Original Article

Plasmin-sensitive Dibasic Sequences in the Third Fibronectin-like Domain of L1–Cell Adhesion Molecule (CAM) Facilitate Homomultimerization and Concomitant Integrin Recruitment

Correspondence to: Anthony M.P. Montgomery, Department of Pediatrics-0983, The Whittier Institute, University of California at San Diego, 9894 Genesee Avenue, La Jolla, CA 92037. Tel:(858) 550-2909 Fax:(858) 558-3495 E-mail:ammontgo{at}ucsd.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

L1 is a multidomain transmembrane neural recognition molecule essential for neurohistogenesis. While moieties in the immunoglobulin-like domains of L1 have been implicated in both heterophilic and homophilic binding, the function of the fibronectin (FN)-like repeats remains largely unresolved. Here, we demonstrate that the third FN-like repeat of L1 (FN3) spontaneously homomultimerizes to form trimeric and higher order complexes. Remarkably, these complexes support direct RGD-independent interactions with several integrins, including _vß₃ and ₅ß₁. A pep- tide derived from the putative C-C' loop of FN3 (GSQRKHSKRHIHKDHV⁸⁵²) also forms trimeric complexes and supports _vß₃ and ₅ß₁ binding. Substitution of the dibasic RK⁸⁴¹ and KR⁸⁴⁵ sequences within this peptide or the FN3 domain limited multimerization and abrogated integrin binding. Evidence is presented that the multimerization of, and integrin binding to, the FN3 domain is regulated both by conformational constraints imposed by other domains and by plasmin- mediated cleavage within the sequence RKHSKRH⁸⁴⁶. The integrin ₉ß₁, which also recognizes the FN3 domain, colocalizes with L1 in a manner restricted to sites of cell–cell contact. We propose that distal receptor ligation events at the cell–cell interface may induce a conformational change within the L1 ectodomain that culminates in receptor multimerization and integrin recruitment via interaction with the FN3 domain.

Key Words: neural CAM, heterophilic ligation, melanoma, _vß₃, ₅ß₁, ₉ß₁

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

Human L1 is a member of a subfamily of phylogenetically conserved neural recognition molecules that share a complex ectodomain structure consisting of multiple immunoglobulin and fibronectin (FN)¹ type III repeats (Hortsch 1996 ). Orthologues of human and mouse L1 have been described, including NILE (rat), NgCAM (chick), E587 (goldfish), L1.1/L1.2 (zebrafish), and neuroglian (Drosophila) (Bock et al. 1985 ; Lemmon and McLoon 1986 ; Bieber et al. 1989 ; Bastmeyer et al. 1995 ; Tongiorgi et al. 1995 ). Pioneering studies implicated the L1 subfamily in a variety of dynamic neurological processes, including neurite fasciculation and outgrowth, as well as cerebellar cell migration (Lindner et al. 1983 ; Martini and Schachner 1986 ; Lagenaur and Lemmon 1987 ). With the recent generation of L1-deficient mice, it has been confirmed that L1 is required for normal corticospinal axon guidance (Cohen et al. 1997 ) and for axonal ensheathment by nonmyelinating Schwann cells (Dahme et al. 1997 ; Haney et al. 1999 ). Many of the neuropathologies now described in L1 knockout mice, including dilated brain ventricles, abnormal dendritic architecture, and developmental defects in the hippocampus and corpus callosum (Dahme et al. 1997 ; Demyanenko et al. 1999 ), are consistent with the manifestations of CRASH, a neurological syndrome associated with mutations in the human L1 gene (Fransen et al. 1997 ; Brummendorf et al. 1998 ).

