EXPERIMENTAL

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RESEARCH

189,100-108

(1990)

Nerve Growth Factor Induces Increased Expression of a Laminin-Binding lntegrin in Rat Pheochromocytoma PC1 2 Cells PAOLA ROSSINO,* ISABELLA GAVAZZI,* RUPERT TIMPL,~ MONIQUE AUMAILLEY,? MARZIA ABBADINI,* FILIPPO GIANCOTTI,~ LORENZO SILENGO,$ PIER CARLO MARCHISIO,* AND GUIDO TARONE$‘~ *Dipartimento

di Scienxe Biamedtihe e Oncologia Umunn; TMax-Plnnck-Institut fiir Bioekemie, 8033 Martinsried, Federal Republic of Germany; and SDipartimento di Genetica Biologia e Chimica Medica, Uniuersita’di Torino, Italy

Rat pheochromocytoma PC12 cells exposed to nerve growth factor differentiate as sympathetic neurons and extend neurites on laminin and to a much lesser extent on Rbronectin. Analysis of laminin fragments indicated that neurite outgrowth occurs mainly on fragment Pl, corresponding to the center of the cross, and only poorly on fragment ES, a long arm structure that is active with other neuronal cells. Integrin antibodies prevented adhesion and neurite sprouting of these cells on laminin, fragment Pl, and flbronectin. By affinity chromatography we isolated an integrin-type receptor for laminin consisting of two subunits with molecular masses of 180 and 136 kDa. The latter is recognized by an antiserum to integrin /31 subunit. The bound laminin receptor could be displaced by EDTA, but not by ArgGly-Asp or Tyr-Ile-Gly-Ser-Arg peptides. Affinity chromatography on laminin fragments showed that the 180/135 kDa receptor binds to Pl. The expression of the 180-kDa (Ysubunit of the laminin receptor at the cell surface was increased lo-fold after NGF treatment. The effect of NGF is specific since the amount of a 160kDa fibronectin-binding integrin a subunit remained unchanged. Moreover, the increased expression of the 180/136 kDa receptor at the cell surface corresponded to a selective increase in cell adhesion to laminin and to fragment Pl. The 180/135-kDa complex is thus an integrin-type receptor for laminin whose expression and binding specificity correlates with the capacity of NGF-induced PC12 cells to extend neurites on lami0 lSS0 Academic Press, Inc. nin.

INTRODUCTION Elongation of neurites requires adhesion of nerve cells to substrates consisting of either glial cells [l] or extracellular matrix proteins secreted by cultured cells [2]. 1 Present address: La Jolla Cancer Research Foundation, La Jolla, CA 92037. 2 To whom correspondence and reprint requests should be addressed at Dipartimentu di Genetica, Biologia e Chimica Medica, Via Santena Sbis, 10126 Torino, Italy.

Analysis of the active components indicates that laminin plays a critical role. This purified protein, in fact, promotes rapid neurite outgrowth [3,4] and potentiates the action of neurotrophic factors [5]. Laminin is a major glycoprotein of basement membranes and consists of three polypeptides folded together to form a typical cross-shaped molecule with a molecular mass of 900 kDa [ 6,7]. Distinct domains possessing specific binding sites for cell surface receptors and for other matrix components, such as collagen type IV, heparin, and nidogen, have been recognized on laminin [7]. The interaction with the cell surface has been investigated to some extent and two laminin fragments interacting with two different cell surface binding sites have been identified [8,9]. Several proteins that can function as cell surface receptors for laminin have been described These include proteins of 180 kDa [lo], 120 kDa [ll], 110 kDa [lo], and 68 kDa [ 12-151. More recently, integrin-type receptors for laminin have been described. Integrins are LY//~ dimers in which the p subunit can associate alternatively with several (Ysubunits, generating a number of different complexes with distinct binding specificities [16-B]. The a3/Bl complex functions as a laminin receptor that can also bind fibronectin and collagen as determined by antibody inhibition of cell adhesion [19-211. A similar complex has been isolated by laminin affinity chromatography from glioblastoma cells [22]. A second integrin-type receptor for laminin consists of a 200/125 complex isolated from rat neuroblastoma cells [23], and a third receptor is represented by the a6/@1 complex identified in platelets [24]. The considerable diversity of laminin receptors could be explained by the existence of more than one cell-binding site in laminin [8, 91 and by the finding that different forms of laminin, characterized by specific tissue distribution, may exist [7,25]. Here we have analyzed the laminin receptor of the rat pheochromocytoma PC12 cell line. These cells, when exposed to the nerve growth factor (NGF) differentiate as sympathetic neurons [26] and extend neurites on laminin substrata. Our results show that neurite outgrowth in PC12 cells occurs mainly in response to a site in the Pl fragment of mouse laminin. We also isolated an integrin-type receptor for laminin with subunits of 180/135 100

0014~4827/90 $3.00 Copyright 0 1990 by Academic All rights of reproduction in

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any form reserved.

