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Vol. 14, Issue 5, 2088-2103, May 2003

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Opposing Roles of Integrin 6A1 and Dystroglycan in Laminin-mediated Extracellular Signal-regulated Kinase Activation

Maria Ferletta ^* , Yamato Kikkawa ^* , Hao Yu , Jan F. Talts , Madeleine Durbeej , Arnoud Sonnenberg , Rupert Timpl ^||, Kevin P. Campbell , Peter Ekblom ^*  ¶ #, and Elke Genersch ^*  ¶

Department of Cell and Molecular Biology, Lund University, SE-22184 Lund, Sweden; ^* Department of Cell and Molecular Biology, Uppsala University, SE-75123 Uppsala, Sweden; Department of Physiology, Biophysics, and Neurology, Howard Hughes Medical Institute, University of Iowa, Iowa City, Iowa; The Netherlands Cancer Institute, Amsterdam, The Netherlands; and ^|| Max-Planck-Institute for Biochemistry, Martinsried, Germany

Submitted January 2, 2003; Accepted January 30, 2003
Monitoring Editor: Richard Hynes

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Laminin–integrin interactions can in some settings activate the extracellular signal-regulated kinases (ERKs) but the control mechanisms are poorly understood. Herein, we studied ERK activation in response to two laminins isoforms (-1 and -10/11) in two epithelial cell lines. Both cell lines expressed 1-containing integrins and dystroglycan but lacked integrin 64. Antibody perturbation assays showed that both cell lines bound to laminin-10/11 via the 31and 61 integrins. Although laminin-10/11 was a stronger adhesion complex than laminin-1 for both cell lines, both laminins activated ERK in only one of the two cell lines. The ERK activation was mediated by integrin 61 and not by 31 or dystroglycan. Instead, we found that dystroglycan-binding domains of both laminin-1 and -10/11 suppressed integrin 61-mediated ERK activation. Moreover, the responding cell line expressed the two integrin 6 splice variants, 6A and 6B, whereas the nonresponding cell line expressed only 6B. Furthermore, ERK activation was seen in cells transfected with the integrin 6A subunit, but not in 6B-transfected cells. We conclude that laminin-1 and -10/11 share the ability to induce ERK activation, that this is regulated by integrin 6A1, and suggest a novel role for dystroglycan-binding laminin domains as suppressors of this activation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Laminins are basement membrane components composed of heterotrimers of , , and chains (Colognato and Yurchenco, 2000). Both laminin-1 (111) and laminin-10/11 (511/521) seem to have important functions in embryogenesis. Laminin-1 is thought to be important for early epithelial morphogenesis in many tissues (Klein et al., 1988; Li et al., 2001, 2002), and mice lacking 5 chain die during midgestation showing multiple morphological abnormalities in several tissues and their compartments (Miner et al., 1998). Laminin 1 has a more restricted tissue distribution than the 5 laminins (Falk et al., 1999; Virtanen et al., 2000). However, both proteins are expressed in the adult stage and may thus have roles in tissue homeostasis.

Laminins bind receptors such as integrins and dystroglycan (Mercurio, 1995; Henry and Campbell, 1999), but the receptor repertoire of all known laminins is not fully clarified. Laminin-1 (111) binds to cells via several 1 integrins, integrin 64, and dystroglycan (Aumailley and Smyth, 1998). Laminin-10/11 is more adhesive than laminin-1 for several cell types (Kikkawa et al., 1998; Gu et al., 1999) and is recognized by integrin 31 (Kikkawa et al., 1998), integrin 61 (Gu et al., 1999; Tani et al., 1999), and integrin 64 (Kikkawa et al., 2000). Yet, binding of two epithelial cells to laminin-10/11 could not be inhibited by antibodies against 3 or 6 integrin subunits when applied singly (Ferletta and Ekblom, 1999). This could be due to complementary functions of integrins or to the presence of other receptors such as dystroglycan (Durbeej et al., 1995, 1998). Dystroglycan binds with high affinity to laminin 1 and 2 chains, mainly to the laminin globular (LG) domains (Timpl et al., 2000), but it does not bind laminin 4 (Talts et al., 2000). Recombinant 5LG domains showed some binding to endothelial dystroglycan (Shimizu et al., 1999), even though a sequence comparison revealed no typical dystroglycan binding sites in the 5 chain (Hohenester et al., 1999).

Laminin–integrin interactions can in some settings activate extracellular signal-regulated kinases (ERKs), but due to the large number of ligands and receptors, information is available only for some interactions. Laminin-5 (332) was reported to activate ERKs in keratinocytes through integrin 64, and it was proposed that 31 and 61 integrins, two major laminin receptors, do not belong to the integrins coupled to the ERK pathway (Wary et al., 1996). This view is still prevailing (Giancotti and Ruoslahti, 1999), although a few reports contradict this view. It has been shown that laminin-5 activates ERK via integrin 31 in keratinocytes, whereas laminin-1 was inactive (Gonzales et al., 1999). Laminin-1 can activate ERK in fibroblasts via unknown receptors (Chen et al., 1994; Fincham et al., 2000). In macrophages, intact laminin-1 does not activate ERK, whereas a shorter laminin 1 peptide does so by yet unknown receptors (Khan and Falcone, 2000). Laminin-1 can activate ERK in macrophages expressing the integrin 6A cytoplasmic splice variant but not in those expressing 6B (Wei et al., 1998). These findings suggest that ERK activation in response to laminins could be regulated at many levels.

Herein, we compared the ability of two laminin isoforms (-1 and -10/11) to activate ERK in two different epithelial cell lines. We first identified integrins 31 and 61 as the major adhesion receptors for laminin-10/11. Mouse laminin-1 was selected as a suitable control because it binds 61 but not 31 (Delwel et al., 1994). It was therefore revealing that both laminins activated ERK in only one of the two cell lines, even though both cell lines showed a similar adhesion behavior on laminin-1 and -10/11. Several assays identified integrin 61 as the mediator of this activation. The cell line responding by ERK activation expressed both integrin 6 splice variants but 6A more prominently, whereas the nonresponding cell line expressed only the 6B variant. These findings demonstrate a crucial role for the cytoplasmic domains of the 6 subunit in ERK activation. We also present evidence for a novel role of dystroglycan as a suppressor of integrin 6A1-mediated ERK activation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Cell Culture
WI-26 VA4 (ATCC CCL-95.1), a human lung epithelial cell line, and WCCS–1, a human kidney epithelial cell line isolated from Wilms' tumor (Talts et al., 1993) were grown as described previously (Ferletta and Ekblom, 1999). Human erythroleukemic K562 cells transfected with integrin 6A or 6B subunits have been described previously (Delwel et al., 1993). For subculturing and for experiments, cells were harvested using 0.05% trypsin and 0.02% EDTA in phosphate-buffered saline (PBS), pH 7.4. Basic fibroblast growth factor was from Roche Diagnostics (Mannheim, Germany). Other cell culture reagents were from Invitrogen (Carlsbad, CA).

