOURNAL OF CELLULAR PHYSIOLOGY 145365-375 (1990)
Interaction of Epidermal Growth Factor Receptors With the Cytoskeleton Is Related to Receptor Clustering NlCO VAN BELZEN, MARCEL SPAARGAREN, ARlE J. VERKLEIJ, AND JOHANNESBOONSTRA* Department of Molecular Cell Biology, University of Utrecht, 3584 CH Utrecht, The M.S., A.I.V., 1.5.); Hubrecht laboratory, Netherlands Institute of Netherlands (N.v.~., Developmental Biology, 3584 CT Utrecht, The Netherlands (M.S.)
Recently it has been established that cytoskeleton-associated epidermal growth factor (EGF) receptors are predominantly of the high-affinity class and that EGF induces a recruitment of low-affinity receptors to the cytoskeleton. The nature of this EGF-induced receptor-cytoskeleton interaction, however, is still unknown. Therefore, we have studied the association of mutated EGF receptors with the cytoskeleton. Receptor deletion mutants lacking almost all intracellular amino acid residues displayed no interaction with the cytoskeleton, demonstrating that the cytoplasmic receptor domain is involved in this interaction. Further analysis revealed that receptor-cytoskeletoninteraction is independent of receptor kinase activity and the C-terminal 126 amino acid residues, which include the autophosphorylation sites. Furthermore, it is shown that the high-affinity receptor subclass is not essential for association of low-affinity receptors to the cytoskeleton. EGF receptor-cytoskeleton interaction was increased, however, by treatment with sphingomyelinase, an enzyme known to induce membrane protein clustering, indicating that EGF receptor clustering may cause the association to the cytoskeleton. Epidermal growth factor (EGF) is a well known mitogen for a wide variety of mammalian cells (for reviews see Carpenter and Cohen, 1979; De Laat et al., 1983; Schlessinger, 1988). It binds specifically to a 170 kDa transmembrane protein, the EGF rece tor. EGF bindin studies indicate that most target ce 1s exhibit two E F receptor subclasses, one class binding EGF with high affinity and low capacity, the other binding with low affinity and high capacity (King and Cuatrecasas, 1982; Blay and Brown, 1985; Boonstra et al., 1985). The most convincing evidence in favor of the existence of two classes of binding sites was obtained in studies using antibodies directed against the EGF receptor (Gregoriou and Rees, 1984; Defize et al., 1989). In articular the monoclonal anti-EGF receptor antibo y 2E9 has been shown to exhibit unique properties in this respect. This antibod blocks specifically the binding of EGF to the low-a inity EGF receptors in a variety of cell lines (Defize et al., 1988, 1989; Boonstra and Ponec, 1989). Using the 2E9 antibody, it was recently demonstrated that activation of the EGF receptor signal transduction cascade, i.e., inositol hosphate production, release of Ca2+ from intrace ular stores, rise in intracellular pH in the absence of HC03-, stimulation of protein kinase C activity, activation of EGF receptor protein tyrosine kinase activity, and induction of c-fos gene expression, can occur completely through exclusive binding of EGF to the high-affinity
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receptor subclass (Defize et al., 1989). Recently these findings were confirmed by means of an antibody that specifically blocked the binding of EGF to the highaffinity subclass (Bellot et al., 1990). The activation of the EGF receptor protein tryosine kinase by binding of EGF has been indicated as a key event in the initiation of signal transduction (Honegger et al., 1987a,b; Moolenaar et al., 1988). Activation of the EGF receptor tyrosine kinase results in phosphorylation of the receptor itself (Carpenter et al., 1979; King et al., 1980) and various other substrates, including a number of cytoskeleton-related proteins, such as cal actidlipocortin (Fava and Cohen, 1984; Sawyer an Cohen, 1985; Pepinsky and Sinclair, 19861, ezrin (Gould et al., 1986; Bretscher, 1989), fodrin, tubulin, tau, and microtubule-associated protein 2 (Akiyama et al., 1986). These latter observations are of particular interest since part of the EGF receptor population has been demonstrated to be associated to the Triton X-100-insoluble cytoskeleton of A431 cells (Landreth et al., 1985; Wiegant et al., 1986). In A431 cells the cytoskeleton-associated fraction of
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Received March 29, 1990; accepted August 1, 1990. *Towhom reprint requestsicorrespondence should be addressed. Nico van Belzen’s present address is Department of Pathology, University of Limburg, Maastricht, The Netherlands.
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the EGF receptor population represents specifically the high-affinity class of receptors (Wiegant et al., 1986; Van Bergen en Henegouwen et al., 1989). Addition of EGF to intact cells results in an additional association of low-affinity receptors t o the cytoskeleton (Van Bergen en Henegouwen et al., 1989). At present the nature of the interaction between EGF receptor and cytoskeleton is unknown, but it seems plausible to suggest that, since EGF causes an increased association of the receptors to the cytoskeleton, EGF binding has to lead to a conformational change of the receptor or of cytoskeletal components, to allow the binding of the receptor to the cytoskeleton. In this respect an important role of the autophosphorylation sites and/or of the kinase activity of the receptor can be proposed, analogous to the platelet-derived growth factor (PDGF) receptor, whose interaction with cellular proteins was shown to depend on receptor autophosphorylation (Kazlauskas and Cooer, 1989). Therefore, we have studied the interaction etween EGF receptor and cytoskeleton in mouse NIH3T3 fibroblasts transfected with cDNA constructs encoding normal or mutated human EGF receptors (Honegger et al., 1987a,b). The cell lines used include NEF cells, expressing the wild-type EGF receptor; CD126 cells, expressing a truncated receptor lacking the C-terminal amino acid residues 1061-1186 (including the major autophosphorylation sites); and the kinasenegative K721A cell line, in which lysine7" in the ATP binding site was replaced by an alanine residue, preventin adenosine triphosphate (ATP) binding and hence inase activity. Using these cell lines, we demonstrate in this paper that the interaction between the EGF receptor and the cytoskeleton does not require the receptor autophosphorylation sites and receptor kinase activity. Furthermore, the C-terminal 126 amino acid residues are not involved in the association. Using the tumor promoter phorbol myristate acetate (PMA), we show that the high-affinity receptors are not essential for binding of low-affinity receptors to the cytoskeleton. On the basis of theoretical considerations, it has been proposed that receptor-cytoskeleton interaction could result from clustering of receptors by multivalent ligands (Brandts and Jacobson, 1983; Jacobson, 1983). The hypothesis is based on the assumption that monomeric plasma membrane proteins contain translational entropy that revents their association to cytoskeletal com onents. of membrane proteins by multiva ent extracellular ligands results in a decrease of translational entropy and hence in an increased affinity for intracellular binding sites. EGF is assumed to bind its receptor monovalently but is nevertheless known to induce receptor clustering (Zidovetski et al., 1981; Hopkins et al., 1985; van Belzen et al., 1988; Torrisi et al., 1988). The EGF-induced receptor clustering could well explain the EGF-driven association of the receptor to the cytoskeleton. To provide evidence for this possible relationship, we have studied the effects of EGF-independent clustering of receptors on their association to the cytoskeleton. Using sphingomyelinase, it is demonstrated that increased clustering of membrane proteins, including the EGF receptor, is accompanied by an increase in the fraction of EGF receptors associated to the cytoskeleton.
