EXPERIMENTAL

CELL

RESEARCH

199,

go-97

(1992)

The Epidermal Growth Factor Receptor Is Associated with Actin Filaments PAULM. P. VANBERGENENHENEGOUWEN,'JANC.DENHARTIGH,PETRAROMEYN, ARIE J. VERKLEIJ,ANDJOHANNESBOONSTRA Department

of Molecular

Cell Biology,

University

of Utrecht, Pudualaan

In this paper we describe our investigations on the association of receptors for the epidermal growth factor (EGF) with the cytoskeleton of A431 cells. In order to determine which filamentous system the EGF receptors are associated to, the cytoskeletal fraction to which these receptors bind was isolated. Second, the possible colocalization of EGF receptors with different cytoskeleta1 elements was examined in A431 cells. By selective extractions of the A431 cytoskeletons, it is shown that more than 90% of the cytoskeleton-associated EGF receptors are removed from the cytoskeletons together with the actin filamentous system. During several cycles of poly- and depolymerization of actin isolated from A431 cells, the EGF receptor precipitates together with the actin containing filaments, indicating that EGF receptors are able to bind in vitro to actin filaments. With immunofluorescence studies we show that EGF receptors especially colocalize with actin filaments. These results demonstrate that the EGF receptor is associated specifically with actin filaments in 0 1992 Academic Press, Inc. A43 1 Cells.

INTRODUCTION The epidermal growth factor (EGF) is a potent mitogen and exerts its effects by binding to surface-located receptors [l, 21. The receptor for EGF is a 170-kDa transmembrane glycoprotein and contains in its intracellular domain a tyrosine kinase whose activity is essential for EGF-induced cell proliferation [3,4]. EGF is able to activate this tyrosine kinase probably by inducing dimerization of the receptor [5-7). Activation of the EGF receptor leads to self-phosphorylation and to the phosphorylation of specific substrates. Among these substrates are enzymes like phospholipase C-y1 and GTPase-activating protein whose activities are thought to be modulated by this phosphorylation [8-121. Furthermore, many components of the cytoskeleton appear to be phosphorylated upon EGF addition, amongst them are p35 or annexin I, ezrin, myosin light chain, and microtubule-associated protein 2 [13-161. 1 To whom reprint

requests should be addressed.

0014.4827/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

8, NL-3584

CH Utrecht, the Netherlands

From EGF binding studies, it has recently been concluded that EGF receptors of Hela cells consist of three types [17]. In this study, EGF receptor subtypes were discriminated on the basis of their EGF binding properties such as affinity for the ligand and rate of ligand association and dissociation. The EGF receptor class exhibiting the highest affinity for EGF was designated as the type I receptor. Low affinity receptors were shown to consist of two types, differing in their association and dissociation rate constants of EGF. One subtype was shown to bind EGF much faster than the other subtype and these subtypes were designated as type II and III, respectively [ 171. As has been shown previously, the type I or high affinity receptors are all associated with the cytoskeleton of different cell types [l&22]. This association between the EGF receptors and the cytoskeleton has been visualized using different electron microscopical methods as dry cleavage and lysis squirting [19, 231. The intracellular domain of the receptor was shown to be responsible for this interaction and the tyrosine kinase activity does not influence this association [al]. Furthermore, it has been shown that EGF treatment of the cells induces an increase in the number of cytoskeleton-associated EGF receptors [22]. These receptors are low affinity receptors and in the end approximately 20% of the total number of EGF receptors becomes associated with the cytoskeleton [22]. The importance of the high affinity receptor in the signal transduction has been demonstrated by using antibodies against the extracellular domain of the receptor. The monoclonal antibody 2E9 was found to prevent EGF binding specifically to low affinity receptors while the monoclonal antibody 108 specifically blocks EGF binding to high affinity receptors [24, 251. It has been shown that the EGF-induced responses were either blocked or remained unaffected by these antibodies, suggesting that the high affinity receptor is primarily involved in the EGF-induced signal transduction process. This association between the type I receptor with the cytoskeleton suggests an important role for the cytoskeleton in the regulation of the EGF-induced signal transduction. In this paper we have investigated to which filamental system the type I or high affinity receptor in A431 cells is associated. For this study, unstimulated A431 cells