Substrates and Antibodies Used in Cell Adhesion Experiments
Mouse laminin-1 was obtained as described previously (Paulsson et al., 1987) or purchased from Invitrogen. The E8 elastase fragment of laminin-1 was prepared as described previously (Paulsson et al., 1987). Laminin-2/4 was from Invitrogen. Laminin-10/11 from human A549 cells was prepared by antibody affinity purification as described previously (Kikkawa et al., 1998). Human laminin-10/11 purified from pepsin digest of placenta by immunoaffinity chromatography by using the anti-5 chain monoclonal antibody (mAb) 4C7 (Tiger et al., 1997; Church and Aplin; 1998; Kikkawa et al., 1998; Ferletta and Ekblom, 1999) was from Invitrogen. Recombinant laminin domains 1LG4-5, 2LG4, 2LG1-3, and 5LG4-5 were produced and purified as described previously (Talts et al., 1999; Yu and Talts, 2003). Dystroglycan from kidney and skeletal muscle was obtained by purification from isolated membranes as described previously (Ervasti et al., 1991; Ohlendieck et al., 1991; Durbeej and Campbell, 1999).

Monoclonal antibodies detecting human integrin subunits were as follows: FB12 against 1, P1E6 against 2, P1B5 against 3 (all from Chemicon International, Temecula, CA), GoH3 against 6, and M13 (BD Biosciences (San Jose, CA) or HA2/5 (BD Biosciences PharMingen, San Diego, CA) against 1. Control monoclonal isotype standards for cell adhesion assays (BD Biosciences PharMingen) were as follows: A112-2 (mouse IgG1,), G155-228 (mouse IgM, ), and R35-95 (rat IgG_2a, ).

Cell Adhesion to Laminin Substrates and Inhibition Assays
Cell adhesion was performed according to Ferletta and Ekblom (1999). For cell adhesion inhibition experiments, cells were preincubated for 20 min at 37°C in suspension in the presence or absence of antibodies at 20 µg/ml and then plated on wells coated with 10 µg/ml laminin-10/11. Cell adhesion was allowed for 10 min at 37°C. Each experiment was performed in triplicate.

Detection of ERK Activation by Western Blotting
ERK activation was detected from cells that had been seeded to confluence (1 x 10⁵ cells/cm²), serum starved for 24 h, detached, and 1) plated on laminin-1 or -10/11 in the presence or absence of antibodies; 2) cultured in suspension in the presence or absence of antibodies; 3) allowed to attach in the presence or absence of recombinant laminin fragments to wells coated with laminin-receptor antibodies overnight at 4°C. For inhibition of ERK phosphorylation, cells in suspension were incubated with PD98059 for 30 min before plating onto laminin-10/11. Extraction of total cellular protein and immunoblotting were performed as described previously (Genersch et al., 2000, 2003). Antibodies were as follows: mouse monoclonal antibodies specific for ERK1, ERK2, mitogen-activated protein kinase kinase (MEK)1, and MEK2 (Transduction Laboratories, Lexington, KY), rabbit polyclonal antibodies against phospho-MEK1/2 (Ser217/221), and mouse monoclonal against phospho-ERK1/2 (T202/Y204) (New England BioLabs, Beverly, MA). An MEK-specific inhibitor of ERK phosphorylation, PD98059, was obtained from Calbiochem. Immunoblots were visualized by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ). The degree of phosphorylation was measured quantitatively by comparing the intensities of bands of blottings made with antibodies detecting kinases irrespective of their phosphorylation status and antibodies detecting the phosphorylated forms. Quantification of data was performed using a Lumi Imager F1 (Roche Diagnostics).

Immunoprecipitation
Biotinylation of cell surface proteins by using the enhanced chemiluminescence protein biotinylation module from Amersham Biosciences was performed according to the manufacturer's instructions. Biotinylated whole cell lysates were precleared with appropriate control IgGs together with protein G plus-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) and subsequently precipitated with antibodies for 2 h at 4°C, and then for 3 h after addition of protein G plus-agarose. Pellets were collected by centrifugation, washed, and boiled in 1x nonreducing Laemmli buffer. Immunoprecipitation reactions were subjected to SDS-PAGE (7.5% polyacrylamide). Electroblotting and detection of biotinylated proteins were performed as described above. The following antibodies were used: integrin 1 antibody P4C10 (Invitrogen), integrin 6 antibody GoH3, integrin 4 antibody ASC-9 (Chemicon International) or -dystroglycan antibody IIH6.

Fluorescence-activated Cell Sorting (FACS) Analysis of Surface Integrins
Half a million of trypsinized and washed cells were resuspended in 50 µl of PBS in microtiter plates at room temperature for 10 min with GoH3 rat anti-human integrin 6 (10 µg/ml) (BD Biosciences PharMingen), or the following mouse antibodies (Chemicon International): FB12 against human integrin 1 (2 µg/ml), AK7 against 2 (10 µg/ml), ASC-6 against 3 (10 µg/ml), and ASC-9 against 4 (10 µg/ml). After 30 min at 4°C, cells were washed three times in PBS and resuspended in 50 µl of goat anti-mouse-Ig-FITC (1/50; DAKO, Glostrup, Denmark) or goat anti-rat-Ig-FITC (1/100; BD Biosciences PharMingen), and incubated in the dark for 10 min before washing and analysis on a FACScan flow cytometer (BD Biosciences). At least 10,000 cells were registered and results analyzed using CellQuest software program (BD Biosciences). Cells incubated with the secondary FITC-conjugated antibody alone were used as controls.

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
Total RNA from WI-26 VA4 and WCCS-1 cells were isolated using RNeasy kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. RT-PCR was performed using Titan One Tube RT-PCR kit (Roche Diagnostics) according to the manufacturer's protocol. Subsequently, a touchdown PCR protocol with a final annealing temperature of 52°C was performed. To detect integrin 6A and 6B splice variants in the same reaction, the following primers were used: 5'-GACTCTTAACTGTAGCGTGA-3' and 5'-ATCTCTCGCTCTTCTTTCCG-3' (Tamura et al., 1991). PCR products were analyzed on a 1.2% agarose gel.