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kDa that binds the Pl fragment. Expression of this receptor at the cell surface is increased lo-fold after exposure of PC12 cells to NGF, suggesting its possible role in neurite outgrowth. MATERIALS

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METHODS

Cells and antisera. PC12 cells, obtained from the stock of Dr. L. A. Greene (New York University, NY), were cultured in RPM1 medium supplemented with 10% horse serum, 5% fetal calf serum, and antibiotics. Nerve ,growth factor 2.59 (NGF), purified from mouse salivary glands, was kindly provided by Dr. P. Calissano (Institute of Neurobiology, CNR, Rome). Cells were seeded on collagen-coated petri dishes and treated with 100 rig/ml NGF in RPM1 medium with 10% horse serum and 5% fetal calf serum for 6 days. Control cells were kept in serum containing RPM1 for 6 days without NGF. During the treatment the medium was not changed nor was NGF added. Treatment with dexamethasone (10 p&f) was performed under the same conditions as those described above. Preparation and characterization of the anti-BHK serum reacting with mouse integrins has been described elsewhere [27]. An antiserum to human placental fibronectin receptor, prepared according to Pytela et al. 1281 has been characterized [29]. A rabbit antiserum to the peptide KKKEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK from the cytoplasmic domain of the human integrin 81 subunit was kindly provided by Erkki Ruoslahti. An antiserum to the last 18 COOH-terminal amino acids of the human a5 subunit was a kind gift of Dr. K. J. Tomaselli [30]. Cell adhesion assays. Laminin from EHS tumor and its cell-binding fragments Pl and ES were isolated following established procedures [31]. The laminin preparation did not contain detectable amounts of collagen IV as judged by amino acid analysis. The purity of the fragments was higher than 98% as determined by specific radioimmuno assays [31,32]. Fibronectin was purified from human plasma by gelatin affinity chromatography as described [33,34]. The peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) was synthesized using an Applied Biosystem Model 430A automated peptide synthesizer by P. Neri (Centro Ricerche Interdipartimentali Scienze Mediche Avanzate, University of Siena). The Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Argamide (CDPGYIGSR-amide) peptide of the laminin Bl chain was synthesized and kindly supplied by S. Henke (Hoechst AG, Frankfurt). Adhesion and inhibition assays with antibodies or with synthetic peptides were performed as described previously [35]. Microtiter plates (96 wells, Nunc Immunoplate 1) were coated with different concentrations of the adhesive proteins and postcoated with bovine serum albumin. PC12 cells were detached from the culture dishes by incubation in PBS with 1 mM EGTA and washed twice in serum-free RPM1 by centrifugation. Cells (1 X 106/well) were plated on coated dishes in serum-free RPM1 medium without NGF for 2 h at 37°C. Serial dilutions of antibodies or preimmune sera were added at the beginning of the test. Adherent cells were fixed with 3% paraformaidehyde and stained with crystal violet. Cell adhesion was evaluated by reading the absorbance at 540 nm. The correspondence between the optical density and the number of cells attached was assured in experiments showing that increasing the cell concentration in the original suspension resulted in proportional increase in the optical density at any coating concentration used. To measure neurite outgrowth NGF-primed cells were detached by EDTA, pipetted to make a single cell suspension, and plated on coated dishes for 2 h in serum-free medium without NGF. Adherent cells were fixed, stained, and photographed. Fifty cells were counted for each sample. Purified laminin Isolation of the hminin andfibrcm~ctin receptors. [31] was coupled to CNBr-activated Sepharose [32], yielding about 1.7-2.3 mg protein bound to 1 g dry adsorbent. Laminin fragments PI and ES were coupled to Sepharose by the same procedure yielding