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MATERIALS AND METHODS Cell culture Mouse NIH-3T3 fibroblasts transfected with human EGF receptor cDNA were kindly provided by Dr. J. Schlessinger (Rorer Biotechnology Inc., King of Prussia, PA). NEF cells contain wild-type human EGF receptors. 3T3-IV cells contain EGF receptors from which all cytoplasmic amino acid residues but nine have been deleted (Prywes et al., 1986; Livneh et al., 1986). K721A cells contain EGF receptors in which amino acid residue Lys721is replaced by Ala721,resulting in a loss of rece tor kinase activity (Honegger et al., 1987a). CD126 cel s contain EGF receptors from which 126 C-terminal amino acid residues 1061-1186 have been deleted (Margolis et al., 1989). Cells were cultured in Dulbecco's modified Ea le's medium (DMEM), supplemented with 7.5% feta calf serum (Inte ro, Zaandam, The Netherlands), and buffered wit 44 mM NaHC03, in a 7% C02 humidified atmosphere. Tissue culture plastics (75 cm2 flasks and 12-wells plates) were from Costar (Cambridge, MA).
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EGF receptor autophosphorylation Total cell lysates were prepared from subconfluent 75 cm2 dishes as described previously (Defize et al., 1989) in a volume of 500 pl. Aliquots from this lysate (40 pl) were incubated in the absence or presence of 100 ng/ml EGF for 20 min at room temperature. The phosphorylation reaction was initiated by addition of 10 p1 phosphorylation assay mix, containing 100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid (HEPES) (pH 7.4), 10 mM MnC12, 50 mM p-nitrophenylphosphate, 200 IJ.MNa3V04, 0.05% bovine serum albumin (BSA), 1%NP-40, and [Y-~~PIATP (75 pM; 5 pCi/nmol). After 10 min at O'C, the reaction was stopped by addition of 50 p1 sample buffer and boiling for 5 min. Samples were analyzed by 5-15% linear gradient SDS-PAGE. Gels were stained by Coomassie blue, dried, and autoradiographed at - 70°C using Kodak X-Omat AR film. Scatchard analysis Cells were grown to near-confluency in gelatincoated 12-wells tissue culture plates. The culture medium was removed, and the cells were incubated for 3 hr at 4°C in binding buffer (25 mM HEPES) (Merck, Darmstadt, Federal Republic of Germany), and 0.1% BSA (Sigma, St. Louis, MO) in DMEM, H (7.4), containing mixtures of labeled and unlabele EGF at concentrations ranging from 0.25 to 301 ng/ml. After two washes with ice-cold phosphate-buffered saline (PBS), cells were lysed in 1M NaOH. To determine the number of cytoskeleton-bound EGF receptors, cells were extracted for 10 min at 4°C with 0.5% Triton X-100 (Sigma), 25 mM HEPES, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), pH 7.4, and washed with PBS after the EGF incubation. This treatment is sufficient to remove soluble proteins and most of the phospholipid (Van Bergen en Henegouwen et al., 1989). The amount of radioactivity in the lysate was determined by y counting. Apparent dissociation constants, number of receptors per cell, and aspecific binding were calculated by means of the LIGAND program (Munson
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and Rodbard, 1980) as described previously (Boonstra et al., 1985). Background labeling ranged between 1% and 10% of the total binding to intact cells. EGF (receptor grade; Collaborative Research, Waltham, Lexington, MA) was radiolabeled with lZ5I(Amersham, Amersham, England) according to the chloramine T (Sigma) method. Phorbol 12-myristate 13-acetate (PMA) treatment Cells were incubated with 50 nglml PMA (Sigma) in binding buffer for 45 min at 37"C, and subjected to Scatchard analysis (in the presence of 50 ng/ml PMA) and detergent extraction as described above. Control cells were treated with 50 n /ml phorbol 12,13-didecanoate (PDD; Sigma) instea of PMA. Quantitative analysis of EGF receptor clustering Cells were grown to near-confluency on thermanox coversli s (Lux Scientific Co oration, Newbury Park, CA). Fo lowing treatment wit 0.75 IU/ml s hingomyelinase (SPHase) for 1h at 37"C, cells were ixed in 2% formaldehyde in PBS at 4°C for 1 hr. After fixation, cells were immunogold labeled as described previously (van Belzen et al., 1988) using 1 pg/ml528 monoclonal (Oncogene Science Inc., antibody and a conjugate of Freezing, fracturing, and replication with PtlC were done as described previously (van Belzen et al., 1988). Electron microscopic images of exoplasmic fracture faces (EFFs) were sampled on-line by means of the Crystal image analysis system (Quantel, Newbury, England). Sample sizes were 1,000 x 700 nm. Each gold particle was assigned an x- and y-coordinate, and the coordinates were transferred to an MS-DOS computer. Cluster sizes were calculated by the Clustersize program (NvB). Gold particles were considered to be clustered if the distance between their centers was less than 35 nm (Van Belzen et al., 1988).
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RESULTS Wild-type EGF receptors from transfected cells are associated with the cytoskeleton To confirm pro er plasmid expression, NIH-3T3 fibroblasts transfecte with wild-type EGF receptor cDNA (NEF cells) were subjected to an EGF receptor autophosphorylation assay. Cell lysates were incubated with [y-3?lATP in the presence or absence of 100 ng/ml EGF, followed by gel electrophoresis and autoradiography. As is shown in Figure 1, lanes 3 and 4, EGF markedly stimulates autophosphorylation of the 170 kDa EGF receptor, demonstrating that the EGF receptor of NEF cells behaves as a wild-type receptor, as has been shown for other transfected cell lines. The number of receptors per cell and the affinity of a receptor for its ligand are usually obtained by determination of the relationship between specific binding and ligand concentration at steady state conditions, followed by analysis via the Scatchard method. Steadystate EGF binding was obtained within 3 hr of incubation in the presence of various concentrations of [1251]EGF(data not shown). The concentration depen-
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Fig. 1. EGF-dependent phosphorylation of EGF receptors. Cell lysates were preincubated for 20 min at room temperature with (+) or without (-) 100 ngiml EGF. The hosphorylation reaction was started by adding [y3'P1ATP phosphoryyation mix as described in Materials and Methods and continued for 10 min on ice. Samples were boiled in sample buffer and analyzed by 5-15% linear gradient SDS-PAGE and autoradiography .