Overlay Assay
Dystroglycan isolated from rabbit skeletal muscle (Ervasti et al., 1991; Ohlendieck et al., 1991) and from rabbit kidney (Durbeej and Campbell, 1999) was separated on 3–12% SDS-PAGE gradient gels and transferred to nitrocellulose membranes. Blots were blocked in laminin binding buffer (LBB) (140 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM triethanolamine, pH 7.6) containing 5% nonfat dry milk and subsequently incubated in LBB containing 3% bovine serum albumin and 0.9 µg/ml laminin-10/11 or -1. Bound laminin-10/11 was detected using monoclonal antibodies specific for human laminin 1 (2E8) and human laminin 5 (4C7) chains, bound laminin-1 was detected using a polyclonal antibody (317) against laminin 1 chain (Durbeej et al., 1996), followed by appropriate peroxidase-coupled secondary antibodies and developed in 4-chloro-1-naphtol and H₂O₂ or enhanced chemiluminescence (SuperSignal; Pierce Chemical, Rockford, IL). For inhibition of -dystroglycan/laminin-10/11 interaction, 1 mg/ml porcine intestinal mucosa heparin (Sigma-Aldrich, St. Louis, MO) tested for its effect on laminin-1 binding to dystroglycan, was included in the LBB.

Solid Phase Assay
Laminin-1 and -10/11 were from Invitrogen. Laminin-5 was purified from the culture medium of a human gastric carcinoma cell line, MKN-45. Cells were cultured in DMEM supplemented with 2.5% fetal bovine serum and 100 ng/ml phorbol 12-myristate 13-acetate. Medium was clarified by centrifugation, supplemented with 5 mM EDTA, 50 µM NEM and 50 µM phenylmethylsulfonyl fluoride, passed over an immunoaffinity column with rabbit polyclonal antilaminin 2 antibodies. The column was washed with 10 mM Tris-buffered saline, pH 7.5, and bound laminin-5 was eluted with 0.1 M glycine-HCl, pH 3.0, and immediately neutralized with 2 M Tris-HCl, pH 7.5. Rat Schwannoma -dystroglycan was purified from RT4 cells. Culture supernatant was passed over a wheat germ agglutinin-Sepharose column (Amersham Biosciences) followed by purification on a laminin-1 affinity column as described previously (Matsumura et al., 1997). Chicken--dystroglycan was a gift from Andrea Brancaccio (Catholic University of Rome, Rome, Italy) and tested as described previously (Talts et al., 1999).

Solid phase assays were carried out with -dystroglycan (5 µg/ml) coated onto the plastic surface of microtiter wells at 4°C. All further incubations were at room temperature. Wells were blocked for 2 h with 50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 5 mM CaCl₂ (wash buffer) with 1% bovine serum albumin. Serial dilutions of laminin-1, -5, and -10/11 were added and incubated for 2 h. After washing, bound ligand was detected with specific antisera against laminin 1 chain (Klein et al., 1988), laminin 3 chain (2B10), or 4C7 mAb against laminin 5 chain (Tiger et al., 1997) diluted to give a maximal absorbance of 1.2–1.6 in enzyme-linked immunosorbent assay. After a further wash, bound antibodies were detected by horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA) followed by addition of 1 mg/ml 5-amino-2-hydroxybenzoic acid (Sigma-Aldrich), 0.001% H₂O₂. Experiments were performed both in the absence and presence of EDTA.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Adhesion of Epithelial Cells to Laminin-10/11 by Cooperation of Integrin 31 and 61
We previously found that laminin-10/11 is a stronger adhesive substrate than laminin-1 for two human epithelial cell lines. Northern blotting showed that both cell types expressed dystroglycan and 1 integrins, and immunofluorescence failed to detect 4 integrins but showed expression of several other integrin chains (Ferletta and Ekblom, 1999). In that study, the strong cell adhesion to laminin-10/11 (Figure 1A) could not be inhibited by antibodies against 3 or 6 integrin subunits in 60-min assays. In shorter 10-min assays, WI-26 VA4 cell attachment to laminin-10/11 could still only partially be inhibited by antibodies directed against either 3 or 6 integrin subunits (Figure 1B). No additional inhibition was seen with any of the other tested combinations of 1, 2, 3, and 6 integrin antibodies (Figure 1B). When both integrin 3 and 6 subunits were blocked, total inhibition of adhesion was achieved in 10-min assays. The combination of 3 and 6 integrin antibodies likewise completely inhibited WCCS-1 cell adhesion to laminin-10/11 (Figure 1C). Blocking the 1 integrin subunit resulted in total inhibition of WCCS-1 cell adhesion to laminin-10/11 (Figure 1C), whereas a residual adhesive activity of WI-26 VA4 cells of 10% was observed (Figure 1B). For both studied epithelial cell lines, either the 31 or the 61 integrin can thus mediate the initial binding to laminin-10/11.

View larger version (14K): [in this window] [in a new window]   Figure 1. Adhesion of WI-26 VA4 and WCCS-1 cells to laminin isoforms and inhibition of cell adhesion to laminin-10/11 by antiintegrin antibodies. (A) WI-26 VA4 (rectangles) and WCCS-1 (circles) cell adhesion to multiwell plates coated with different concentrations of laminin-1 (open symbols) or -10/11 (closed symbols) was measured after 1 h by using a colorimetric reaction. Adhesion of WI-26 VA4 cells (B) or WCCS-1 cells (C) to laminin-10/11 in the presence or absence of integrin antibodies. In B and C, plates were coated with laminin-10/11 at 10 µg/ml and cells were then incubated in the presence or absence of 20 µg/ml either anti-1 integrin antibody (FB12), anti-2 (P1E6), anti-3 (P1B5), anti-6 (GoH3), or anti-1 (Mab13), or combinations of antibodies as indicated in the figure. Cell adhesion was measured after 10 min. Adhesion of control cells in the absence of antibodies was set as 100%, and values reported in the bar graph express the percentage of adherent cells in presence of antibodies. Each point represents the mean of triplicate wells (± SD). The experiments were repeated multiple times with similar results for A, B, and C.