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respectively 5 and 1.7 mg protein per gram of resin. The fibronectinSepharose matrix was a generous gift of Dr. R. Pytela (Department of Medicine, University of California, San Francisco) and was prepared as described [36]. Columns were prepared from 5 ml of swollen protein adsorbent. PC12 cells cultured in 5 450-cm2 Nunc plates were treated with NGF for 6 days. Cells were collected in phosphate-buffered saline (PBS) and radiolabeled with 2 mCi of iz61as described [34]. Labeled cells were extracted with 4 ml of 20 mM Tris-HCl, pH 7.4,150 n&f NaCl (TBS) with 200 m&f p-octylglucoside, 1 mAf CaClz, 1 mhf MgClz, 1 mM M&l,, 10 pg/ml leupeptin, 4 wg/ml pepstatin, and 0.1 TIU/ml aprotinin. After centrifugation at lO,OOOg, soluble material was incubated overnight by agitation with the affinity matrix at 4’C. The suspension was poured into a column and washed with 5 vol of 50 mM @-octylglucoside in TBS with cations and eluted sequentially with 2 vol of 10 mMEDTA in TBS, 50 mAf&octylglucoside, and 2 vol of 0.1 M sodium carbonate, pH 11, 50 n&f j3octylglucoside. When indicated, elution was performed in TBS with cations, 50 n&f fl-octylglucoside, and 1 mg/ml of the indicated synthetic peptide. The column flow was kept at 40 ml/h through all steps of the chromatography. Relevant fractions were dialyzed against water, concentrated by lyophilization, and analyzed by electrophoresis. Zmmunoprecipitation of membraneproteins. Integrins were precipitated from radioiodinated or metabolically labeled cells. Radioiodination of surface proteins was performed as described above. Metabolic labeling was achieved by overnight incubation in complete medium containing 50 &i/ml of [3H)6-D-glucosamine (20 Ci/mM, Amersham) or in methionine-free medium with 10% horse and 5% fetal calf sera and 25 &i/ml of [35S]methionine (800 Ci/mM, Amersham). Labeled cells were suspended by EGTA treatment, washed by centrifugation and incubated with antibodies for 1 h at 4°C with gentle agitation [35]. Unbound antibodies were removed by washing and cells extracted for 20 min at 4°C with 0.5% Triton X-100 (Pierce) in TBS with divalent cations and protease inhibitors (see above). In some experiments, 50 r&f /3-octylglucoside was used instead of Triton X-100 to solubilize membrane proteins without affecting the results. After centrifugation at 10,OOOgfor 30 min, soluble immunocomplexes were bound to Protein A-Sepharose beads (Pharmacia) and recovered by centrifugation. After a wash, bound material was eluted by boiling beads in 1% sodium dodecyl sulfate and analyzed by electrophoresis. A slightly different protocol was followed when antisera to cytoplasmic peptides were used. In this case to make epitopes accessible to antibodies, cells were first extracted with detergent and antisera were then added. Ekctrophetic analysis of proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed under nonreducing conditions unless specified. Gels were made of 6% acrylamide using the protocol described by Laemmli [37]. When necessary, gels were processed for fluorography 1381, dried, and placed in contact with an Amersham MP Hyperfilm. Molecular mass markers (under reducing conditions) were: myosin (200 kDa), phosphorylase B (93 kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa), and lysozyme (14 kDa). Analysis of the electrophoretic mobility of reduced protein was performed as described [35]. After separation under nonreducing conditions, bands were visualized on the gel by autoradiography and cut with a razor blade. Each band was divided in two pieces of the same size and swollen in buffer either with or without 1% 2-/3-mercaptoethanol. After boiling, samples were separated by SDS-PAGE. RESULTS

Adhesion of Undifferentiated and NGF-Primed PC12 Cells to Different Substrates Previous experiments have indicated that integrins are involved in adhesion and neurite outgrowth of PC12 cells [30, 391. We investigated here the adhesive prop-

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FIG. 1. Adhesion of undifferentiated and NGF-primed PC12 cells to laminin, laminin fragments, and fibronectin. PC12 cells grown in the absence of NGF (dashed lines) or exposed to 100 rig/ml NGF for 6 days (continuous lines) were suspended by EGTA treatment and plated on coated dishes in serum-free RPM1 medium without NGF. Adhesion was determined after 120 min (a and b) or after 30 min (c and d) of incubation at 37%. Attached cells were fixed and stained with crystal violet and adhesion is expressed as absorbance at 540 nm. The number of undifferentiated and differentiated cells plated on coated dishes was identical within the same test but differed in the two experiments (upper and lower panels). Each point is the mean value from a triplicate. The experiments were repeated a minimum of three times with reproducible results. Coating substrates are fibronectin (Cl) laminin (m), laminin fragment Pl (O), and laminin fragment E8 (0).

erties of PC12 cells before and after differentiation by NGF. Undifferentiated PC12 cells adhere to laminin and to a much lesser extent to fibronectin (Fig. la). It was previously shown that various cells which adhere to laminin recognize structures on laminin fragments Pl and E8 [8, 91. We thus analyzed the activity of these two fragments and found that PC12 cells adhered strongly to fragment Pl, but much more weakly to three different batches of fragment ES (Fig. lb). This finding was unexpected since it was previously reported that neurons preferentially adhere to and extend neurites on the ES fragment [5]. Control experiments were thus performed in parallel with PC12 and the human neuroblastoma cell line GIME-N [40]. The latter cell line, but not PC12 cells, adhered to fragment ES, confirming the data reported in the literature and indicating that our E8 preparations were active.