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were used because in this situation only type I receptors are cytoskeleton associated. The cytoskeleton of A431 cells consists of three different filamental systems, actin filaments or microfilaments, microtubules, and intermediate filaments. The intermediate filaments in A431 cells were previously shown to consist of 10 different molecules of which the cytokeratins 8 (52.5 kDa, p1 6.1) and cytokeratin 18 (45 kDa, ~15.7) are the most prominent [26]. Vimentin is not present in A431 cells as previously reported by Franke et al. [26]. The three filamental systems were fractionated and the presence of EGF receptors in these fractions was measured using lz51-EGF. We have found that the type I EGF receptor is coextracted with actin filaments and that these receptors also bind in vitro to these filaments during several cycles of actin polymerization. Second, by performing double immunolabeling studies we show that EGF receptors colocalize with actin filaments in A431 cells. MATERIALS

AND

METHODS

Antibodies. Rabbit antibodies against actin were obtained from Amersham International Plc. (Amersham, England), mouse monoclonal anti-tubulin was from Sera-Lab Ltd. (Sussex, England), and mouse monoclonal antibodies against different cytokeratins (CK18.2, RCKlOZ and RGE53) were a kind gift from Prof. Dr. F. C. S. Ramaekers (University of Limburg, the Netherlands). The anti-keratins were characterized as described previously (CK18.2 [27], RCKlO2 (281, and RGE53 [29]). Applied antibodies against the EGF receptor were 2E9 and 281-7 which were characterized as described elsewhere [24]. Secondary antibodies, goat anti-mouse, goat anti-rabbit, and goat anti-rat conjugated with alkaline phosphatase, fluorescein isothiocyanate (FITC), or Texas red were from Jackson ImmunoResearch (West Grove, PA). Cell culture. A431 epidermoid carcinoma cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Paisley, Scotland) supplemented with 7.5% fetal calf serum (Integro, Zaandam, the Netherlands) in a 7% CO,-humidified atmosphere at 37°C. Cellulur extractions. A431 cells were grown in 75cm2 flasks (Costar, Cambridge, MA) to a final density of 75,000-100,000 cells/cm’ and detached by an incubation in 2 mM ethylene glycol [email protected] ether) N,N’-tetraacetic acid (EGTA) in phosphate-buffered saline (PBS; 8.0 mM Na,HPO,, 1.5 mA4 KH,PO,, 150 mM NaCl, 3 mM KCl, pH 7.2) for 10 min at 37°C. The cells were collected, centrifuged at 8OOg for 5 min, and resuspended in a cytoskeleton stabilizing buffer (CSK-buffer; 10 n&f piperazine-N,N’-bis(2-ethanesulfonic acid) (Pipes), pH 6.8, 250 m&f sucrose, 3 mM MgCl,, 150 mM KCl, 1 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF)) [30]. Cytoskeletons were isolated by an incubation for 5 min in 0.15% Triton X-100 in CSK-buffer at room temperature. Tubulin was depolymerized by chilling the samples to 4°C and removed by two washes with cold CSK-buffer, using centrifugation steps for 10 min at 14,000g. Actin was depolymerized by extracting the cytoskeletons with 0.6 A4 KI in CSK-buffer for 20 min at 4°C. The actin was removed by two washes with 0.6 MKI containing CSK-buffer by centrifugation steps at 40,OOOgfor 20 min. SDS-PAGE and immunoblotting. Protein determination was done using the BCA reagent (Pierce Chemical Co., Rockford, IL) with BSA as standard. Proteins were solubilized in sample buffer (33% glycerol, 300 m&f dithiothreitol, 6.7% sodium dodecyl sulfate, 0.01% bromophenol blue, and 20 m&f Tris-HCl, pH 6.8), separated on 10% polyacrylamide gels, and blotted onto nitrocellulose (Schleicher and Schuell, Dassel, Germany) as described previously [31]. The nitrocellulose was blocked with 0.3% milkpowder in PBS for 45 min at 37°C.