dency of EGF binding was measured as described in Materials and Methods. As has been found in other cell lines transfected with wild-type EGF receptors (Livneh et al., 1986; Prywes et al., 19861, Scatchard analysis of [1251]EGFbinding to NEF cells yielded a curvilinear graph indicating the presence of two receptor populations differing in their affinity for EGF (Fig. 2A, Table 1).The high-affinity receptor subclass, comprising 20,000 receptorskell, bound EGF with an apparent dissociation constant (Kd) of 0.05 nM, whereas 280,000 receptorskell displayed a lower affinity for EGF (Kd 2.8 nM). Subsequently, cytoskeletons were isolated following EGF binding using 0.5% Triton X-100 as described in Materials and Methods. The Triton X-100-insoluble cytoskeletons were washed with ice-cold PBS, and the remaining [1251]EGFbinding was determined by Scatchard analysis. As is shown in Figure 2A, a curvilinear Scatchard plot was obtained, indicating that both highand low-affinity receptors were associated to the cytoskeleton. Analysis of the data revealed that cytoskeletons isolated from EGF-treated NEF cells contained 27% of the high-affinity EGF receptor subclass present
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Fig. 2. EGF-induced association of EGF receptors to the cytoskeleton. Cells were incubated with mixtures of labeled and unlabeled EGF (0.25-301 ng/ml) a t 4°C for 3 hr, followed by two washes in ice-cold PBS to remove unbound ligand. Cytoskeletons were prepared by extracting the cells with 0.5%Triton X-100, 25 mM HEPES, 1 mM PMSF (pH 7.4) at 4°C for 10 min and washed with PBS just after the EGF treatment. The amount of radioactivity present on cells or cytoskeletons was determined by y counting. The binding data were interpreted by means of the LIGAND program (Munson and Rodbard,
1980) and plotted according to the method of Scatchard (1949).Circles represent control cells not extracted with detergent. Triangles show the amount of EGF bound to cytoskeletons isolated from EGF-treated cells. Results shown represent the mean of several independent experiments (see Table 1). Note that the lower panels are enlar e ments of the upper panels. A: NEF cells that express wild-type E8F receptors. B: CD126 cells that express EGF receptors lacking the 126 C-terminal amino acids. C: K721A cells that express kinase-deficient EGF receptors.
TABLE 1. EGF binding characteristics of cells and cytoskeletons containing wild-type, kinase-deficient, or truncated EGF receptors'
Intact cells Cell line
Kdl
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Cytoskeletons isolated from EGF-treated cells R1 Kdz Rz 5,400
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'Cells were incubated with mixtures of labeled and unlabeled EGF (0.25-301 ng/ml) for 3 hr at 4OC, washed twice in ice-cold PBS to remuve unbound ligand, and then either lysed directly in 1 M NaOH or extracted with 0.6% Triton X-100,25 mM HEPES, 1mMPMSF(pH7.4)for10min at4"Ctoisolate thecytoskeleton,priortolysis.Theamountofcell-orcytoskeletonasociated radioactivityin the lysate was determined by y counting. Analysis of the binding data by means of the LIGAND program (Munson and Rodbard, 1980) yielded the apparent dissociation constants (expressed in nM) and the number of receptors per cell of the high. and low-affinity EGF receptors (Kdl, Kdz, R1, and Rz. respectively) n denotes the number of rxperiments.
in intact cells and 4% of the low-affinity receptor subclass (Fig. 2A, Table 1).Qualitatively similar results (Van Belzen, unpublished) were obtained with HER14, another mouse fibroblast cell line transfected with wild-type human EGF receptor cDNA. These data indicate that EGF receptors from NEF and HER14 cells resemble EGF receptors from the nontransfected A431 cell line with respect to their association with the cytoskeleton. A striking difference, however, is the observation that in A431 cells all high-affinity receptors appeared to be associated to the cytoskeleton (Van Bergen en Henegouwen et al., 1989), whereas in NEF cells only a fraction is cytoskeleton bound. EGF receptor-cytoskeleton interaction depends on the cytoplasmic receptor domain EGF receptors may associate to the cytoskeleton either via their cytoplasmic domain or via their extra-
cellular domain by interaction with a cytoskeletonbound transmembrane molecule. Therefore, we investigated the cytoskeleton binding properties of EGF receptor mutants that contain only nine cytoplasmic amino acid residues (Livneh et al., 1986).Cells expressin this mutant receptor, designated 3T3-IV cells, were su jected to Scatchard analysis of [lz5I1EGFbinding at 4"C, followed by extraction with 0.5% Triton X-100 for 10 min at 4°C to isolate the cytoskeleton. In agreement with Livneh et al. (1986),we observed only one class of EGF binding sites in these cells. Intact 3T3-IV cells exhibited 153,000 EGF receptors, which bound EGF with a Kd of 0.49 nM. In contrast, cytoskeletons isolated from EGF-treated 3T3-IV cells contained only background levels of [lz5I1EGFbinding (Fig. 3), indicating that EGF receptors interact with the cytoskeleton via their cytoplasmic domain, not indirectly via their extracellular domain.
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Fig. 3. Deletion of the cytoplasmic domain abolishes cytoskeleton association. 3T3dV cells were treated as described for Figure 2. Open circles represent intact cells; filled circles represent cytoskeletons. Results shown are the mean of three independent experiments performed in duplicate.
Deletion of EGF receptor autophosphorylation sites does not impair interaction with the cytoskeleton The structural interaction between EGF receptors and the cytoskeleton and its EGF-induced increase suggest that an EGF-induced conformational change of the receptor or of a cytoskeletal component causes the interaction with the cytoskeleton. An attractive possibility in this respect appears a conformational change of the receptor molecule induced by rece tor selfphosphorylation. Therefore, we have studie EGF receptor-cytoskeleton association in CD126 cells, a transfected cell line that expresses EGF receptors from which amino acid residues 1061-1186, including the major auto hosphorylation sites, have been deleted. As expected, t is cell line does not contain the 170 kDa phosphorylated EGF receptor (Fig. 1, lanes 5 and 6). However, a faint signal of a slightly smaller phosphoprotein, probably the truncated EGF receptor, is present. This mutant receptor has been shown to be phosphorylated on novel sites not used in wild-type EGF receptors (Margolis et al., 1989). As is shown in Figure 2B, EGF binding in this cell line is characterized by a curvilinear Scatchard graph, indicating the presence of both high- and low-affinity binding sites, comparable to NEF cells. Analysis of the data revealed a high-affinity receptor class with a Kd of 0.02 nM and 3,400 receptorsicell and a low-affinity class with a Kd of 4.1 nM and 171,000 receptorskell (Table 1). Cytoskeletons isolated from EGF-treated CD126 cells contained 47% of the number of highaffinity EGF receptors present on intact cells and 8%of the low-affinity receptor population (Fig. 2B, Table 1). These results clearly demonstrate that neither the autophosphorylation sites nor the 126 C-terminal amino acid residues of the EGF receptor are involved in its interaction with the cytoskeleton. Kinase-deficient EGF receptor mutants are capable of cytoskeleton binding The EGF receptor possesses intrinsic tyrosine kinase activity, which is considered to be of vital importance