Adhesion of NGF-differentiated PC12 cells was then studied. NGF treatment increased adhesion of PC12 cells and a differential effect was observed on different substrata depending on the time of incubation and the substrate concentration. In the first set of experiments, adhesion was measured after 120 min of incubation on coated dishes at 37°C. Under these conditions at high coating concentrations (5-20 pg/ml) (Figs. la and lb), NGF treatment was found to induce a modest increase (0.3- to l-fold) of adhesion to all substrata. At lower coating concentrations (0.6 to 2.5 pg/ml) NGF treatment induced a selective and large increase of adhesion (3- to lo-fold) to laminin and fragment Pl, while adhesion to fibronectin and laminin fragment E8 remained unchanged. In the second set of experiments at 30 min a selective increase in adhesion of NGF-primed PC12 to laminin and Pl fragment was also observed (Figs. lc and Id). In conclusion, when cell adhesion was tested under

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GRGDSP or CDPGYIGSR-amide peptides when used up to a concentration of 1 mg/ml (not shown). The former peptide corresponds to the cell-binding sequence of fibronectin [41] and the latter to a laminin Bl chain sequence reported to inhibit cell adhesion to laminin [42]. These data indicate that integrin complexes are involved in PC12 cell adhesion and neurite extension and suggest that NGF may alter the expression of these receptors. They also indicate that Pl fragment of laminin is the principal fragment active in both attachment and neurite outgrowth. To identify the integrin proteins involved in adhesion to laminin, we performed affinity chromatography and immunoprecipitation experiments. Isolation of an Integrin Complex That Binds to the PI Fragment of Laminin

FIG. 2. Morphology of NGF-treated PC12 cells adhering to different substrata. PC12 cells primed for 6 days with 100 rig/ml of NGF were removed from the culture plate by EGTA treatment and plated on coated dishes in serum-free RPM1 medium without NGF for 2 h at 3’7°C. Attached cells were fixed, stained with crystal violet, and photographed. Substrates were coated with 10 pg/ml of (a) laminin, (b) laminin fragment Pl, (c) laminin fragment E8, and (d) fibronectin. Bar corresponds to 50 Wm.

NGF-primed PC12 cells were surface labeled with lz51 and immunoprecipitated with adhesion-blocking antibodies to mouse integrins (Fig. 4, lane a) or with antibodies specific for a carboxy-terminal peptide of the integrin @l subunit (Fig. 4, lane b). The two sera gave an identical pattern consisting of 180,150, and 135 kDa bands which are likely to correspond to (Y(180,150 kDa) and /3 (135 kDa) integrin subunits. This pattern is comparable to that previously described by Tomaselli et al. for PC12 cells [30]. To determine the binding specificity of these integrin complexes, chromatography experiments were performed on laminin and fibronectin affinity columns. A detergent extract of ‘251-labeled NGF-treated PC12 cells was applied to a laminin-Sepharose column. Manganese ions were included in the extraction and column buffers since these cations increase the affinity of integrins for their ligands [22, 23, 431. The column was sequentially eluted with EDTA and with a pH 11 buffer to release cation-stabilized integrins [ 18, 361 and residual bound material. EDTA elution of the laminin matrix re-

conditions-low substrate concentration or short incubation time-a selective increase in adhesion of differentiated cells to laminin and fragment Pl was detected. NGF also induced the ability to extend neurites on laminin, but not on fibronectin. As shown in Fig. 2 and Table 1, 85% of NGF-treated cells extend processes of about 35 pm in length within 2 h of incubation at 37°C on laminin. On fibronectin, on the other hand, neurite TABLE 1 outgrowth was much less pronounced both in terms of Neurite Outgrowth of NGF-Treated PC12 Cells percentage of cells with processes and process length on Various Substrates (Fig. 2 and Table 1). The difference in neurite length Mean neurite between cells plated on fibronectin or laminin was highly 5%of cells length significant with a P < 0.005. In all cases undifferentiated Substrate with neurites” (pm + SD) * cells remained round and did not elongate processes on any of the substrates tested. Analysis of laminin frag- Laminin” 85 35f 9 ments revealed that NGF-treated PC12 cells were able Fibronectin 50 25+ 6 5 23f 7 to extend neurites on fragment Pl as effectively as on Laminin fragment E8 Laminin fragment Pl 82 33+10 intact laminin, but on fragment E8 process outgrowth was negligible (Fig. 2 and Table 1). These data are con’ Experimental conditions were like those in Fig. 2. Neurites were sistent with the results of the adhesion assays described defined as processes with length equivalent to or longer than one cell above. diameter(10 pm).The percentages of cells with neurites were calcuAn antiserum reacting with mouse integrins [27] in- lated by counting 200 adherent cells per sample. Each figure is a mean hibited attachment of NGF-treated PC12 to laminin and value from a duplicate. * The mean neurite length was calculated by analyzing 50 cells with fibronectin. The antibodies also inhibited adhesion to neurites per sample. Pl and to ES laminin fragments (Fig. 3). No inhibition ’ Culture dishes were coated with 10 pg/ml of the indicated protein of adhesion to laminin or Pl fragment was observed with or fragment and postcoated with bovine serum albumin. limiting