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The filters were incubated with the different antibodies for 60 min diluted in 0.03% milkpowder in PBS at room temperature. After washing in the same buffer the filter was incubated with secondary antibodies conjugated with alkaline phosphatase. The antibody reaction was visualized in 100 mM Tris-HCl, pH 9.5, 100 m&f NaCl, 50 mM MgCl, containing 0.33 mg/ml nitroblue tetrazolium salt and 0.17 mg/ml 5-bromo-4-chloro-3-indolyl phosphate toluidinium salt (Sigma, St. Louis, MO). EGF binding studies. Intact A431 cells or differentially extracted A431 cytoskeletons were spun down and resuspended in binding buffer (DMEM, 0.1% BSA, 20 n&f N-2-hydroxyethylpiperazine-N’2-ethanesulfonic acid (Hepes), pH 7.4) at a concentration of 10scellsl ml. EGF (Collaborative Research, Waltham, MA) was iodinated using the chloramine-T method yielding a specific activity of 300,000900,000 cpm/ng as described previously [17]. Different EGF concentrations were incubated with lo5 cells/ml for 60 min on ice in siliconized Eppendorf cups. After incubation, intact cells and cytoskeletons were centrifuged at 14,000g for 5 min and 0.6 M KI extracted cytoskeletons were centrifuged 20 min at 40,OOOg.The supernatant was removed and radioactivity in the pellet was measured in a gamma counter (Crystal 5412, United Technologies Packard, Downers Grove, IL). Background labeling was determined in the presence of 500 rig/ml unlabeled EGF and was less than 10% of the total binding to intact cells. The binding data were analyzed using the LIGAND program [32] as described previously [22,33]. Zmmuno/luorescence. A431 cells were grown on coverslips to 50% confluency. For actin and tubulin staining, cells were fixed with 3% paraformaldehyde, 0.25% glutaraldehyde, 0.25% Triton X-100 in PBS for 30 min at room temperature. The cells were treated two times for 5 min with 1 mg/ml NaBH, in PBS and subsequently with PBS containing 0.2% gelatin. For cytokeratin staining, the cells were fixed 2 min with methanol at -2O’C and washed in PBS containing 0.2% gelatin. Actin filaments were stained with Phalloidin-FITC (Sigma). Tubulin, keratin, and the EGF receptors were stained with specific antibodies for 60 min at room temperature. For secondary antibodies goat anti-mouse or rabbit conjugated with FITC or Texas red was used. Finally the cells were embedded in Mowiol as described previously [31] and examined in a Leitz Orthoplan microscope equipped with epi-illumination. Actin polymerization. Suspended A431 cells were extracted with 0.5% Triton X-100 in CSK-buffer for 20 min at 4°C. After extraction the resulting cytoskeletons were washed with cold CSK-buffer and centrifuged at 40,OOOg for 20 min. The pellet was further extracted with 0.6 M KI in CSK-buffer for 20 min and centrifuged at 40,OOOgfor 20 min. Subsequently, the supernatant was dialyzed against 10 mM Pipes, pH 6.8,l mM EGTA, 2 mM MgCl,, 1 mM PMSF, and 5 pg/ml colchicine (Sigma), for at least 3 h at 4°C. During this dialysis the actin polymerized and was spun down at 40,OOOg for 20 min. The pellet was resuspended in 0.6 M KI in CSK-buffer and incubated for l-2 h at 4°C. The solution was clarified by centrifugation (40,OOOgfor 20 min) and dialyzed against the same buffer as described above which induced actin polymerization [34]. The actin polymers were again spun down at 40,OOOgfor 20 min, solubilized in CSK-buffer, and analyzed for the presence of EGF receptors and cytoskeletal components.