Laminin-1 and -10/11 Induce Mitogen-activated Protein Kinase Signaling in One of the Cell Lines
Whereas human laminin-1 binds both integrin 31 and 61 (Virtanen et al., 2000), mouse laminin-1 binds 61 but not 31 (Delwel et al., 1994). We therefore compared ERK activation initiated by cell binding to mouse laminin-1 and human laminin-10/11. Serum-starved epithelial cells were detached and either kept in suspension or plated on dishes coated with laminin-1. No ERK activation was seen when cells were seeded on plastic. In WI-26 VA4 cells, adhesion to both laminin-1 and laminin-10/11 strongly increased the amount of phosphorylated isoforms of ERK1/2 compared with control cells in suspension (Figure 2A), indirectly suggesting involvement of integrin 61 in ERK signaling in this cell type. In contrast, adhesion of WCCS-1 cells to either laminin did not result in detectable phosphorylation of ERK1/2, although WCCS-1 cells readily responded by ERK activation to basic fibroblast growth factor (Figure 2B). Hence, variability of the ERK activation was not due to different laminin isoforms tested, but rather due to differential ability of cell types to respond to the ligands. Furthermore, firm WCCS-1 cell adhesion to laminin-10/11, mediated by 6 and 3 integrins, was not sufficient to activate ERK.

View larger version (29K): [in this window] [in a new window]   Figure 2. ERK phosphorylation in WI-26 VA4 and WCCS-1 cells attached to laminin-1 or laminin-10/11. (A). Serum-starved cells were detached and kept in suspension or cultured for 60 min on plates coated with laminin-1 or -10/11 at 10 µg/ml as indicated in the figure. Total cellular protein extracts (20 µg) were subjected to Western blot analysis by using antibodies specific for the phosphorylated isoforms of ERK1/2. To evaluate loading efficiency, membranes were stripped and reprobed with antibodies recognizing ERK2 irrespective of its phosphorylation status. The positions of phospho-ERK1/2 and ERK2 are indicated. The same results were seen in more than three experiments for both cell lines. (B) WCCS-1 cells were cultured in medium containing 10% fetal calf serum for 48 h. ERK activation was then measured as in A from cells cultured for an additional 5 min in the presence or absence of basic fibroblast growth factor at 5 nM. (C) Serum-starved cells were detached and then cultured with or without the MEK-specific inhibitor PD98059 (50 µM, PD) for 30 min in suspension and then for 30 min on wells coated with laminin-10/11 at 10 µg/ml. The presence of kinases and their phosphorylated forms was detected by immunoblotting as described above. The positions of phospho-MEK1/2, phospho-ERK1/2, MEK2, and ERK1 are indicated. (D) Quantitative measurements of the phosphorylation status of the kinases. Phosphorylation of MEK1/2 and ERK1/2 in nontreated control cells attached to laminin-10/11 was set as 100%. The same results were seen in three independent experiments.

In the classical mitogen-activated protein kinase signaling pathway, ERK1/2 is phosphorylated through MEK1/2. PD98059, a specific inhibitor for MEK1/2, significantly prevented ERK phosphorylation in response to laminin-10/11 compared with noninhibited control cells (Figure 2, C and D), suggesting that phosphorylation of ERK in WI-26 VA4 cells plated on laminins is achieved through activity of MEK1/2. Time-course experiments showed significant activation of MEK at 40 min on both substrates. Activation peaked at 60 min and was still detectable at 120 min. Activation of ERK showed similar kinetics (Figure 3, A and B).

View larger version (18K): [in this window] [in a new window]   Figure 3. ERK1/2 and MEK1/2 phosphorylation in WI-26 VA4 cells attached to laminin-1 or laminin-10/11. (A) Serum-starved cells were detached and seeded onto wells coated with laminin-1 or laminin-10/11 at 10 µg/ml. Each lane was loaded with 20 µg of total protein extracted at indicated time points and subsequently analyzed for the presence of ERK1/2, MEK1/2, and their phosphorylated forms by immunoblotting. (B) Quantitative measurements of the phosphorylation status of the kinases. Duplicate experiments gave the same results. (C) Serum-starved WI-26 VA4 cells were cultured for 80 min in the presence or absence of 3 or 6 integrin antibodies at 10 µg/ml, first for 20 min in suspension, and then for 60 min attached to wells coated with laminin-1 or -10/11. The presence of ERK1 and phosphorylated forms of ERK1/2 and MEK1/2 was measured by immunoblotting. During the 60-min attachment period, the presence of 3 and 6 antibodies does not influence the number of attached cells (Ferletta and Ekblom, 1999). Cells kept in suspension without antibodies served as an additional control. The positions of phospho-MEK1/2, phospho-ERK1/2, and ERK1 are indicated. The same results were seen in two experiments.

Integrin 61 Is Responsible for Mitogen-activated Protein Kinase Activation
To define receptors involved in ERK activation, we first examined the phosphorylation status of MEK1/2 and ERK1/2 of WI-26 VA4 cells bound to laminin-1 or -10/11 in the presence of 6 or 3 integrin antibodies. Significant but not complete inhibition of MEK and ERK phosphorylation was observed in the presence of 6 antibodies but not in the presence of 3 antibodies (Figure 3C). The incomplete inhibition of signaling by 6 antibodies was most likely due to the ability of these antibodies to induce ERK activation, but more weakly than the natural ligands. As a stringent test of this possibility, we analyzed the ability of antibodies to induce ERK activation in cells kept in suspension. Of the tested antibodies, only integrin 6 antibodies increased phosphorylation of MEK1/2 and ERK1/2 (Figure 4A). Several experiments suggested that natural ligands induce a stronger ERK activation than antibodies, possibly due to ligation of several receptor types. Yet, the IIH6 -dystroglycan antibody decreased ERK activation mediated by cell attachment to plates coated with the 6 antibody, with maximal reduction at 10 µg/ml (Figure 4B). In contrast, IIH6 slightly increased ERK activation at 100 µg/ml, and at 200 µg/ml a two- to threefold increase was noted (our unpublished data). ERK activation by attachment to laminin-10/11 was also decreased by 10 µg/ml IIH6, and increased by 100 µg/ml IIH6 (our unpublished data). IIH6 is an IgM and might at high concentrations cause significant cross-linking of receptors or have unspecific effects. The influence of 10 µg/ml anti-dystroglycan antibodies on cells and on integrin 6-mediated ERK activation suggested that natural dystroglycan ligands might decrease integrin-mediated signaling pathways. However, these data must be cautiously interpreted, because high concentrations of IIH6 caused a reverse effect. Furthermore, in these experiments exposure times were longer than in experiments with natural ligands, revealing some residual ERK activation also in cells in suspension.