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serum dilution FIG. 3. Inhibition of PC12 cell adhesion by integrin antibodies. PC12 cells were exposed to 100 rig/ml of NGF for 6 days and removed from the culture plate by EGTA treatment. Cells were plated on coated dishes in RPM1 medium without NGF for 2 h at 37°C in the presence of serial dilution of antibodies to mouse integrins (anti-BHK). Adhesion is expressed as percentage of the number of cells adhering in the presence of RPM1 medium with serial dilutions of nonimmune serum. Each point is the mean value from a triplicate. The experiments were repeated a minimum of three times with reproducible results. Coating substrates (10 pg/ml) are fibronectin (O), laminin (W), laminin fragment Pl (O), and laminin fragment E8 (0).

leased about 20% of bound radioactivity which was resolved by SDS-PAGE in two ‘251-labeledproteins of 180 and 135 kDa comigrating with the 180 and 135kDa molecules immunoprecipitated by integrin antisera from the cell extracts (Fig. 4, lane c). Further elution with pH 11 buffer released material that could not be resolved in distinct components (Fig. 4, lane d). The 180 and 135 kDa proteins could not be eluted with the synthetic hexapeptide GRGDSP, known to release fibronectin and vitronectin receptors from their ligands [18], nor with the CDPGYIGSR-amide, a laminin Bl chain sequence, thought to bind to the 6%kDa receptor [42]. When the chromatography was performed in the absence of Mn2+, but with Ca2+and Mg+, the pattern of molecules bound and eluted from the laminin column with EDTA was identical to that shown in Fig. 4, but the yield of receptor molecules was 10 times lower.

ET AL.

The 180/135-kDa receptor was specific for laminin and did not bind to fibronectin since the 180-kDa band was not seen in the eluates from the fibronectin-sepharose column (Fig. 4, lane g). This was the case even when the PC12 cell extract was first passed through the fibronectin affinity matrix and then through the laminin column. The integrin nature of the proteins eluted from the laminin column was confirmed by antisera to mouse integrins [27,35,44] and to the carboxy-terminal peptide of the @l integrin subunit. If the cell extract was quantitatively immunodepleted with the mouse integrin antiserum before performing affinity chromatography, EDTA elution of the laminin column no longer yielded the 180/135-kDa proteins. Furthermore, the 180/135kDa protein complex present in the EDTA eluate from the laminin column was immunoprecipitated by the antiserum to the @l peptide (Fig. 4, lane e). In addition to the 180/135-kDa components, a less intense band of about 150-kDa was also eluted with EDTA from the laminin column (Fig. 4, lane c). The nature of this component is uncertain, but it may represent either a second integrin (Ysubunit binding to laminin or a distinct molecule. Compared to the MO-kDa protein, however, the 150-kDa is either a minor component or it binds laminin with lower affinity. To understand which region of the laminin molecule is recognized by the 180/135kDa receptor, affinity chromatography on laminin fragments Pl and E8 were performed. While chromatography on E8 did not yield a significant amount of bound material, Pl chromatography resulted in the isolation of the 180 and 135~kDa proteins previously shown to bind the whole laminin molecule (Fig. 4, lane f). Isolation

of a Fibronectin-Binding

Integrin

After laminin chromatography, the flowthrough fraction was further adsorbed on a fibronectin-Sepharose column. EDTA elution of this column released two labeled bands of 150 and 135-kDa comigrating with the 135 and 150-kDa proteins recognized by the integrin antisera (Fig. 4, lane g). These proteins could also be released from the affinity matrix with buffer containing synthetic GRGDSP peptide (Fig. 4, lane i). The affinitypurified fibronectin receptor was immunoprecipitated by the antiserum to the carboxy-terminal peptide of the 01 subunit (Fig. 4, lane j), indicating that the laminin and fibronectin receptors share immunologically related @subunits. An antiserum to the carboxy-terminal peptide of the a5 human fibronectin receptor subunit [30] did not precipitate the purified PC12 fibronectin receptor (not shown), while it reacted with the receptor from rat fibroblasts [30]. This data is in agreement with the results previously reported by Tomaselli et al. [30] and indicates that the (Ysubunit of the PC12 fibronectin receptor is not related to the human (~5subunit.