RESULTS

Fractionation

of A431 Cytoskektons

A431 cytoskeletons were isolated by an extraction of suspended A431 cells with the nonionic detergent Triton X-100 in a cytoskeleton stabilizing buffer [27]. Suspended A431 cells were used in order to be able to remove the different fractions by centrifugation. A431 cytoskeletons attached to a substrate are not suitable for

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% Triton X-100

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EN HENEGOUWEN

Time (min)

FIG. 1. Effect of Triton X-100 extraction on EGF binding to A431 cells. Suspended A431 cells were extracted for 10 min at room temperature with different concentrations of Triton X-100 in CSKbuffer (A) or with 0.15% Triton X-100 in CSK-buffer for different time intervals (B). EGF binding was determined by incubation of intact or extracted cells in the presence of ‘*‘I-EGF (20 rig/ml, 50,000 cpm/ng) for 60 min at room temperature. Separation of free EGF from bound was done by centrifugation as described under Materials and Methods.

these experiments since extractions of cytoskeletal subunits also interfere with cellular attachment. The effect of extraction time with Triton X-100 on the isolation of the cytoskeleton-associated receptors was monitored with ‘251-EGF. In Fig. 1A the effect of various concentrations of Triton X-100 on the extraction of cytoskeleton bound EGF receptors is shown. EGF binding dramatically decreases after the addition of detergent and it reaches a minimum level when 0.15% Triton X-100 is used. Higher detergent concentrations do not alter this level. Extraction time is determined using 0.15% Triton X-100 and a minimum of EGF binding is obtained after 2 min of extraction. Based on these studies, we used an extraction of 0.15% Triton X-100 for 5 min for the isolation of A431 cytoskeletons. Extraction of suspended A431 cells with 0.15% Triton X-100 at room temperature removes 70% + 17 (SD) of total cellular proteins (Table 1). Coomassie blue staining of the proteins of intact cells and cytoskeletons separated on SDS-PAGE shows that a specific set of proteins is remained after the extraction (Fig. 2, CB, lane 1, 2). The presence of cytoskeletal subunits was tested by immunoblotting. For cytokeratins the fractions were tested for both cytokeratin 8 and cytokeratin 18. Isolated A431 cytoskeletons contain the cytoskeletal proteins actin, tubulin, and cytokeratins 8 and 18 which are also present in intact A431 cells (Fig. 2, lanes 1, 2). No difference is visible between the staining intensity of both cytokeratin bands of intact cells and those of cytoskeletons, indicating that no cytokeratin is extracted. In contrast, most of the tubulin and some of the actin is extracted during the cytoskeleton isolation. Scatchard analysis of EGF binding reveals that intact cytoskeletons contain 15.6 f 0.8 (SD) X lo3 receptors, which is less than 1% of the total number of EGF receptors of A431 cells (Table 1). Adherent cells were previously shown to contain approximately 5% of cytoskeletal-as-

ET AL.

sociated receptors [22]. Obviously, suspension of A431 cells leads to a reduction in the number of cytoskeletonassociated EGF receptors, which is in agreement with the observations made by Roy et al. [20]. Subsequently, tubulin was removed from the A431 cytoskeletons by chilling the cytoskeletons to 4°C. By this treatment approximately 6% of the proteins is extracted (Table 1) and all tubulin is extracted, leaving only actin and intermediate filaments as cytoskeletal filaments (Fig. 2, lanes 3). As determined with Scatchard analysis, only 6% of the cytoskeletal-associated receptors is coextracted with the tubulins (Fig. 3, Table 1). Since all tubulin is extracted, it can be concluded that the main fraction of the type I EGF receptor is not associated with microtubules. For the removal of actin, high salt washing steps are needed [35]. We have tested the effect of both KC1 and KI on the extraction of actin from the cold cytoskeletons and both extraction methods yielded the same results. In Fig. 2, the effect of an extraction with 0.6 M KI for 20 min at 4°C is shown which was found to be suflicient for the removal of virtually all actin. The remaining cytoskeletons still contained 10% +- 2 (SD) of the original cellular proteins (Table 1). These cellular residues mainly consist of intermediate filaments of which the presence of cytokeratins 8 and 18 is shown Fig. 2, lane 4. During this extraction, more than 90% of the cytoskeleton-associated EGF receptors is extracted (Fig. 3, Table 1). This high salt extraction did not alter the affinity of the receptor for EGF, which remains on the residue (Fig. 3). In conclusion, by performing a selective extraction procedure, we are able to remove step-by-step the different cytoskeletal subunits of the A431 cells and second we have shown that the EGF receptors are coextracted specifically with the actin filaments.