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for proper receptor functioning. To determine whether this kinase activity is involved in the association of EGF receptors with the cytoskeleton, possibly due to conformational changes of cytoskeletal components by EGF-induced phosphorylation, we studied the [1251]EGF binding characteristics of K721A cells and cytoskeletons. As demonstrated in Figure 1, lanes 1 and 2, these cells express mutant EGF receptors that do not possess intrinsic kinase activity (Honegger et al., 1987a).As is shown in Figure 2C, Scatchard analysis of [12511EGFbinding to these cells indicates the presence of high- and low-affinity receptors, as was reported previously (Hone er et al., 1987a). K721A cells exhibit 5,800 high ( d 0.08 nM) and 51,000 low (Kd 3.6 nM) EGF receptors (Table 1). Cytoskeletons isolated from EGF-treated K721A cells also contained both classes of binding sites (Fig. 2C). From the data presented in Table 1, it can be concluded that 1 4 9 of the high-affinity and 5% of the low-affinity receptors are induced by EGF to bind the K721A cytoskeleton. These results clearly demonstrate that the kinase activity of the EGF receptor is not involved in its interaction with the cytoskeleton, implying that EGF-induced phosphorylation of cytoskeletal elements is not involved in the association of the receptor to the cytoskeleton. Role of high-affinity receptors in receptorcytoskeleton interaction Characterization of the interaction between EGF receptors and the cytoskeleton in the transfected cell lines, as well as in A431 cells (Van Bergen en Henegouwen et al., 1989), revealed that this receptor population is enriched in high-affinity receptors. Since the high-affinity receptors have been indicated as the most important receptor subclass in the EGF-induced responses, it is tempting to speculate that the highaffinity state of the EGF receptor is responsible for its interaction with the cytoskeleton. Therefore, we have studied the effect of the tumor promoter PMA on EGF receptor-cytoskeleton interaction in NEF cells. PMA is known to affect specifically the high-affinity EGF receptors in a wide variety of cell lines (King and Cuatrecasas, 1982; Boonstra et al., 1985). Incubation of NEF cells with 50 nglml PMA for 45 min a t 37°C resulted in a loss of high-affinity binding (Fig. 4A), without a significant effect on low-affinity binding. Thus these data are in agreement with the effects of PMA on EGF binding in other cells. When these cells were consecutively treated with PMA, ['2511EGF, and Triton X-100, the resulting cytoskeletons contained only low-affinity EGF receptors (Fig. 4B), indicating that the hi h-affinity site of the EGF receptor is not essential or the interaction of low-affinity receptors with the cytoskeleton. Similar results were found in K721A and CD126 cells (data not shown). Relationship between clustering and cytoskeleton association of EGF receptors The data described above demonstrate that receptorcytoskeleton interaction does not result from EGF receptor self-phosphorylation, kinase activity, or high affinity. A clue to the cause of receptor-cytoskeleton association comes from the observation that concanava-
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Fig. 4. Effect of PMA on the EGF binding characteristics of intact cells and on the EGF-induced association of EGF receptors to the cytoskeleton. NEF cells were treated with 50 ngiml PMA (or PDD as a control) for 45 min at 37°C and subjected to Scatchard analysis of EGF binding in the continued presence of the respectivephorbol esters for 3 hr at 4°C. Subsequently, cells were either lysed directly (A) or extracted with Triton X-100 prior to lysis (B) (see Materials and Methods for details of the procedure).The amount of cell- or cytoskeleton-associated radioactivity was determined and plotted as described for Figure 2. Filled circles represent control cells (A) and cytoskeletons (B),open circles represent cells (A) and cytoskeletons (B) treated with PMA. Data shown represent the results obtained from three independent experiments.
lin A (Con A) increases the fraction of EGF receptors cou led to the cytoskeleton of pheochromocytoma cells (Va e and Shooter, 1983). These findings are in a reement with a model proposed by Brandts and Jaco son (19831, who suggest that association of plasma membrane proteins with the cytoskeleton is due to protein clustering. To examine the effect of Con A on the binding of wild-type EGF receptors to the cytoskeleton, we used the NEF cell line. Cells were labeled with EGF at 4"C, followed by a treatment with increasing concentrations of Con A at 4°C for 1 hr and isolation of the cytoskeleton. In agreement with previous findings (Vale and Shooter, 1983), Con A was found to induce a concentration-dependent increase in the binding of EGF to the NEF cytoskeleton, up to five times control values at the highest Con A concentration tested. In contrast, the amount of EGF bound to intact cells was not affected by Con A. However, similar results were obtained in cells expressing the 3T3-IV receptor mutant that lacks the cytoplasmic receptor domain. Apparently Con A cross links the truncated EGF receptors to cytoskeletonbound membrane proteins, inducing an artificial interaction with the cytoskeleton. These findings indicate
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that Con A, due to its cross linking properties, is unsuitable to demonstrate specific membrane proteincytoskeleton interaction and that its use as such is incorrect. Other studies have led to similar conclusions (Jung et al., 1984; Vale et al., 1985; Patton et al., 1989). To establish whether clustering of EGF receptors is related to their association to the cytoskeleton, we have sought for an alternative method that causes clustering of EGF receptors in an EGF-independent manner, without cross linking of the receptor to other membrane proteins. Clustering of membrane proteins has been reported to occur as a result of treatment of cells with SPHase. This enzyme, which degrades sphingomyelin into ceramides and phosphorylcholine, has been shown to induce intramembrane particle aggregation in erythrocytes (Verkleij et al., 1973). To investigate whether extracellularly applied SPHase degraded sphingomyelin in our cell system, NEF cells were treated with 0-4 IUlml SPHase (purified according to Zwaal et al., 1975) for 1 hr at 37°C in a buffer containing 150 mM NaC1, 5 mM KC1, 1 mM MgC12, 1 mM CaC12, 5 mM glucose, 20 mM Tris, pH 7.4 (Verkleij et al., 1973). Following the li ase incubation, the incubation medium was checked or lactate dehydrogenase activity, which turned out to be insi nificant, indicating that no cell lysis had occurre . Subsequently, cellular lipid was extracted, li id species were separated by means of thin-layer cp1romatography (TLC), and the amount of phosphorus present in the individual TLC spots was determined. The SPHase treatment resulted in degradation of sphingomyelin from 11.4% in control cells to below 3% in cells incubated with 0.5 IU/ml SPHase or more. The SPHaseresistant sphin omyelin apparently is located in the inner leaflet o f t e plasma membrane. The other phospholipid species examined (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol) were not degraded, indicating that the SPHase preparation was free of other lipases. Effect of sphingomyelinase on plasma membrane protein aggregation To ascertain whether sphingomyelin degradation results in membrane protein clustering, NEF cells were treated with 0.75 IU/ml SPHase for 1 hr at 3TC, followed by fixation, immunogold labeling with monoclonal anti-EGF receptor antibody, and freeze-fracturing as described in Materials and Methods. As is shown in Figure 5, this treatment resulted in a strong aggregation of intramembrane particles. The effect of sphingomyelin degradation on the lateral distribution of EGF receptors was studied uantitatively using the approach developed previous y (Van Belzen et al., 1988) as described in Materials and Methods. As is shown in Figure 5C, SPHase treatment induced an increase in the number of small (two or three) and larger (four or more) EGF receptor clusters. Thus, degradation of sphingomyelin results in aggregation of plasma membrane proteins, including the EGF receptor. Effect of sphingomyelinase on EGF receptorcytoskeleton interaction Subsequently, the effect of SPHase on EGF receptorcytoskeleton interaction was studied. Treatment of
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Interaction of Epidermal Growth Factor Receptors With the Cytoskeleton Is Related to Receptor Clustering NlCO VAN BELZEN, MARCEL SPAARGAREN, ARlE J. VERKLEIJ, AND JOHANNESBOONSTRA* Department of Molecular Cell Biology, University of Utrecht, 3584 CH Utrecht, The M.S., A.I.V., 1.5.); Hubrecht laboratory, Netherlands Institute of Netherlands (N.v.~., Developmental Biology, 3584 CT Utrecht, The Netherlands (M.S.)