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FIG. 4. Immunoprecipitation and affinity isolation of the laminin (A) and fihronectin (B) receptors from NGF-treated PC12 cells. (A) *%Ilabeled PC12 cell extract was immunoprecipitated with an antiserum to mouse integrins (a) and with antibodies to the carboxy-terminal peptide of 01 subunit (b). The three proteins with molecular weight of 180, 150, and 135 kDa represent the integrin complexes expressed in NGFor on a Pl-Sepharose column. After removing primed PC12 cells. A detergent extract of ‘251-labeled cells was applied on a laminin-Sepharose unbound material, the columns were eluted in sequence with 10 mMEDTA and pH 11 buffer. The relevant fractions were pooled and analyzed by SDS-PAGE under nonreducing conditions. ‘251-labeled proteins were eluted with EDTA (lane c) and pH 11 buffer (lane d) from the laminin column. An aliquot of EDTA-eluted material was immunoprecipitated with @l carboxy-terminal peptide antiserum (lane e). The detergent extract from a parallel stock of labeled cells was chromatographed on the Pl affinity matrix and the column was eluted with EDTA (lane f). (B) The iz51-labeled cell extract not bound to the laminin-Sepharose was applied on a fibronectin-Sepharose column to isolate the fibronectin receptor. iz51-labeled proteins eluted with EDTA (lane g), pH 11 buffer (lane h), or GRGDSP peptide (lane i) from the fibronectin column. Material immunoprecipitated from the EDTA eluate by the @l carboxy-terminal peptide antiserum (lane j). The positions of molecular mass standards (kDa) is indicated on the right side of each panel. Integrin bands are identified on the left side of each panel.

Expression of the 180/135kDa Laminin Receptor Is Regulated by NGF Adhesion experiments indicated a selective increase in attachment of NGF-primed PC12 to laminin. To investigate whether this corresponds to an altered expression of the laminin receptor, the integrin pattern of undifferentiated and differentiated PC12 cells was compared by immunoprecipitation. When undifferentiated, metabolically labeled PC12 cells were immunoprecipitated with the mouse integrin antiserum, only two bands of 150 and 135-kDa (Fig. 5, lanes a, c, e, and g) corresponding to the fibronectin binding integrin were detected. The relative labeling intensity of the two bands was reversed compared to surface radioiodination (Fig. 4, lanes a and b). These cells express only barely detectable amounts of the 180-kDa laminin binding subunit that is present on NGF differentiated cells (Fig. 5, lanes b, d, and f; Fig. 4, lanes a and b). This difference was observed after labeling with [35S]methionine or [3H]glucosamine and all these proteins were also immunoprecipitated with an antiserum to the human fibronectin receptor (Fig. 5).

Densitometric analysis of the fluorograms indicated that the amount of the 180-kDa subunit increased at least lo-fold after NGF treatment. Treatment of cells with NGF for different time periods showed that increased expression of the 180-kDa subunit occurred within 24 h (Fig. 5, lanes g and h) and preceded neurite outgrowth that occurs only somewhat later. Dexamethasone is known to induce differentiation of PC12 into chromaffine cells [26]. Under these conditions, cells do not show up-regulation of the 180~kDa band and display a normal pattern of 150 and 135 kDa subunits (not shown), suggesting that the increased expression of the 180-kDa subunit is related to the onset of the neuronal phenotype. Trinity chromatography experiments confumed these data, showing that undifferentiated PC12 cells contain normal levels of the fibronectin receptor, but only barely detectable amounts of the laminin receptor.

Effect of Reduction on Electrophoretic Mobility of Integrin Subunits Reduction of disulfide bonds is known to affect the electrophoretic mobility of some integrin subunits [ 181.

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analysis of integrins in PC12 cells before FIG. 5. Electrophoretic and after differentiation by NGF. Membrane proteins of cells labeled with [35S]methionine (lanes a and b and e-h) or [3H]glucosamine (lanes c and d) were immunoprecipitated with an antiserum to mouse integrins (a-d, g and h) or a goat antiserum to human fibronectin receptor (lanes e and f), and radioactive proteins were analyzed by SDSPAGE and fluorography. Integrins from control PC12 cells (-) are compared to integrins from PC12 cells treated with NGF (+) for 6 days (lanes b, d, and f) or for 1 day (lane h). The molecular mass (kDa) of each subunit is indicated on the left.