Although designated a neural cell adhesion molecule (CAM), both murine and human L1 homologues have been described on cells of diverse histological origin including epithelial cells associated with kidney collecting ducts (Debiec et al. 1998 ) and with the intestinal and urogenital tract (Thor et al. 1987 ; Kujat et al. 1995 ). Interestingly, the L1 expressed by renal epithelium has been shown to be important for normal branching morphogenesis (Debiec et al. 1998 ). Cells of lymphoid and myelomonocytic origin also express L1 (Ebeling et al. 1996 ; Pancook et al. 1997 ), however, the functional significance of L1 within the immune system remains to be determined. In this regard, Di Sciullo et al. 1998 have shown that L1 is important for maintaining normal lymph node architecture during an immune response and suggest a mechanism based on the expression of L1 by reticular fibroblasts. A potential function for L1 in tumor progression is also suggested by widespread expression on many tumor cell lines including neuroectodermal tumors (melanoma and neuroblastoma), carcinomas (lung, renal, and skin), and monocytic leukemias (Mujoo et al. 1986 ; Linnemann et al. 1989 ; Reid and Hemperly 1992 ; Katayama et al. 1997 ; Pancook et al. 1997 ). Supporting a role for L1 in tumor progression, Linnemann et al. 1989 reported finding elevated levels of L1 on a metastatic variant of a melanoma cell line. Indeed, a recent study by Ohnishi et al. 1998 suggests that L1 may promote metastasis by facilitating tumor cell invasion or migration.

Structure–function studies have defined multiple interactive moieties within L1 that facilitate either homophilic or heterophilic interactions (Hortsch 1996 ). Thus far, many of the interactions defined involve one or more of the six Ig-like domains that constitute the NH₂-terminal portion of the L1 ectodomain. An antiparallel alignment of the first four Ig-like domains of L1 is proposed to facilitate homophilic L1–L1 binding (Su et al. 1998 ). The chondroitin sulfate proteoglycan, neurocan, binds with high affinity to the NH₂-terminal Ig-like domain of L1 (Oleszewski et al. 1999 ). An arginine-glycine-aspartate (RGD) motif in the sixth Ig-like domain of human L1 supports heterophilic interactions with multiple members of the integrin superfamily, including _vß₃ and ₅ß₁ (Montgomery et al. 1996 ; Felding-Habermann et al. 1997 ; Oleszewski et al. 1999 ). Axonin-1/TAG-1, a neuron-specific CAM, undergoes a cis-interaction with the chick L1-orthologue NgCAM via multiple moieties including the first and second Ig-like domains as well as the third FN-like repeat (Kunz et al. 1998 ). Other cis-interacting elements include the tetraspan signaling molecule CD9 (Schmidt et al. 1996 ), NCAM (Feizi 1994 ), and the heat-stable antigen CD24 (Kadmon et al. 1995 ), although the regions of L1 responsible for these interactions have yet to be determined.

The functional significance of the FN-like repeats that constitute the membrane-proximal portion of the L1 ectodomain remains largely unresolved. An antibody directed to an epitope between FN-like repeats 2 and 3 has been shown to promote signal transduction and concomitant neurite extension (Appel et al. 1995 ). Based on this observation the authors proposed that distal recognition events may induce conformational changes that are funneled to a region within the FN-like repeats, which then represents the ultimate site for the induction of signaling events. Recent rotary shadow analysis of the purified L1 ectodomain showed that the FN-like repeats assume a tight globular configuration (Drescher et al. 1996 ). Any conformational lability within this structure may, therefore, provide a mechanism for translating distal ligation events into signaling events. In a further study, Holm et al. 1995 demonstrated that certain FN-like domain fragments have a capacity for homoaggregation, suggesting that one or more of the FN-like domains may have the potential for self-association, perhaps leading to the clustering of L1 at the cell surface; such clustering may in turn be subject to conformational constraints imposed by the globular configuration of the FN-like repeats.

In this study, we have identified moieties within the third FN-like repeat of L1 (FN3) that can simultaneously potentiate receptor clustering and integrin recruitment. Both multimerization of, and integrin binding to, the FN3 domain are shown to be critically regulated by conformational constraints imposed by other domains and by proteolysis. Several integrins are implicated in binding to the FN3 domain including _vß₃, ₅ß₁, and ₉ß₁. Based on our findings, we propose that ligation events at the cell–cell interface may induce a conformation change within the L1 ectodomain that culminates in receptor multimerization and integrin recruitment via the FN3 domain. This paradigm has important implications for L1 signaling and for the modulation of integrin activity during cell–cell interactions.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