Recently it has been established that cytoskeleton-associated epidermal growth factor (EGF) receptors are predominantly of the high-affinity class and that EGF induces a recruitment of low-affinity receptors to the cytoskeleton. The nature of this EGF-induced receptor-cytoskeleton interaction, however, is still unknown. Therefore, we have studied the association of mutated EGF receptors with the cytoskeleton. Receptor deletion mutants lacking almost all intracellular amino acid residues displayed no interaction with the cytoskeleton, demonstrating that the cytoplasmic receptor domain is involved in this interaction. Further analysis revealed that receptor-cytoskeletoninteraction is independent of receptor kinase activity and the C-terminal 126 amino acid residues, which include the autophosphorylation sites. Furthermore, it is shown that the high-affinity receptor subclass is not essential for association of low-affinity receptors to the cytoskeleton. EGF receptor-cytoskeleton interaction was increased, however, by treatment with sphingomyelinase, an enzyme known to induce membrane protein clustering, indicating that EGF receptor clustering may cause the association to the cytoskeleton. Epidermal growth factor (EGF) is a well known mitogen for a wide variety of mammalian cells (for reviews see Carpenter and Cohen, 1979; De Laat et al., 1983; Schlessinger, 1988). It binds specifically to a 170 kDa transmembrane protein, the EGF rece tor. EGF bindin studies indicate that most target ce 1s exhibit two E F receptor subclasses, one class binding EGF with high affinity and low capacity, the other binding with low affinity and high capacity (King and Cuatrecasas, 1982; Blay and Brown, 1985; Boonstra et al., 1985). The most convincing evidence in favor of the existence of two classes of binding sites was obtained in studies using antibodies directed against the EGF receptor (Gregoriou and Rees, 1984; Defize et al., 1989). In articular the monoclonal anti-EGF receptor antibo y 2E9 has been shown to exhibit unique properties in this respect. This antibod blocks specifically the binding of EGF to the low-a inity EGF receptors in a variety of cell lines (Defize et al., 1988, 1989; Boonstra and Ponec, 1989). Using the 2E9 antibody, it was recently demonstrated that activation of the EGF receptor signal transduction cascade, i.e., inositol hosphate production, release of Ca2+ from intrace ular stores, rise in intracellular pH in the absence of HC03-, stimulation of protein kinase C activity, activation of EGF receptor protein tyrosine kinase activity, and induction of c-fos gene expression, can occur completely through exclusive binding of EGF to the high-affinity
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receptor subclass (Defize et al., 1989). Recently these findings were confirmed by means of an antibody that specifically blocked the binding of EGF to the highaffinity subclass (Bellot et al., 1990). The activation of the EGF receptor protein tryosine kinase by binding of EGF has been indicated as a key event in the initiation of signal transduction (Honegger et al., 1987a,b; Moolenaar et al., 1988). Activation of the EGF receptor tyrosine kinase results in phosphorylation of the receptor itself (Carpenter et al., 1979; King et al., 1980) and various other substrates, including a number of cytoskeleton-related proteins, such as cal actidlipocortin (Fava and Cohen, 1984; Sawyer an Cohen, 1985; Pepinsky and Sinclair, 19861, ezrin (Gould et al., 1986; Bretscher, 1989), fodrin, tubulin, tau, and microtubule-associated protein 2 (Akiyama et al., 1986). These latter observations are of particular interest since part of the EGF receptor population has been demonstrated to be associated to the Triton X-100-insoluble cytoskeleton of A431 cells (Landreth et al., 1985; Wiegant et al., 1986). In A431 cells the cytoskeleton-associated fraction of
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Received March 29, 1990; accepted August 1, 1990. *Towhom reprint requestsicorrespondence should be addressed. Nico van Belzen’s present address is Department of Pathology, University of Limburg, Maastricht, The Netherlands.
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the EGF receptor population represents specifically the high-affinity class of receptors (Wiegant et al., 1986; Van Bergen en Henegouwen et al., 1989). Addition of EGF to intact cells results in an additional association of low-affinity receptors t o the cytoskeleton (Van Bergen en Henegouwen et al., 1989). At present the nature of the interaction between EGF receptor and cytoskeleton is unknown, but it seems plausible to suggest that, since EGF causes an increased association of the receptors to the cytoskeleton, EGF binding has to lead to a conformational change of the receptor or of cytoskeletal components, to allow the binding of the receptor to the cytoskeleton. In this respect an important role of the autophosphorylation sites and/or of the kinase activity of the receptor can be proposed, analogous to the platelet-derived growth factor (PDGF) receptor, whose interaction with cellular proteins was shown to depend on receptor autophosphorylation (Kazlauskas and Cooer, 1989). Therefore, we have studied the interaction etween EGF receptor and cytoskeleton in mouse NIH3T3 fibroblasts transfected with cDNA constructs encoding normal or mutated human EGF receptors (Honegger et al., 1987a,b). The cell lines used include NEF cells, expressing the wild-type EGF receptor; CD126 cells, expressing a truncated receptor lacking the C-terminal amino acid residues 1061-1186 (including the major autophosphorylation sites); and the kinasenegative K721A cell line, in which lysine7" in the ATP binding site was replaced by an alanine residue, preventin adenosine triphosphate (ATP) binding and hence inase activity. Using these cell lines, we demonstrate in this paper that the interaction between the EGF receptor and the cytoskeleton does not require the receptor autophosphorylation sites and receptor kinase activity. Furthermore, the C-terminal 126 amino acid residues are not involved in the association. Using the tumor promoter phorbol myristate acetate (PMA), we show that the high-affinity receptors are not essential for binding of low-affinity receptors to the cytoskeleton. On the basis of theoretical considerations, it has been proposed that receptor-cytoskeleton interaction could result from clustering of receptors by multivalent ligands (Brandts and Jacobson, 1983; Jacobson, 1983). The hypothesis is based on the assumption that monomeric plasma membrane proteins contain translational entropy that revents their association to cytoskeletal com onents. of membrane proteins by multiva ent extracellular ligands results in a decrease of translational entropy and hence in an increased affinity for intracellular binding sites. EGF is assumed to bind its receptor monovalently but is nevertheless known to induce receptor clustering (Zidovetski et al., 1981; Hopkins et al., 1985; van Belzen et al., 1988; Torrisi et al., 1988). The EGF-induced receptor clustering could well explain the EGF-driven association of the receptor to the cytoskeleton. To provide evidence for this possible relationship, we have studied the effects of EGF-independent clustering of receptors on their association to the cytoskeleton. Using sphingomyelinase, it is demonstrated that increased clustering of membrane proteins, including the EGF receptor, is accompanied by an increase in the fraction of EGF receptors associated to the cytoskeleton.