We thus examined the SDS-PAGE pattern of the 180, 150, and 135 kDa subunits under nonreducing and reducing conditions. As shown in Fig. 6, the NGF-induced laminin receptor 180-kDa (Ysubunit did not change migration significantly after reduction (lanes a and d). The 150-kDa protein was resolved in two components of apparent molecular mass of 155 and 135-kDa (lanes b and e), while the common 135-kDa fi subunit showed a slightly decreased mobility (lanes c and f). Increased mobility of integrin (Ysubunit is due to the dissociation of a disulfide-linked light chain (about 20 kDa) that is seldom detected on gels [18]. Not all integrin cx subunits consist of light and heavy chains, and in this case their electrophoretic mobility does not decrease upon reduction. While the l&JO-kDa subunit seem to belong to the latter category, the 150-kDa band seems to consist of two distinct (Ysubunits which are either intact or cleaved into heavy and light chain. Both the molecular weight and the nonreduced/reduced gel pattern indicate that the 180-kDa protein is similar to the (~1 subunit of human integrins [16, 181. A definitive proof awaits the availability of antibodies reactive with both human and rat proteins.

ET AL.

grin is based on the following evidence: (i) its subunits are immunoprecipitated by two different antisera to rodent and human integrins, as well as by an antiserum to the carboxy-terminal peptide of the /31 subunit; (ii) its binding to laminin requires divalent cations, a property common to many integrins [ 181; (iii) electrophoretic behavior of its subunits is typical of integrins. This 180/135-kDa integrin binds to laminin and not to fibronectin. In this respect it is distinct from the (~3/ pl complex and the glioblastoma receptor that interacts also with fibronectin [ 19-221 and collagens [al]. In addition the (Ysubunit of the laminin receptor on differentiated PC12 cells has a molecular mass and electrophoretie behavior under reducing and nonreducing conditions clearly different from those of the (r3 [16] or the glioblastoma receptor (Ychain [22]. The 180/135-kDa receptor is also distinct from the (u6/@1laminin receptor of platelets [24] as judged by differences in molecular mass and electrophoretic behavior of the (Ychains. At present we are unable to unequivocally ascertain the correspondence of the 180-kDa (Ysubunit of the rat PC12 with the known human (Ychains. However, its size and electrophoretic mobility are similar to those of the cul [16] subunit. The laminin receptor described here is likely to be identical to a laminin-binding 200/120-kDa integrin complex isolated from a rat neuroblastoma cell line [23]. Tomaselli et al. [30, 391 have previously deNR

R

DISCUSSION

In this paper we show that NGF induces increased expression of an integrin-type laminin receptor at the surface of PC12 cells. We also show that this receptor binds to EHS mouse tumor laminin fragment Pl that is the major site in laminin-supporting neurite outgrowth of differentiated PC12 cells. The laminin receptor consists of two different subunits of 180 and 135-kDa. The identification as an inte-

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FIG. 6. Effect of reduction on the electrophoretic mobility of integrin subunits. Integrins immunoprecipitated from [36S]methionine-labeled, NGF-treated PC12 cells were separated by SDS-PAGE and exposed to X-ray film without fluorography. Each band was cut and divided in two samples and run in SDS-PAGE under nonreducing (NR) or reducing conditions (R). (Lanes a and d) 160-kDa (Ysubunit; (lanes b and e) 150~kDa a! subunit; (lanes c and f) 135-kDa 0 subunit. Molecular mass standards (kDa) are shown on the right.