TABLE Determination Protein Content tal Fractions Fraction Whole cells TX-loo, RT TX-loo, 4 KI

1

of Total Receptor Numbers and Relative of A431 Cells, Cytoskeletons, and Cytoskele-

RT (X103) 1840 15.6 14.6 1.5

+ 158 F 0.8 ? 0.7 -c 0.1

R% 100 + 9

0.85 -c 0.04 0.80 + 0.04 0.08 jr 0.01

P% 100 + 17 29 +- 1

23 f 4 10 f 2

Note. Receptor numbers per cell or cytoskeleton were determined by Scatchard analysis as shown in Fig. 2. Relative protein contents were determined as described under Materials and Methods. Data are presented as the mean f SD of duplicate experiments and each data point was determined in duplo. TX-100, RT: cells extracted with 0.15% Triton X-100 at room temperature; TX-100,4: cells extracted with 0.15% Triton X-100 at room temperature and chilled to 4’C; KI: cells extracted with 0.15% Triton X-100, chilled to 4°C and extracted with 0.6 M KI. RT, number X103 receptors per cell; R%, percentage of EGF receptors; P%; percentage amount of protein.

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Actin

Tubulin

CB

AND

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CK-18

20097

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89

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48-

1234

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2

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3

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FIG. 2. Immunoblot analysis of different cytoskeletal fractions of A431 cells. A431 cells were not extracted (l), extracted with 0.15% Triton X-100 in CSK-buffer for 5 min at room temperature (2), extracted with 0.15% Triton X-100 and chilled to 4°C (3), or extracted with 0.15% Triton X-100 at 4°C and followed by an extraction with 0.6 M KI in CSK-buffer (4). These fractions (lo5 cells and cytoskeletons) were separated on 10% SDS-PAGE either stained with Coomassie blue (CB) or transferred onto nitrocellulose and probed for the presence of tubulin, actin, cytokeratin 8 (CK-8), and cytokeratin 18 (CK-18) as described under Materials and Methods.

EGF Receptors Bind in Vitro to Actin Filaments The data presented above suggest that type I EGF receptors are associated with actin filaments. However, when the interaction between the EGF receptor and the other cytoskeletal elements is sensitive for high salt, the receptor would also be extracted together with the actin filaments. In order to see whether the EGF receptor is indeed associated with the coextracted actin filaments, we have polymerized the actin in vitro and investigated whether the EGF receptor associates with these filaments in vitro. A431 cytoskeletons without microtubules were extracted with high salt. After centrifugation, actin polymerization was induced by removing the high salt by dialysis against a low salt buffer containing 2 r&f MgCl, as described by Burn et al. [34]. Colchicin was included in this buffer to prevent polymerization of

possible traces of cold stable tubulin. After 3 h, the actin polymers were pelleted by centrifugation and the pellet was dissolved again in high salt buffer. After two of such cycles of polymerization and depolymerization, the actin filaments were analyzed for the presence of cytoskeleta1 subunits and the EGF receptor. Proteins from whole A431 cells were used as a positive control in this experiment (Fig. 4, lane 1). To see whether all present EGF receptors bind to the actin filaments, proteins from the supernatant from the first polymerization cycle were also monitored for the presence of EGF receptors. As expected this cytoskeletal fraction contains only actin as a cytoskeletal component and tubulin and both

CB

EGF-R

Tubulin

Actin

CK-8

M-18

200 -

B/F

97 -

I 0.02 1 k

89 48-

0.01

30 -

‘\ \\

0L 0

1

0, 2

1212121212

3

EGF Bound (f Mel/ lo5 cytoskeletons) FIG. 3. Scatchard analysis of EGF binding to A431 cytoskeletons. A431 cells were extracted with 0.15% Triton X-100 in CSKbuffer at room temperature (O), extracted with 0.15% Triton X-100 and chilled to 4°C (Cl), or extracted tvith Triton X-100 at 4’C and subsequently extracted with 0.6 M KI in CSK-buffer (A). Different concentrations of i2’I-EGF were allowed to bind to these cytoskeletons and unbound EGF was removed by centrifugation as described under Materials and Methods. Binding data were analyzed according to the method of Scatchard. B/F, ratio of bound and free EGF.