Reagents
Antiintegrin antibodies used in this study include the following: anti-ß₁ mAbs P4C10, LM534, B44, and Cl. 18; anti-₅ß₁ mAbs P1D6 and NKI-SAM-1, anti-₅ mAb Cl.1, anti-₉ß₁ mAb Y9A2; anti-_vß₃ mAb LM609; anti-ß₃ antibody AP3; anti-ß₅ antibody 11D1; and an anti-_v/anti-ß₃ (anti-VNR) polyclonal antibody (pAb). mAbs B44, P1D6, and Y9A2 were purchased from Chemicon International. mAbs Cl. 18 and Cl.1 were purchased from Transduction Laboratories. mAb NKI-SAM-1 was purchased from Southern Biotechnology. mAb P4C10 was provided by Dr. E.A. Wayner (University of Minnesota, Minneapolis, MN). LM609, the anti–human L1 mAb 5G3 (Mujoo et al. 1986 ), the anti–L1-Ig6 mAb LP1B9, the anti-VNR pAb, and the anti-5G3 Ag pAb were generated within the Scripps Research Institute. Antibodies AP3 and 11D1 were provided by Dr. David Cheresh (The Scripps Research Institute). An anti–L1 ectodomain (anti–L1-ECD) pAb was generated against, and affinity-purified using the L1 ectodomain fusion protein. A pAb specific for glutathione S-transferase (GST) was purchased from Upstate Biotechnology Inc. L1 peptides were synthesized on an ABI 430A peptide synthesizer within The Scripps Research Institute Core Facility as described previously (Felding-Habermann et al. 1997 ). For the purpose of immobilization, most peptides were made with NH₂-terminal cysteine residues. RGD and RGE control peptides were as follows: GRGDSPC and GRGESPC. Human plasmin was purchased from Calbiochem.

Cell Lines and Culture
M21 human melanoma cells were derived from the UCLA-SO-M21 cell line, which was provided by Dr. D.L. Morton (University of California, Los Angeles, CA). Variant v-integrin–deficient cells (M21-L) were negatively selected from M21 cells by FACS at The Scripps Research Institute. All cells were maintained in RPMI-1640 supplemented with 10% FCS. Transfected CHO cells bearing the human ₉ integrin subunit were generated in the laboratory of Dr. Sheppard and cultured as described (Taooka et al. 1999 ).

Construction and Expression of L1 Fusion Proteins
The recombinant L1 fusion proteins shown in Table 1 (schematic) were generated by PCR amplification of appropriate coding sequences and restriction cloned into the appropriate fusion vector based upon the required tag and reading frame. Primer sequences and their corresponding L1 amino acid start (sense oligos) or stop (antisense oligos) translation sites, as well as the restriction enzymes used for insertion of the respective PCR products into the respective fusion protein vectors, are shown in Table 1. Plasmids were purchased from Amersham Pharmacia Biotech or GIBCO BRL for pGEX GST fusion vectors or pProEX 6xHis fusion vectors, respectively. Mutagenesis of the FN3 construct was performed essentially as described previously (Nayeem et al. 1999 ). In brief, mutagenic sense and antisense oligonucleotides (Table 1) were annealed and extended with Pfu polymerase for a total of 18 cycles. Nonmutant starting material was digested with DpnI and the final product was transformed into supercompetent Escherichia coli. Final constructs were confirmed by dideoxy sequencing.

Purification of the recombinant fusion proteins was performed from log phase BL21 strain E. coli induced with either 100 µM (GST) or 600 µM isopropyl-ß-D-thiogalactopyranoside (6xHis). GST fusion protein purification was performed as previously described (Nayeem et al. 1999 ). For His fusion protein purification, cultures were resuspended in lysis buffer (50 mM Tris-HCl, pH 8.5, 300 mM KCl, 20 mM imidazole, and 0.1% Triton X-100–containing protease inhibitors) and incubated with 100 µg/ml lysozyme at 4°C. Lysates were clarified by centrifugation and fusion proteins were immobilized on Ni-NTA agarose (Qiagen) before extensive washing of the matrix with lysis buffer, followed by washing with 50 mM Tris-HCl, pH 8.5, 500 mM KCl, 40 mM imidazole, and elution with 20 mM Tris-HCl, pH 8.5, 300 mM KCl, 250 mM imidazole. Purified GST and His fusion proteins were dialyzed extensively against PBS.