View larger version (25K): [in this window] [in a new window]   Figure 4. ERK activation mediated by antibodies in WI-26 VA4 cells. (A) Serum-starved WI-26 VA4 cells were detached and incubated in suspension in serum-free medium in the absence or presence of 3 integrin antibody (anti-3), 6 integrin antibody (anti-6), or anti-dystroglycan antibody (anti--DG) at 10 µg/ml, as indicated. The presence of ERK1 and phosphorylated forms of ERK1/2 and MEK1/2 was detected by immunblotting. (B) Influence of -dystroglycan antibodies on ERK activation induced by cell attachment to the integrin 6 antibody. Serum-starved WI-26 VA4 cells were preincubated in suspension for 20 min in the presence of different concentrations of antibodies against -dystroglycan as indicated, and then seeded on wells coated with antibodies against integrin 6 (10 µg/ml) and allowed to attach for 1 h. The phosphorylated isoforms were analyzed as described in text. The lower panels show quantitative measurements of the phosphorylation status. The positions of phospho-MEK1/2, phospho-ERK1/2, and ERK1 are indicated. The experiments were repeated multiple times and all gave similar results.

We also compared ERK activation in cells allowed to attach to wells coated with anti-6, anti-3, or anti-dystroglycan antibodies. ERK activation was increased only in cells attached to the 6 antibodies (Figure 5, A and B). Inspection of cultures suggested that an equal number of cells attached to each of these three antibody-coated wells, whereas cells did not attach to wells coated with their respective control antibodies. Interestingly, the cells attached to wells coated with IIH6 dystroglycan antibody but remained rounded and did not spread. Quantification revealed equal binding to wells coated with 3 or 6 antibodies, but it was not possible to count cells bound to the IIH6 antibody. During the more harsh washing procedures required for quantification of cell adhesion compared with the assays for ERK activation, essentially all cells bound to IIH6-coated wells detached (Figure 5C).

View larger version (13K): [in this window] [in a new window]   Figure 5. Comparison of ERK activation induced by attachment of WI-26 VA4 cells to wells precoated with integrin 3, 6, or dystroglycan antibodies. (A) ERK activation in cells allowed to attach for 1 h to wells that had been precoated overnight at 4°C with the indicated antibody at 10 µg/ml. Activation was determined by immunoblotting of protein extracts from cells. The positions of phospho-ERK1/2, and ERK1 are indicated. (B) Quantitative measurements of the phosphorylation status. The same results were obtained in three experiments. (C) Quantification of WI-26 VA4 cell adhesion to antibodies. Cells were allowed to attach for 60 min to multiwell plates that were coated overnight at 4°C with 20 µg/ml P1B5 mouse IgG antibody against integrin 3 (anti-3), rat IgG antibody GoH3 against integrin 6 (anti-6), or mouse IgM antibody IIH6 against -dystroglycan (anti--DG), antibody against 1 integrin (anti-1), or appropriate control antibodies. After stringent washings, the amount of bound cells was measured using a colorimetric reaction. Each point represents the mean of triplicate wells (± SD).

To further test the possibility that dystroglycan ligands counteract integrin 6-mediated ERK activation, we tested recombinant laminin fragments with highly varying affinities to dystroglycan. Recombinant laminin 1LG4–5, which binds with high affinity to dystroglycan (Talts et al., 1999) but does not bind any known integrins, reduced ERK activation mediated by the 6 integrin antibody. Inhibition was observed at 11 nM 1LG4–5, with no further inhibition at 100-fold higher concentrations (Figure 6A). In agreement with the hypothesis that integrin 6 ligation increases and dystroglycan ligation decreases ERK activation, recombinant 2LG1-3 decreased 6 antibody-mediated ERK activation less efficiently than 1LG4–5 (Figure 6A). Although recombinant 2LG1-3 binds dystroglycan with higher affinity than 1LG4-5, it also binds integrins (Talts et al., 1999; Talts and Timpl, 1999). Finally, recombinant 2LG4, which has no dystroglycan-binding activity (Talts et al., 1999), slightly increased 6 integrin antibody-mediated ERK activation. This is shown herein for 40-fold higher concentrations than those of 1LG4–5 sufficient to reduce ERK activation (Figure 6A). The influence of purified recombinant proteins is thus not due to unspecific toxic effects.

View larger version (21K): [in this window] [in a new window]   Figure 6. Modulation of ERK signaling by recombinant laminin LG domains. The presence of ERK1 and phosphorylated forms of ERK1/2 and MEK1/2 was measured by immunoblotting. The quantification of results is shown below the gels. (A) WI-26 VA4 cells were cultured for 80 min in the presence or absence of recombinant laminin fragments, first preincubated in suspension for 20 min, and then allowed to attach for 60 min to wells precoated overnight with integrin 6 antibodies (10 µg/ml). Cells kept in solution served as control. Similar results were seen in three experiments. (B) ERK activation after 30 min in response to attachment of WI-26 VA4 cells to integrin 6 antibodies, laminin-1, -10/11, or E8 fragment of laminin-1 at a concentration of 10 µg/ml. The same results were seen in two experiments. (C) The cells were preincubated in the presence of soluble recombinant laminin LG modules as indicated and then allowed to attach to the E8 fragment of laminin-1 (10 µg/ml). The ERK activation was detected after 30 min of attachment. Similar results were seen in three experiments.

The E8 fragment of laminin-1 binds integrin 6 but lacks the dystroglycan binding site of E3 (Delwel et al., 1994). Binding of cells to E8 was used to test whether signaling through dystroglycan has an effect on ERK activation resulting from an interaction of 61 with laminin-1. Cells adhered avidly within 30 min to E8, but detached during subsequent 30 min of culture. Cell attachment to E8 at 30 min activated ERK (Figure 6B). Recombinant 1LG4-5 at 100 nM significantly decreased ERK activation mediated by cell attachment to E8, whereas 100 nM 2LG4 and 5LG4-5 failed to do so in these 30-min assays (Figure 6C). To test whether 5LG4-5 nevertheless has activity, cells were plated on laminin-10/11 for 60 min in the presence or absence of the recombinant proteins. In these assays, 5LG4-5 very significantly reduced ERK activation (Figure 7).

View larger version (25K): [in this window] [in a new window]   Figure 7. Influence of recombinant laminin LG modules on laminin-10/11 mediated ERK activation. WI-26 VA4 cells were pretreated with the indicated LG modules as described in Figure 6. The cells were then allowed to attach on laminin-10/11 for 1 h in the presence or absence of the indicated LG modules. The presence of ERK1 and phosphorylated pERK1/2 is indicated and quantitative measurements of the phosphorylation status are shown in the lower panels. Two of the three experiments with similar results are shown.