12

FIG. 4. Immunoblot analysis of actin filaments from A431 cells. Actin filaments were isolated from A431 cells by an extraction of A431 cytoskeletons lacking microtubules with 0.6 M KI in CSK buffer. This fraction was cleared by centrifugation at 40,OOOgand the supernatant was dialyzed against 10 n&f Pipes, pH 6.8,l n&f EGTA, 2 mA4 MgCl,, 1 m&f PMSF, and 5 pg/ml colchicine at 4”C, which promotes actin polymerization. After two subsequent cycles of polymerization and depolymerization, the actin filaments were separated on 10% SDS-PAGE (CB, Coomassie blue-stained gel; lo5 cells and 5-pg actin filaments), blotted onto nitrocellulose, and analyzed for the presence of EGF receptors and cytoskeletal subunits as tubulin, actin, cytokeratin 8 (CK-8). and cytokeratin 18 (CK-18).

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keratins are absent in this fraction (Fig. 4). Analysis for the presence of EGF receptors in this fraction clearly shows that the EGF receptor associates with actin filaments during several cycles of polymerization and depolymerization of actin filaments. Increasing the number of polymerization cycles to four did not lead to the loss of EGF receptors (data not shown). No EGF receptors were found in the supernant, indicating that all type I receptors are capable of binding to the actin filaments (data not shown). With these results we clearly show that the EGF receptor associates in vitro with actin filaments. Colocalization of EGF Receptors with Cytoskeletal Components In order to investigate whether EGF receptors are also associated with actin filaments in the in uiuo situation, we have performed double immunolabeling studies. Unstimulated A431 cells were simultaneously fixed and permeabilized and all EGF receptor subtypes and the different cytoskeletal subunits were labeled with specific antibodies. As shown in Figs. 5C and 5D, EGF receptors strongly colocalize with the actin filaments. Especially at cell-cell contacts a similar distribution of actin and EGF receptors can be seen. Remarkably, EGF receptors do not colocalize with stress fibers. Colocalization of EGF receptors with intermediate filaments is virtually absent (Figs. 5E, 5F). As shown in Figs. 5A and 5B some colocalization of EGF receptors and microtubules can be seen around the nucleus, which is possibly due to the fact that intracellular transport of endocytotic vesicles containing EGF-EGF receptor complexes may translocate along the microtubules [36]. As shown with video-enhanced microscopy, endocytosis of goldlabeled EGF receptors is blocked by nocodazole, an inhibitor of tubulin polymerization [36]. This observation makes it very likely that intracellular transport of the EGF receptor is occurring along microtubules, which may cause the intracellular colocalization of EGF receptors with microtubules. In conclusion, this experiment suggests that EGF receptors are also in the in uiuo situation associated with the actin filamental system. DISCUSSION

Until now, a large number of receptors has been described to be associated with the cytoskeleton including receptors for growth factors as the nerve growth factor and the platelet-derived growth factor [37-401. Only for

ET AL.