Adhesion Assays
Adhesion assays were performed essentially as described previously (Felding-Habermann et al. 1997 ). In brief, purified L1 fusion proteins (100–250 nM) were spotted (2-µl spots) or coated (100 µl) onto the bottom of 96-well Titertek plates (ICN Biomedicals) and allowed to coat for 1–2 h at 37°C before blocking with 5% BSA. For adhesion studies involving immobilized peptides, wells were precoated overnight with murine IgG2a antibody before incubation with the heterobifunctional cross-linker SPDP (Pierce Chemical Co.), washing and incubation with peptides at 100–200 µg/ml for 2–3 h before blocking with 5% BSA. Control wells received antibody and SPDP alone without peptide. Cells were harvested and resuspended in adhesion buffer (HBSS, 10 mM Hepes, 0.5% BSA, pH 7.4) containing divalent cations (0.4 mM MnCl₂, 1 mM MgCl₂, 1 mM CaCl₂) with or without antiintegrin function-blocking antibodies. For assays with _v(-)M21-L cells, adhesion was determined in the presence of 0.4 mM MnCl₂ alone. Cells were added at 10⁵ cells/well in the presence or absence of antibodies, and the plates were spun at 700 rpm to give a continuous monolayer. After 15–40 min at 37°C wells were washed with PBS, and the remaining adherent cells were fixed with 1% paraformaldehyde before counting the number of cells per high power field using a 40x objective and an ocular grid at a minimum of four areas per well. Experimental treatments were performed in triplicate.

Fractionation and Detection of L1-His Fusion Proteins
L1-FN3 (His) fusion proteins (5 µg) were fractionated at a flow rate of 0.180 ml/min using a 40-ml bed volume Sephacryl S-200 column (Amersham Pharmacia Biotech). Fractions of 250 µl were collected, and 100 µl of each fraction was applied per well of a Ni-NTA HisSorb plate (Qiagen) for overnight immobilization at 4°C. Wells were subsequently washed with 0.5% BSA in PBS (BSA/PBS) before detection of bound His fusion protein as follows. Wells were incubated with anti–L1-ECD pAb for 1 h with constant shaking before being washed at least five times with BSA/PBS and subsequently incubated with HRP-conjugated goat anti–rabbit secondary antibody (Jackson ImmunoResearch Laboratories). Wells were washed further and bound antibody was detected colorimetrically with TMB (Bio-Rad Laboratories). Color development was arrested with H₂SO₄, and the plates were read at 450 nm on a microplate reader (Kinetic Microplate Reader; Molecular Devices).

Integrin-binding Assays
Purified _vß₃ and ₅ß₁ integrin heterodimers were purchased from Chemicon International. Integrin _vß₃ was biotinylated using NHS-LC-biotin (Pierce Chemical Co.). L1 fusion proteins (10–40 µg/ml) were adsorbed overnight at 4°C onto 96-well Titertek plates. Alternatively, 20 µg/ml of rabbit Ig was adsorbed before incubation with SPDP and immobilization of various peptides as described above for adhesion assays. After coating, the wells were washed and blocked with 0.5% BSA in TBS buffer. Purified integrin heterodimers were added at 1 µg/ml in TBS supplemented with 0.4 mM MnCl₂ and 0.5% BSA. After washing, bound _vß₃ was detected with an HRP-conjugated antibiotin mAb (Sigma Chemical Co.). Bound ₅ß₁ was detected with anti-₅ß₁ mAb NKI-SAM-1, followed by HRP-conjugated goat anti–mouse Ig (Jackson ImmunoResearch Laboratories). Color was developed with TMB or OPD (Sigma Chemical Co.) and plates were read at 450 nm. Control wells received no integrin or were not coated with the L1 fusion protein. To assess the equivalent coating of immobilized GST fusion proteins, parallel wells were blocked with BSA/PBS, incubated with an anti-GST pAb, washed further, and incubated with an HRP-conjugated goat anti–rabbit antibody before colorimetric detection with either TMB or OPD at 450 nm.

Double Immunofluorescence
Aggregated nonadherent _v(-)M21-L melanoma cells from routine tissue culture were harvested, washed, and resuspended in ice-cold FACS buffer (BSA/PBS with 0.05% NaN₃) for incubation with both anti-₉ß₁ mAb Y9A2 and the anti–L1-ECD pAb. Cell aggregates were washed and double stained with fluorochrome-conjugated affinity-purified donkey F(ab')-specific for mouse IgG (Texas red) or rabbit IgG (FITC), which had been preadsorbed to minimize cross-reactivity (Jackson ImmunoResearch Laboratories). Stained cell aggregates were mounted and analyzed using an MRC 1024 confocal microscope (Bio-Rad Laboratories) or a Nikon Eclipse E800 fluorescent microscope. Some stained cell aggregates were gently disrupted by pipetting in the presence of 1% paraformaldehyde, and single cells were assessed for L1 and ₉ß₁ colocalization. In further studies, all of the experimental steps described above were performed at 37°C in the absence of sodium azide.