Expression of Integrin Subunits and Dystroglycan by WI-26 VA4 and WCCS-1 Cells
To distinguish whether laminin-induced ERK activation was due to 61 or 64 integrins in WI-26 VA4 cells, protein complexes precipitated with antibodies against 1, 4, or 6 integrin subunits were analyzed by SDS-PAGE. Due to alternative splicing or proteolysis, 4 can exist as variants with different molecular masses, but none of these were precipitated with antibodies against 4 subunits. In contrast, four bands were detected in 1 immunoprecipitates (Figure 8A). Based on the known molecular masses, the 180-kDa protein was identified as 1 integrin, the broad 160- to 150-kDa protein complex as a likely mixture of 2, 3, and 6 integrins, and the 120-kDa protein as 1 integrin. The identity of the 90-kDa protein remained unclear. Immunoprecipitation with 6 antibodies revealed two proteins typical for the 6 and 1 subunits, but no apparent 4 subunits (Figure 8A). One major -dystroglycan form of 120 kDa was detected in WI-26 VA4 cells (Figure 8A).

View larger version (38K): [in this window] [in a new window]   Figure 8. Detection of cell surface receptors expressed by WI-26 VA4 and WCCS-1 cells. (A) Immunoprecipitation of integrins and dystroglycan from WI-26 VA4 cells. Whole cell lysates of surface labeled cells were precipitated with antibodies to 1, 6, 4 integrin subunits, or -dystroglycan, as indicated on top. Precipitates were separated by SDS-PAGE under nonreducing conditions. Biotinylated proteins were detected as described under MATERIALS AND METHODS. The known migration patterns of some integrin subunits are indicated at left. The positions of molecular weight markers and the major band precipitated with the -dystroglycan antibody IIH6 are indicated at right. (B) Surface expression levels of some integrin subunits on WI-26 VA4 and WCCS-1 cells, shown by FACS profiles. Cells were stained with antibodies to integrin 1, 2, 3, 6, or 4 integrin subunits before analysis with flow cytometry (black profiles). The negative control (white profiles) shows background levels of staining with the secondary antibody alone. Duplicate experiments gave the same results. (C) Detection of the mRNA for the two splice variants of 6-integrin with different cytoplasmic domains in WI-26VA4 and WCCS-1 cells. Total RNA from WI-26 VA4 and WCCS-1 cells was extracted and subjected to RT-PCR analysis. A primer pair that detects both the 550-base pair amplicon corresponding to variant 6A and the 420-base pair variant corresponding to 6B was used. A control PCR reaction was performed in the absence of templates. PCR products were analyzed by agarose gel electrophoresis. The positions of three marker bands as well as of the 6A- And 6B-amplicons are indicated.

Fluorescence-activated cell sorting showed that all analyzed chains (1, 2, 3, and 6) were expressed on the surface of WI-26 VA4 cells and that WCCS-1 expressed only the 3 and 6 subunits (Figure 8B). Of note, both cell types expressed similar levels of integrin 6. Fluorescence-activated cell sorting confirmed that neither cell type expressed 4 subunits (Figure 8B). The failure of WCCS-1 cell adhesion to laminins to activate ERK is thus neither due to low cell surface expression of 6, nor its association with the 4 subunit. The data also show that integrin 6-mediated ERK activation in WI-26 VA4 cells is mediated by 61 rather than 64. To explain why laminins induce ERK activation in one but not in the other cell line, it was of interest to study the expression of the integrin 6A and 6B splice variants, which have different cytoplasmic tails. Consistent with the results that forced expression of integrin subunit 6A in macrophages confers laminin-1 the ability to induce ERK activation (Wei et al., 1998), we found that the responding cell line WI-26 VA4 expressed both splice variants but 6A more prominently, whereas the nonresponding cell line WCCS-1 only expressed the 6B variant (Figure 8C).

To further substantiate these findings, we analyzed laminin-10/11–mediated ERK activation in human erythroleukemic K562 cells, transfected either with the 6A or 6B variants. Integrin activation increases 6-dependent attachment of these cells to laminin-1 (Delwel et al., 1993), but activation is not required for binding to laminin-10/11 (Delwel et al., 1994; Kikkawa et al., 2000). Experiments with K562 cells and laminin-10/11 were therefore carried out without integrin activation. Whereas untransfected K562 cells bind poorly to laminin-1 or -10/11 (Gu et al., 2003), the K562 6A or 6B variants bound equally well to laminin-10/11 or to integrin 6 antibodies (Figure 9A). Selective MEK/ERK activation was seen in the K5626A cells bound to laminin-10/11 (Figure 9, B–D) or to the integrin 6 antibody (our unpublished data).

View larger version (28K): [in this window] [in a new window]   Figure 9. ERK and MEK activation in K562 cells expressing integrin 6A or 6B variants. (A) Cell adhesion at 1 h to the indicated laminin isoforms, integrin 6 antibodies or to control antibodies (20 µg/ml) was measured colorimetrically. Each point represents the mean of triplicate wells (± SD). The same results were obtained in two different experiments. (B) K5626A and K5626B cells were attached to wells precoated with laminin-10/11 (10 µg/ml) for 1 h. The presence of kinases and their phosphorylation status was examined by immunoblotting as described previously. Quantitative measurements of the phosphorylation status of MEK (C) and ERK (D). Phosphorylation of MEK1/2 and ERK1/2 in cells kept in suspension were set as 100%. Triplicate experiments gave the same results.

Laminin-10/11 as a Ligand for -Dystroglycan
Because both the known -dystroglycan ligand 1LG4-5 and the corresponding laminin-10/11 fragment 5LG4-5 reduced integrin 6A1-induced ERK activation, it was of interest to test whether intact laminin-10/11 from adult tissues is a ligand for dystroglycan. Skeletal muscle and kidney -dystroglycan were therefore separated in SDS-PAGE and incubated with purified laminin-10/11. Analysis of bound laminin-10/11 by antibodies specific for laminin 1 and 5 chains revealed that -dystroglycan is able to bind laminin-10/11 (Figure 10A). Binding of laminin-1 to -dystroglycan was inhibited by heparin as expected (Gee et al., 1993; Pall et al., 1996), but the interaction of laminin-10/11 with -dystroglycan was heparin insensitive (Figure 10B).