a few receptors has this association been investigated in more detail. The fibronectin receptor has been shown to be associated to actin via talin, vinculin, and a-actin [41]. The fhIet-Leu-Phe receptor of human neutrophils has been shown to be associated with actin via a GTP binding protein [42]. Human platelets contain several glycoproteins which are receptors for different kinds of ligands such as thrombin and von Willebrand factor (GIb) and fibronectin, vitronectin, and fibrinogen (GIIb-IIIa) [43,44]. These receptors are associated with the actin filamental system directly (GIIb-IIIa) or via an actin binding protein of 540,000 Da (GIb) [43,44]. Furthermore, the acetylcholine receptors have been reported to be associated with actin but the exact nature of this association is not yet clear [45]. In this paper we have shown that receptors for the epidermal growth factor are associated with the actin filamental system. EGF receptors are extracted together with actin and by different cycles of polymerization we have demonstrated that EGF receptors bind also in vitro to actin filaments. Evidence for an EGF receptoriactin association in the in uiuo situation has been obtained with immunofluorescence studies since we found a strong colocalization of these receptors with the actin filaments. The actin containing fraction is free from the other cytoskeletal subunits of these cells but contains a large number of actin binding proteins. Therefore it is not clear at the moment whether the interaction of EGF receptors with actin filaments is a direct or indirect linkage. For an indirect actin association several anchoring proteins can be suggested. Annexin I is a substrate of the EGF receptor which binds to actin and phosphatidylserine [13]. In addition, ezrin, another substrate of the EGF receptor, might be a candidate. It is a spectrinlike protein and as a component of the membrane skeleton it is capable of binding actin [ 141. Alternatively, the EGF receptor might be directly associated with actin as has been described for the glycoprotein IIb-IIIa in human platelets [44]. On the other hand, the part of the receptor which is involved in the actin association is not yet known. As described by Van Belzen et al. [21], an intact intracellular domain of the receptor is necessary for this attachment, since a mutant which lacks almost the whole intracellular part (from amino acid 655) is unable to bind the cytoskeleton. Also the tyrosine autophosphorylation sites containing domain of the receptor (amino acid 1060 to 1186) is not involved in this association, indicating that the actin filament-asso-

FIG. 5. Localization of EGF receptors and cytoskeletal subunits in A431 cells. A431 cells were fixed with 3% paraformaldehyde, 0.25% glutaraldehyde, and 0.25% Triton X-100 in PBS for actin and tubulin staining or with -20°C methanol for intermediate filament staining. EGF receptors were stained with either FITC or Texas red, allowing simultaneous visualization in the same cell: (A, C and E) distribution of EGF receptors and distribution of tubulin (B), actin (D), and keratin (F). Note the strong colocalization of EGF receptors with actin filaments which is absent for stress fibers (arrowheads). Magnification 850X.

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ciated domain can be expected between residue 653 and 1060 [21]. An interesting question concerns the biological role of the association of the EGF receptor with actin filaments. As described previously, the high affinity receptor or type I receptor is the cytoskeleton-associated receptor [19, 221 and its kinase is fully active [18]. The high affinity receptor is shown to have a pivotal role in the EGF-induced signal transduction process [ 24,251. It is tempting to suggest that the cytoskeletal association of the EGF receptor induces the high affinity state of the receptor by a thus far unknown process. In this way, the cytoskeleton would play a regulatory role in the sensitivity of the cell toward EGF. Alternatively, the actin filament association might be an intermediate step in the internalization of EGF/EGFR complexes as has been similarly suggested for the met-Leu-Phe receptor in human neutrophils [46]. This would imply that the cytoskeleton association of EGF receptors in unstimulated cells would have a completely different function than after stimulation when it is possibly involved in down regulation of the receptors. However, a consequently preferential internalization of the type I receptor after EGF addition has not been observed so far [47]. Recently we have found that several phosphatidylinositol (PI) kinases such as PI- and PIP kinase, diacylglycerol kinase, and also phospholipase C are associated with the actin filaments in A431 cells [48]. Such enzymes are among the first activated after EGF addition, leading to the production of IP, and DAG, which is an activator of protein kinase C [ 11. Obviously, these enzymes are together with the EGF receptor associated to actin filaments. Actin filaments may function as a matrix providing for a local high concentration of enzymes which are involved in the signal transduction process together with the receptor which is the key enzyme in the activation of the above-described enzymes. In this way the cytoskeleton would function as a matrix in the assembly of the so-called signal transduction particle which could improve the efficiency in the signal transduction cascade.

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The epidermal growth factor receptor is associated with actin filaments.

In this paper we describe our investigations on the association of receptors for the epidermal growth factor (EGF) with the cytoskeleton of A431 cells...
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