1
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MATERIALS AND METHODS Cell culture Mouse NIH-3T3 fibroblasts transfected with human EGF receptor cDNA were kindly provided by Dr. J. Schlessinger (Rorer Biotechnology Inc., King of Prussia, PA). NEF cells contain wild-type human EGF receptors. 3T3-IV cells contain EGF receptors from which all cytoplasmic amino acid residues but nine have been deleted (Prywes et al., 1986; Livneh et al., 1986). K721A cells contain EGF receptors in which amino acid residue Lys721is replaced by Ala721,resulting in a loss of rece tor kinase activity (Honegger et al., 1987a). CD126 cel s contain EGF receptors from which 126 C-terminal amino acid residues 1061-1186 have been deleted (Margolis et al., 1989). Cells were cultured in Dulbecco's modified Ea le's medium (DMEM), supplemented with 7.5% feta calf serum (Inte ro, Zaandam, The Netherlands), and buffered wit 44 mM NaHC03, in a 7% C02 humidified atmosphere. Tissue culture plastics (75 cm2 flasks and 12-wells plates) were from Costar (Cambridge, MA).
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EGF receptor autophosphorylation Total cell lysates were prepared from subconfluent 75 cm2 dishes as described previously (Defize et al., 1989) in a volume of 500 pl. Aliquots from this lysate (40 pl) were incubated in the absence or presence of 100 ng/ml EGF for 20 min at room temperature. The phosphorylation reaction was initiated by addition of 10 p1 phosphorylation assay mix, containing 100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid (HEPES) (pH 7.4), 10 mM MnC12, 50 mM p-nitrophenylphosphate, 200 IJ.MNa3V04, 0.05% bovine serum albumin (BSA), 1%NP-40, and [Y-~~PIATP (75 pM; 5 pCi/nmol). After 10 min at O'C, the reaction was stopped by addition of 50 p1 sample buffer and boiling for 5 min. Samples were analyzed by 5-15% linear gradient SDS-PAGE. Gels were stained by Coomassie blue, dried, and autoradiographed at - 70°C using Kodak X-Omat AR film. Scatchard analysis Cells were grown to near-confluency in gelatincoated 12-wells tissue culture plates. The culture medium was removed, and the cells were incubated for 3 hr at 4°C in binding buffer (25 mM HEPES) (Merck, Darmstadt, Federal Republic of Germany), and 0.1% BSA (Sigma, St. Louis, MO) in DMEM, H (7.4), containing mixtures of labeled and unlabele EGF at concentrations ranging from 0.25 to 301 ng/ml. After two washes with ice-cold phosphate-buffered saline (PBS), cells were lysed in 1M NaOH. To determine the number of cytoskeleton-bound EGF receptors, cells were extracted for 10 min at 4°C with 0.5% Triton X-100 (Sigma), 25 mM HEPES, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), pH 7.4, and washed with PBS after the EGF incubation. This treatment is sufficient to remove soluble proteins and most of the phospholipid (Van Bergen en Henegouwen et al., 1989). The amount of radioactivity in the lysate was determined by y counting. Apparent dissociation constants, number of receptors per cell, and aspecific binding were calculated by means of the LIGAND program (Munson
t;
CYTOSKELETON-ASSOCIATED EGF RECEPTORS
and Rodbard, 1980) as described previously (Boonstra et al., 1985). Background labeling ranged between 1% and 10% of the total binding to intact cells. EGF (receptor grade; Collaborative Research, Waltham, Lexington, MA) was radiolabeled with lZ5I(Amersham, Amersham, England) according to the chloramine T (Sigma) method. Phorbol 12-myristate 13-acetate (PMA) treatment Cells were incubated with 50 nglml PMA (Sigma) in binding buffer for 45 min at 37"C, and subjected to Scatchard analysis (in the presence of 50 ng/ml PMA) and detergent extraction as described above. Control cells were treated with 50 n /ml phorbol 12,13-didecanoate (PDD; Sigma) instea of PMA. Quantitative analysis of EGF receptor clustering Cells were grown to near-confluency on thermanox coversli s (Lux Scientific Co oration, Newbury Park, CA). Fo lowing treatment wit 0.75 IU/ml s hingomyelinase (SPHase) for 1h at 37"C, cells were ixed in 2% formaldehyde in PBS at 4°C for 1 hr. After fixation, cells were immunogold labeled as described previously (van Belzen et al., 1988) using 1 pg/ml528 monoclonal (Oncogene Science Inc., antibody and a conjugate of Freezing, fracturing, and replication with PtlC were done as described previously (van Belzen et al., 1988). Electron microscopic images of exoplasmic fracture faces (EFFs) were sampled on-line by means of the Crystal image analysis system (Quantel, Newbury, England). Sample sizes were 1,000 x 700 nm. Each gold particle was assigned an x- and y-coordinate, and the coordinates were transferred to an MS-DOS computer. Cluster sizes were calculated by the Clustersize program (NvB). Gold particles were considered to be clustered if the distance between their centers was less than 35 nm (Van Belzen et al., 1988).
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RESULTS Wild-type EGF receptors from transfected cells are associated with the cytoskeleton To confirm pro er plasmid expression, NIH-3T3 fibroblasts transfecte with wild-type EGF receptor cDNA (NEF cells) were subjected to an EGF receptor autophosphorylation assay. Cell lysates were incubated with [y-3?lATP in the presence or absence of 100 ng/ml EGF, followed by gel electrophoresis and autoradiography. As is shown in Figure 1, lanes 3 and 4, EGF markedly stimulates autophosphorylation of the 170 kDa EGF receptor, demonstrating that the EGF receptor of NEF cells behaves as a wild-type receptor, as has been shown for other transfected cell lines. The number of receptors per cell and the affinity of a receptor for its ligand are usually obtained by determination of the relationship between specific binding and ligand concentration at steady state conditions, followed by analysis via the Scatchard method. Steadystate EGF binding was obtained within 3 hr of incubation in the presence of various concentrations of [1251]EGF(data not shown). The concentration depen-
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Fig. 1. EGF-dependent phosphorylation of EGF receptors. Cell lysates were preincubated for 20 min at room temperature with (+) or without (-) 100 ngiml EGF. The hosphorylation reaction was started by adding [y3'P1ATP phosphoryyation mix as described in Materials and Methods and continued for 10 min on ice. Samples were boiled in sample buffer and analyzed by 5-15% linear gradient SDS-PAGE and autoradiography .