View larger version (17K): [in this window] [in a new window]   Figure 10. Binding of laminin-10/11 to -dystroglycan. (A) Purified -dystroglycan from skeletal muscle and kidney was subjected to 3–12% gradient SDS-PAGE, blotted onto nitrocellulose membranes, and subsequently incubated in the presence (left lane) or absence (right lane) of purified laminin-10/11. Bound laminin chains were detected by antibodies specific for laminin 5 and 1 chains (mAbs 4C7 and 2E8) together with appropriate secondary horseradish peroxidase-coupled antibodies. Position of -dystroglycan/laminin-10/11 complex is indicated. (B) Purified -dystroglycan from rabbit skeletal muscle was subjected to 3–12% gradient SDS-PAGE, blotted onto nitrocellulose membranes, and subsequently incubated with purified laminin-1 or laminin-10/11 in the presence or absence of 1 mg/ml heparin as indicated in the figure. Bound laminin-1 was detected by a polyclonal antibody against laminin 1 chain (polyclonal antibody 317), and bound laminin-10/11 was detected by antibodies specific for laminin 5 and 1 chains (mAbs 4C7 and 2E8, respectively). Positions of -dystroglycan/laminin-1 and -dystroglycan/laminin-10/11 complexes are indicated. (C) Rat Schwannoma -dystroglycan binds to laminin-1 and -10/11, but not to laminin-5, as detected by solid phase assays. Absorbance was monitored at 490 nm as a function of whole laminin concentration. Binding of soluble laminin-1 (), -10/11 (), or laminin-5 () to -dystroglycan adsorbed to plates was measured as described under MATERIALS AND METHODS. Experiments were also performed in the presence of 10 mM EDTA with soluble laminin-1 () or -10/11 (). Data are shown as semilogarithmic titration curves. The same results were obtained in three experiments.

Quantitative differences in laminin-1, -5, and -10/11 binding to -dystroglycan from rat Schwannoma cells were tested in a solid phase binding assay. Laminin-1 showed a distinct binding profile with 30 nM laminin-1 required for half-maximal binding (Figure 10C). Laminin-5 did not bind at any of the concentrations tested (maximum 400 nM). Laminin-10/11 showed distinct binding. However, although recombinant 5LG4-5 efficiently reduced laminin-mediated ERK activation, binding was weak compared with the laminin-1/dystroglycan interaction. Due to the low binding, half-maximal binding could not be measured exactly. Very similar binding profiles were obtained with chicken kidney -dystroglycan as the immobilized ligand (our unpublished data). Dystroglycan binding to both laminin-1 and laminin-10/11 was completely inhibited by EDTA, showing a dependence on divalent cations for both interactions (Figure 10C).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Cell adhesion to laminins does not invariably activate the ERK pathway, but the control mechanisms are poorly understood. The 61 integrin is a common adhesion receptor for many laminins and its chain can be alternatively spliced to generate the 6A and 6B cytoplasmic domain variants. We found that laminin-1 and -10/11 activated ERK only in cells expressing 6A1. The integrin 31 was a potent adhesion receptor for laminin-10/11 for both cell lines, but it could not be linked to ERK activation. We also provide evidence for a novel role of dystroglycan as a suppressor of integrin-mediated ERK activation. The recombinant laminin-1 fragment 1LG4–5, known to bind with high affinity to dystroglycan, as well as dystroglycan antibodies at low concentrations, suppressed ERK activation mediated by the integrin 6 antibody. A similar inhibition of laminin-10/11-mediated ERK activation was seen with a recombinant laminin 5 fragment, which also binds dystroglycan.

ERK Activation by Laminin-1 and -10/11
Both tested cell lines expressed the laminin receptors, integrins 31 and 61, and dystroglycan. Also, both cell lines bound laminin-10/11 via the 31 and 61 integrins. We previously showed that both cell lines bound more efficiently to laminin-10/11 than to laminin-1 (Ferletta and Ekblom, 1999). An obvious possibility was that the more adhesive substrate activates ERK more profoundly. However, regardless of the substrate tested, only one of the cell lines responded by increased phosphorylation of MEK1/2 and ERK1/2. The observed prolonged ERK activation is typically initiated by cell–matrix interactions and is distinct from the strong but more transient activation by many growth factors (Aplin et al., 1998). Sustained rather than a transient ERK phosphorylation may be necessary for ERK-mediated changes in gene expression (McCawley et al., 1999; Zeigler et al., 1999; Genersch et al., 2000).

All results consistently defined integrin 61 as the mediator of ERK activation in WI-26 VA4 cells. Mouse laminin-1, a ligand of integrin 61 but not 31, stimulated ERK phosphorylation in these cells as efficiently as laminin-10/11. The ability of WI-26 VA4 cells to remain fully attached to laminin-10/11 when confronted with either the 6 or 3 antibody singly in 60-min assays (Ferletta and Ekblom, 1999) was used to more directly dissect the role of these two integrin subunits for ERK activation. In this assay, 6 but not 3 antibodies decreased laminin-mediated ERK activation. This inhibition was incomplete, suggesting that 6 antibodies themselves cause some stimulation of the MEK/ERK pathway. Indeed, in the absence of extracellular ligands, the 6 antibody activated ERK both in cells kept in solution and in cells allowed to attach to 6 antibodies. No such stimulation was observed with antibodies against integrin 3 or dystroglycan.

WCCS-1 cells adhered to both laminins were shown to use the same receptors as the WI-26 VA4 cells for adhesion to laminin-10/11, expressed the same amount of integrin 6 on the cell surface as WI-26 VA4 cells, could respond to growth factor activation by ERK activation, but neither laminin isoform activated ERK in WCCS-1 cells. The integrin 6 subunit can be alternative spliced to generate 6A and 6B cytoplasmic domain variants (de Melker and Sonnenberg, 1999). The responding cell line WI-26 VA4 expressed both splice variants but 6A more prominently, whereas the nonresponding cell line WCCS-1 only expressed the 6B variant. Similar findings have been reported for laminin-1 in macrophages forced to express these variants (Wei et al., 1998) and was herein demonstrated with laminin-10/11 and K562 cells forced to express either integrin 6A or 6B. It may thus be a general rule that alternative splicing of the cytoplasmic domains of 6 can determine whether the integrin 61 can activate ERK, regardless of the type of extracellular ligands. The cytoplasmic domains of the 6A and 6B variants are almost completely different (de Melker and Sonnenberg, 1999) so they should have distinct intracellular functions.