NERVE

GROWTH

FACTOR

AND

PC12 CELL

scribed an integrin pattern of PC12 cells similar to that reported in this paper. These integrins were purified as a mixture of different complexes, inserted into liposomes, and shown to bind to laminin and collagen type IV [30]. Our data clearly identify the 180/120-kDa bands as the laminin receptor. Recent studies [8,9] have shown that various nonneuronal cells attach to high-affinity cell-binding sites present on laminin fragment Pl, originating from the center of the cross-shaped molecule, and on fragment E8 corresponding to the end of the long arm of laminin. Neurons also exhibit a dual recognition of laminin since they respond by strong neurite outgrowth when exposed to fragment E8 and by weaker reaction when exposed to a structure (El-4) similar to but larger than fragment Pl [5]. We found that PC12 cells are peculiar with respect to other nerve cells since they attach strongly to fragment Pl, but only weakly to fragment ES. In addition neurite extension occurred on Pl, but was insignificant on E8. In agreement with these data, we found that the 180/135-kDa receptor binds to the Pl fragment in affinity chromatography experiments. We thus conclude that differentiated PC12 interact primarily with the Pl region of laminin via the 180/135kDa integrin complex. It was recently demonstrated that fragment Pl contains a single RGD sequence in its A chain component and that RGD peptides can efficiently block a latent cell binding site present in fragment Pl [45, 461. This sequence is apparently not involved in PC12 cell-binding as shown by the failure to inhibit adhesion to Pl by a corresponding synthetic peptide. Since Pl would also not promote neurite extension of neurones [46], the site recognized by PC12 seems distinct from that present on fragment El-4 [5]. The weak interaction of PC12 cells with fragment E8 should also involve integrin complexes as indicated by antibody inhibition experiments (see Fig. 3). However, we were unable to obtain significant binding of proteins on E8 affinity chromatography. Whether this interaction is also mediated by the 180/135-kDa complex or by the minor 150-kDa component isolated on the laminin column is unknown. It may be relevant to this point to mention that the a3/@1 and the (~6//31 laminin receptors primarily recognize sites present in fragment E8 [47,48]. Several studies [12-151 have implicated a 68-kDa protein as cellular receptor for laminin fragment Pl. It was also indicated that synthetic peptides containing the YIGSR sequence of the Bl chain component in fragment Pl are able to elute this receptor from an affinity matrix [42]. The laminin receptor described here is distinct both in size and in lack of sensitivity toward YIGSR peptide. If the 68-kDa receptor is expressed in PC12 cells it might have been released from the laminin matrix by elution with alkaline buffer. Unfortunately, this material contained a complex mixture of proteins which was difficult to resolve into single components by electrophoresis.

ADHESION

TO LAMININ

107

A major finding of this work is the ability of NGF to increase the expression of the 180/135-kDa laminin receptor. NGF selectively increased the expression of the 180~kDa Q! subunit without affecting the levels of the 150-kDa (Y subunit of t.he fibronectin-binding integrin. To our knowledge this is the first example showing that a physiologically relevant differentiation factor can induce a selective alteration in the expression of extracellular matrix receptors at the cell surface. The transforming growth factor /3 was previously shown to up-regulate the expression of integrin complexes in several cell types [49]. In this case, however, TGF-/3 indiscriminately upregulated all integrin complexes expressed by the cell. The increased expression of the laminin receptor correlates with increased cell adhesion to laminin and with their ability to elongate neurites on this substrate, suggesting that this receptor might have a role in this process. This hypothesis is further supported by the fact that this receptor binds to laminin fragment Pl that was found to be the major laminin fragment supporting neurite extension of differentiated PC12 cells. In addition, the increased expression of the receptor occurs within 24 h of NGF treatment before neurite outgrowth can be morphologically appreciated (usually 72-96 h); thus the kinetics of induction is compatible with a role of the receptor in neurite extension. The expression of basal levels of the 180/135-kDa laminin receptor in undifferentiated cells can explain their ability to adhere to laminin and fragment Pl when these substrates are provided in high concentrations (see Figs. la and lb). These cells, however, do not extend neurites, suggesting that the expression of this receptor per se in not sufficient to support neurite outgrowth. It is likely that induction of other biochemical changes, such as alterations of the cytoskeleton, are necessary to make NGF-differentiated PC12 cells fully competent for neurite elongation. A previous report has failed to detect a distinct fibronectin receptor on PC12 cells [30]. This discrepancy could perhaps be explained by the different procedures used to detect binding to fibronectin-affinity chromatography, in our case, and binding of reconstituted liposomes in the case of Tomaselli et al. [30] In fact, the fibronectin receptor of PC12 cells is rather inefficient in promoting adhesion to coated dishes (see Fig. 1) and has structural features distinct from those of the a5/pl complex present in many different cell types. The laminin and fibronectin receptors are likely to have different functions in PC12 cells since neurite extension occurs well on laminin, but weakly on fibronectin (see also Ref. [38]). This indicates that the two receptors generate different responses after binding to their respective ligands. While adhesion through the laminin receptor may generate a signal leading to the organization of neurites, adhesion through the fibronectin receptor does not allow this process. Since the two receptors differ in the (Y subunit, it is likely that structural differ-

108

ROSSINO

ences in these subunits functional responses.

are responsible

for the different

ET AL.

24. Sonnenberg, A., Modderman, 25.

The authors thank Dr. P. Calissano for the generous supply of NGF and Dr. E. Ruoslahti and K. Tomaselli for the gift of the antisera to 61 and ~y5 cytoplasmic peptides. This work was supported by grants from the Consiglio Nazionale delle Ricerche “Progetto Finalizzato Oncologia,” from the Associazione Italiana per la Ricerca sul Cancro (AIRC), from the Italian Ministry of Education, and from the Deutsche Forschungsgemeinschaft.

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