dency of EGF binding was measured as described in Materials and Methods. As has been found in other cell lines transfected with wild-type EGF receptors (Livneh et al., 1986; Prywes et al., 19861, Scatchard analysis of [1251]EGFbinding to NEF cells yielded a curvilinear graph indicating the presence of two receptor populations differing in their affinity for EGF (Fig. 2A, Table 1).The high-affinity receptor subclass, comprising 20,000 receptorskell, bound EGF with an apparent dissociation constant (Kd) of 0.05 nM, whereas 280,000 receptorskell displayed a lower affinity for EGF (Kd 2.8 nM). Subsequently, cytoskeletons were isolated following EGF binding using 0.5% Triton X-100 as described in Materials and Methods. The Triton X-100-insoluble cytoskeletons were washed with ice-cold PBS, and the remaining [1251]EGFbinding was determined by Scatchard analysis. As is shown in Figure 2A, a curvilinear Scatchard plot was obtained, indicating that both highand low-affinity receptors were associated to the cytoskeleton. Analysis of the data revealed that cytoskeletons isolated from EGF-treated NEF cells contained 27% of the high-affinity EGF receptor subclass present
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Fig. 2. EGF-induced association of EGF receptors to the cytoskeleton. Cells were incubated with mixtures of labeled and unlabeled EGF (0.25-301 ng/ml) a t 4°C for 3 hr, followed by two washes in ice-cold PBS to remove unbound ligand. Cytoskeletons were prepared by extracting the cells with 0.5%Triton X-100, 25 mM HEPES, 1 mM PMSF (pH 7.4) at 4°C for 10 min and washed with PBS just after the EGF treatment. The amount of radioactivity present on cells or cytoskeletons was determined by y counting. The binding data were interpreted by means of the LIGAND program (Munson and Rodbard,
1980) and plotted according to the method of Scatchard (1949).Circles represent control cells not extracted with detergent. Triangles show the amount of EGF bound to cytoskeletons isolated from EGF-treated cells. Results shown represent the mean of several independent experiments (see Table 1). Note that the lower panels are enlar e ments of the upper panels. A: NEF cells that express wild-type E8F receptors. B: CD126 cells that express EGF receptors lacking the 126 C-terminal amino acids. C: K721A cells that express kinase-deficient EGF receptors.
TABLE 1. EGF binding characteristics of cells and cytoskeletons containing wild-type, kinase-deficient, or truncated EGF receptors'
Intact cells Cell line
Kdl
R1
Kd2
Rz
n
Kdl
NEF
0.05
0.08
7 10
0.02
3,400
2.8 3.6 4.1
280,000
K721A CD126
20,000 5,800
0.03 0.03 0.09
51,000 171,000
3
Cytoskeletons isolated from EGF-treated cells R1 Kdz Rz 5,400
800 1,600
n
3.4 3.2
12,000 2,400
7 8
3.0
14,000
3
'Cells were incubated with mixtures of labeled and unlabeled EGF (0.25-301 ng/ml) for 3 hr at 4OC, washed twice in ice-cold PBS to remuve unbound ligand, and then either lysed directly in 1 M NaOH or extracted with 0.6% Triton X-100,25 mM HEPES, 1mMPMSF(pH7.4)for10min at4"Ctoisolate thecytoskeleton,priortolysis.Theamountofcell-orcytoskeletonasociated radioactivityin the lysate was determined by y counting. Analysis of the binding data by means of the LIGAND program (Munson and Rodbard, 1980) yielded the apparent dissociation constants (expressed in nM) and the number of receptors per cell of the high. and low-affinity EGF receptors (Kdl, Kdz, R1, and Rz. respectively) n denotes the number of rxperiments.
in intact cells and 4% of the low-affinity receptor subclass (Fig. 2A, Table 1).Qualitatively similar results (Van Belzen, unpublished) were obtained with HER14, another mouse fibroblast cell line transfected with wild-type human EGF receptor cDNA. These data indicate that EGF receptors from NEF and HER14 cells resemble EGF receptors from the nontransfected A431 cell line with respect to their association with the cytoskeleton. A striking difference, however, is the observation that in A431 cells all high-affinity receptors appeared to be associated to the cytoskeleton (Van Bergen en Henegouwen et al., 1989), whereas in NEF cells only a fraction is cytoskeleton bound. EGF receptor-cytoskeleton interaction depends on the cytoplasmic receptor domain EGF receptors may associate to the cytoskeleton either via their cytoplasmic domain or via their extra-
cellular domain by interaction with a cytoskeletonbound transmembrane molecule. Therefore, we investigated the cytoskeleton binding properties of EGF receptor mutants that contain only nine cytoplasmic amino acid residues (Livneh et al., 1986).Cells expressin this mutant receptor, designated 3T3-IV cells, were su jected to Scatchard analysis of [lz5I1EGFbinding at 4"C, followed by extraction with 0.5% Triton X-100 for 10 min at 4°C to isolate the cytoskeleton. In agreement with Livneh et al. (1986),we observed only one class of EGF binding sites in these cells. Intact 3T3-IV cells exhibited 153,000 EGF receptors, which bound EGF with a Kd of 0.49 nM. In contrast, cytoskeletons isolated from EGF-treated 3T3-IV cells contained only background levels of [lz5I1EGFbinding (Fig. 3), indicating that EGF receptors interact with the cytoskeleton via their cytoplasmic domain, not indirectly via their extracellular domain.
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Fig. 3. Deletion of the cytoplasmic domain abolishes cytoskeleton association. 3T3dV cells were treated as described for Figure 2. Open circles represent intact cells; filled circles represent cytoskeletons. Results shown are the mean of three independent experiments performed in duplicate.
Deletion of EGF receptor autophosphorylation sites does not impair interaction with the cytoskeleton The structural interaction between EGF receptors and the cytoskeleton and its EGF-induced increase suggest that an EGF-induced conformational change of the receptor or of a cytoskeletal component causes the interaction with the cytoskeleton. An attractive possibility in this respect appears a conformational change of the receptor molecule induced by rece tor selfphosphorylation. Therefore, we have studie EGF receptor-cytoskeleton association in CD126 cells, a transfected cell line that expresses EGF receptors from which amino acid residues 1061-1186, including the major auto hosphorylation sites, have been deleted. As expected, t is cell line does not contain the 170 kDa phosphorylated EGF receptor (Fig. 1, lanes 5 and 6). However, a faint signal of a slightly smaller phosphoprotein, probably the truncated EGF receptor, is present. This mutant receptor has been shown to be phosphorylated on novel sites not used in wild-type EGF receptors (Margolis et al., 1989). As is shown in Figure 2B, EGF binding in this cell line is characterized by a curvilinear Scatchard graph, indicating the presence of both high- and low-affinity binding sites, comparable to NEF cells. Analysis of the data revealed a high-affinity receptor class with a Kd of 0.02 nM and 3,400 receptorsicell and a low-affinity class with a Kd of 4.1 nM and 171,000 receptorskell (Table 1). Cytoskeletons isolated from EGF-treated CD126 cells contained 47% of the number of highaffinity EGF receptors present on intact cells and 8%of the low-affinity receptor population (Fig. 2B, Table 1). These results clearly demonstrate that neither the autophosphorylation sites nor the 126 C-terminal amino acid residues of the EGF receptor are involved in its interaction with the cytoskeleton. Kinase-deficient EGF receptor mutants are capable of cytoskeleton binding The EGF receptor possesses intrinsic tyrosine kinase activity, which is considered to be of vital importance