The integrin 31 could not be linked to activation of the MEK/ERK pathway in either of the tested cell lines, in agreement with the proposals of Wary et al. (1996). However, this integrin may activate ERK in some settings (Gonzales et al., 1999). It should be noted that we did not test the influence of laminin-5 or antibodies against one of its chains, as was done by Gonzales et al. (1999). It is possible that only some ligands for 31 integrin can activate ERK or that the 3A and 3B cytoplasmic splice variants differ in their signaling capacity. These possibilities should be analyzed further with cells of defined expression of such variants (DiPersio et al., 2001). Modest ERK activation and more prominent AKT activation in response to cell attachment to laminin-10/11 was recently reported (Gu et al., 2002) for cells entirely dependent on integrin 31 for cell binding to laminin-10/11, but the receptor responsible for ERK activation was not identified. The current data showing a prominent role of 6A in laminin-mediated ERK activation is supported by recent findings in human ECV304 cells. These cells use 31 as the major and 61 as a minor laminin-10/11 adhesion receptor, yet ERK is activated strongly by 6 antibodies and less efficiently by 3 antibodies (Genersch et al., 2003).

Activated ERK has many targets, ranging from transcription factors to diverse cytoplasmic components (Aplin et al., 1998). In fibroblasts, ERK activated by fibronectin or laminin-1 becomes localized both to the nucleus and focal adhesion complexes (Miyamoto et al., 1995; Fincham et al., 2000). Our findings of distinct roles for 3 and 6 integrin subunits in postadhesion intracellular signaling cascades are interesting in view of reports that 3 and 6 integrin subunits stimulate the formation of different types of focal adhesion complexes in fibroblasts (Dogic et al., 1998) and that 31 regulates cytoskeletal assembly as an inhibitor of other integrins in keratinocytes (Hodivala-Dilke et al., 1998). One future issue is therefore whether integrin 6A1 activated ERK is recruited to focal adhesion macroaggregates in fibroblasts (Laplantine et al., 2000) or to discrete focal adhesion complexes in epithelial cells.

Suppression of Integrin-mediated ERK Activation by Dystroglycan
Recently, Chen et al. (2001) hypothesized that the presence of coreceptors might be necessary for integrin 61-mediated ERK activation. Herein, we demonstrate suppression of this activation by a coreceptor. The dystroglycan antibody IIH6 suppressed integrin 6A1-induced ERK activation in WI-26 VA4 cells. A similar decrease was obtained by recombinant laminin fragment 1LG4-5, which binds dystroglycan with high affinity but lacks integrin-binding sites (Talts et al. 1999). Recombinant laminin fragments with capacity to bind both dystroglycan and integrin 61 (Talts et al., 1999) were not as efficient inhibitors of ERK activation as the dystroglycan-specific 1LG4-5 module. In this context, it is noteworthy that Cyr61, a small nonlaminin ligand for 61 presumably not binding dystroglycan, causes a strong and even more sustained ERK activation than laminins in fibroblasts (Chen et al., 2001).

Dystroglycan Binding to Laminin-10/11
Laminin 1 or 2 LG domains bind dystroglycan with high affinity in a strictly calcium-dependent manner, but lack of similar calcium-binding sites in LG domains of 3, 4, and 5 chains suggest that these should bind poorly to dystroglycan (Hohenester et al., 1999; Timpl et al., 2000). Nevertheless, binding of dystroglycan from an endothelial cell line to recombinant fragment 5LG1-5 has been reported, and 5LG1-5 could even displace laminin-1 attachment to dystroglycan (Shimizu et al., 1999). These domains were produced in bacteria so they might lack important posttranslational modifications. Herein, we demonstrate that dystroglycan from several sources can bind intact laminin-10/11 in overlay and solid phase assays.

Solid phase assays demonstrated no binding of the 3-containing laminin-5 to dystroglycan, as predicted by Timpl et al. (2000). However, some binding to the 5-containing laminin-10/11 was noted, but the binding was weak. Binding of laminin-10/11 could be abolished by EDTA, suggesting divalent cation dependence. Overlay assays also demonstrated binding of laminin-10/11 to dystroglycan isolated both from muscle and a tissue rich in epithelium (kidney). Binding of laminin 1LG4 to dystroglycan can be blocked by heparin (Talts et al., 1999), and a heparin-sensitive cell binding site was recently mapped to mouse 5LG4 (Nielsen et al., 2000). Yet, laminin-10/11 binding to dystroglycan in overlay assays was not perturbed by heparin, suggesting that heparin and dystroglycan binding requires distinct sites. Heparin-insensitive binding to dystroglycan has been shown also for laminin-2/4 (Pall et al., 1996; Talts et al., 1999).

The quantitative binding studies, showing a clear hierarchy among laminin isoforms for -dystroglycan binding are in reasonable agreement both with structural predictions (Hohenester et al., 1999; Timpl et al., 2000) and the report that 5LG1-5 can interact with dystroglycan (Shimizu et al., 1999). Measured binding affinities in cell free assays of some integrins to laminins are also rather low, although these interactions are of obvious biological importance. For instance, integrin 31 had a low binding activity of >600 nM for laminin-5 in conditions reflecting those found in tissues, and bound laminin-10/11 even less efficiently (Eble et al., 1998). Recombinant 5LG4-5 was recently shown to contain the dystroglycan-binding site in another study (Yu and Talts, 2003) and was in the present study shown to be a potent inhibitor of laminin-10/11–mediated ERK activation. This was evident in 60-min assays, but not in 30-min assays carried out with laminin E8 as the substratum. The differences may be explained by the low affinity of laminin-10/11 modules to dystroglycan, or other unknown differences in the binding mechanisms. The finding is notable considering the low affinity of the interaction, but strongly supports the view that the dystroglycan-binding domains of laminins can suppress ERK activation. Hence, the recognition of laminin-10/11 by -dystroglycan might play a significant role in the modulation of signaling cascades initiated by laminins and integrins.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We thank Dr. T. Olofsson (Department of Medicine, Lund University, Lund, Sweden) for the help with FACS analyses. This work was supported by a postdoctoral stipend from Wenner-Gren Foundation to Y.K., and a postdoctoral stipend to M.D. and the fellowship program (to P.E. and K.P.C.) of the Swedish Foundation for International Cooperation in Research and Higher Education, the Swedish Cancer Society, the Swedish Natural Science Research Council, Barncancerfonden, and Crafoordska Stfitelsen. K.P.C. is an Investigator of the Howard Hughes Medical Institute.

	Footnotes

 
Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E03-01-0852. Article and publication date are at www.molbiolcell.org/cgi/doi/10.1091/mbc.E03-01-0852.

Abbreviations used: ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase kinase; LG, laminin globular module.

^¶ These authors contributed equally to this study.

^# Corresponding author. E-mail address: peter.ekblom{at}medkem.lu.se.

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