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for proper receptor functioning. To determine whether this kinase activity is involved in the association of EGF receptors with the cytoskeleton, possibly due to conformational changes of cytoskeletal components by EGF-induced phosphorylation, we studied the [1251]EGF binding characteristics of K721A cells and cytoskeletons. As demonstrated in Figure 1, lanes 1 and 2, these cells express mutant EGF receptors that do not possess intrinsic kinase activity (Honegger et al., 1987a).As is shown in Figure 2C, Scatchard analysis of [12511EGFbinding to these cells indicates the presence of high- and low-affinity receptors, as was reported previously (Hone er et al., 1987a). K721A cells exhibit 5,800 high ( d 0.08 nM) and 51,000 low (Kd 3.6 nM) EGF receptors (Table 1). Cytoskeletons isolated from EGF-treated K721A cells also contained both classes of binding sites (Fig. 2C). From the data presented in Table 1, it can be concluded that 1 4 9 of the high-affinity and 5% of the low-affinity receptors are induced by EGF to bind the K721A cytoskeleton. These results clearly demonstrate that the kinase activity of the EGF receptor is not involved in its interaction with the cytoskeleton, implying that EGF-induced phosphorylation of cytoskeletal elements is not involved in the association of the receptor to the cytoskeleton. Role of high-affinity receptors in receptorcytoskeleton interaction Characterization of the interaction between EGF receptors and the cytoskeleton in the transfected cell lines, as well as in A431 cells (Van Bergen en Henegouwen et al., 1989), revealed that this receptor population is enriched in high-affinity receptors. Since the high-affinity receptors have been indicated as the most important receptor subclass in the EGF-induced responses, it is tempting to speculate that the highaffinity state of the EGF receptor is responsible for its interaction with the cytoskeleton. Therefore, we have studied the effect of the tumor promoter PMA on EGF receptor-cytoskeleton interaction in NEF cells. PMA is known to affect specifically the high-affinity EGF receptors in a wide variety of cell lines (King and Cuatrecasas, 1982; Boonstra et al., 1985). Incubation of NEF cells with 50 nglml PMA for 45 min a t 37°C resulted in a loss of high-affinity binding (Fig. 4A), without a significant effect on low-affinity binding. Thus these data are in agreement with the effects of PMA on EGF binding in other cells. When these cells were consecutively treated with PMA, ['2511EGF, and Triton X-100, the resulting cytoskeletons contained only low-affinity EGF receptors (Fig. 4B), indicating that the hi h-affinity site of the EGF receptor is not essential or the interaction of low-affinity receptors with the cytoskeleton. Similar results were found in K721A and CD126 cells (data not shown). Relationship between clustering and cytoskeleton association of EGF receptors The data described above demonstrate that receptorcytoskeleton interaction does not result from EGF receptor self-phosphorylation, kinase activity, or high affinity. A clue to the cause of receptor-cytoskeleton association comes from the observation that concanava-
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Fig. 4. Effect of PMA on the EGF binding characteristics of intact cells and on the EGF-induced association of EGF receptors to the cytoskeleton. NEF cells were treated with 50 ngiml PMA (or PDD as a control) for 45 min at 37°C and subjected to Scatchard analysis of EGF binding in the continued presence of the respectivephorbol esters for 3 hr at 4°C. Subsequently, cells were either lysed directly (A) or extracted with Triton X-100 prior to lysis (B) (see Materials and Methods for details of the procedure).The amount of cell- or cytoskeleton-associated radioactivity was determined and plotted as described for Figure 2. Filled circles represent control cells (A) and cytoskeletons (B),open circles represent cells (A) and cytoskeletons (B) treated with PMA. Data shown represent the results obtained from three independent experiments.
lin A (Con A) increases the fraction of EGF receptors cou led to the cytoskeleton of pheochromocytoma cells (Va e and Shooter, 1983). These findings are in a reement with a model proposed by Brandts and Jaco son (19831, who suggest that association of plasma membrane proteins with the cytoskeleton is due to protein clustering. To examine the effect of Con A on the binding of wild-type EGF receptors to the cytoskeleton, we used the NEF cell line. Cells were labeled with EGF at 4"C, followed by a treatment with increasing concentrations of Con A at 4°C for 1 hr and isolation of the cytoskeleton. In agreement with previous findings (Vale and Shooter, 1983), Con A was found to induce a concentration-dependent increase in the binding of EGF to the NEF cytoskeleton, up to five times control values at the highest Con A concentration tested. In contrast, the amount of EGF bound to intact cells was not affected by Con A. However, similar results were obtained in cells expressing the 3T3-IV receptor mutant that lacks the cytoplasmic receptor domain. Apparently Con A cross links the truncated EGF receptors to cytoskeletonbound membrane proteins, inducing an artificial interaction with the cytoskeleton. These findings indicate
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that Con A, due to its cross linking properties, is unsuitable to demonstrate specific membrane proteincytoskeleton interaction and that its use as such is incorrect. Other studies have led to similar conclusions (Jung et al., 1984; Vale et al., 1985; Patton et al., 1989). To establish whether clustering of EGF receptors is related to their association to the cytoskeleton, we have sought for an alternative method that causes clustering of EGF receptors in an EGF-independent manner, without cross linking of the receptor to other membrane proteins. Clustering of membrane proteins has been reported to occur as a result of treatment of cells with SPHase. This enzyme, which degrades sphingomyelin into ceramides and phosphorylcholine, has been shown to induce intramembrane particle aggregation in erythrocytes (Verkleij et al., 1973). To investigate whether extracellularly applied SPHase degraded sphingomyelin in our cell system, NEF cells were treated with 0-4 IUlml SPHase (purified according to Zwaal et al., 1975) for 1 hr at 37°C in a buffer containing 150 mM NaC1, 5 mM KC1, 1 mM MgC12, 1 mM CaC12, 5 mM glucose, 20 mM Tris, pH 7.4 (Verkleij et al., 1973). Following the li ase incubation, the incubation medium was checked or lactate dehydrogenase activity, which turned out to be insi nificant, indicating that no cell lysis had occurre . Subsequently, cellular lipid was extracted, li id species were separated by means of thin-layer cp1romatography (TLC), and the amount of phosphorus present in the individual TLC spots was determined. The SPHase treatment resulted in degradation of sphingomyelin from 11.4% in control cells to below 3% in cells incubated with 0.5 IU/ml SPHase or more. The SPHaseresistant sphin omyelin apparently is located in the inner leaflet o f t e plasma membrane. The other phospholipid species examined (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol) were not degraded, indicating that the SPHase preparation was free of other lipases. Effect of sphingomyelinase on plasma membrane protein aggregation To ascertain whether sphingomyelin degradation results in membrane protein clustering, NEF cells were treated with 0.75 IU/ml SPHase for 1 hr at 3TC, followed by fixation, immunogold labeling with monoclonal anti-EGF receptor antibody, and freeze-fracturing as described in Materials and Methods. As is shown in Figure 5, this treatment resulted in a strong aggregation of intramembrane particles. The effect of sphingomyelin degradation on the lateral distribution of EGF receptors was studied uantitatively using the approach developed previous y (Van Belzen et al., 1988) as described in Materials and Methods. As is shown in Figure 5C, SPHase treatment induced an increase in the number of small (two or three) and larger (four or more) EGF receptor clusters. Thus, degradation of sphingomyelin results in aggregation of plasma membrane proteins, including the EGF receptor. Effect of sphingomyelinase on EGF receptorcytoskeleton interaction Subsequently, the effect of SPHase on EGF receptorcytoskeleton interaction was studied. Treatment of
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