Immunoprecipitation
For analysis of the coprecipitation of L1 with integrin ₉ß₁, _v(-)M21-L cells were cultured until the nonadherent fraction contained numerous aggregates, at which time the adherent population was composed largely of independent cells. The nonadherent population was harvested, washed, and lysed in 50 mM Tris 7.6, 300 mM NaCl, 0.5% Triton X-100–containing protease inhibitors. Adherent cells were washed and lysed on the plate, and both adherent and nonadherent samples were clarified of Triton-insoluble material. Equal quantities of adherent and nonadherent cell lysate (3.5 mg) were precleared with protein G–Sepharose (Pierce Chemical Co.) before overnight incubation with 5 µg anti-₉ß₁ mAb Y9A2. Antibody–antigen complexes were precipitated with protein G–Sepharose, washed with lysis buffer, and boiled in reducing SDS-PAGE sample buffer before being subjected to SDS-PAGE and immunoblotting with an anti–L1-ECD pAb and a combination of anti–ß₁ integrin mAbs B44 and Cl. 18 as described below.

For studies on the coimmunoprecipitation of integrin subunits with L1, M21 or M21-L cells were aggregated by rotation, harvested, allowed to settle, and lysed as described above for the nonadherent M21-L population. After clarification of the Triton-insoluble fraction, equal quantities of lysate (7.5 mg) were precleared with protein A–Sepharose (Sigma Chemical Co.) before overnight incubation with 15 µg of either anti-5G3Ag pAb or a control anti-GST pAb. Antibody–antigen complexes were precipitated with protein A–Sepharose and washed extensively with lysis buffer (M21-L cells) or RIPA buffer (M21 cells) before boiling in nonreducing SDS-PAGE buffer and separation by SDS-PAGE and immunoblotting as described below. Precipitated L1 and associated integrins were detected with the appropriate antibody as follows: L1, mAb 5G3; ß₁ integrin, mAb LM534; integrin ₅, mAb Cl. 1; integrin ß₃, mAb AP3; integrin ß₅, mAb 11D1.

SDS-PAGE and Immunoblotting
Peptides, purified proteins, or immunoprecipitates were prepared as described and separated in the presence or absence of 2-mercaptoethanol (as required) by SDS-PAGE on precast Tris-glycine gels (Novex). Separated proteins were electroblotted as required to a PVDF membrane (Immobilon-P; Millipore), which was subsequently blocked with 5% milk in PBS. Appropriate primary antibodies were incubated for 1–2 h in TBS containing 0.1% Tween 20 (TBS-T) and 0.5% milk, and bound primary antibody was detected with either an HRP-conjugated goat anti–rabbit Ig (Southern Biotechnology) or donkey anti–mouse Ig (Jackson ImmunoResearch Laboratories) preadsorbed to minimize cross-reactivity with the precipitating immunoglobulins. Antibody complexes were visualized with the chemiluminescent substrate PS-3 (Lumigen Inc.). Alternatively, gels were fixed and processed for staining.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

The Third FN-like Repeat of L1 Promotes RGD-independent ß1 Integrin Interaction with the L1 Ectodomain
Previously, we have shown that the single RGD motif in the sixth Ig-like domain (Ig6) of human L1 is recognized by multiple integrin heterodimers, with the contribution of each of these integrins being dictated by cell type and the cation environment (Felding-Habermann et al. 1997 ). In a physiological cation environment, the adhesion of M21 melanoma cells to the Ig6 domain was found to be solely dependent on integrin _vß₃ (Montgomery et al. 1996 ). While these studies established the importance of the RGD motif in the context of individual domain fragments, integrin recognition of the intact L1 ectodomain was not addressed.

Significant dose-dependent adhesion of M21 cells was observed on immobilized fusion proteins consisting of either the Ig6 domain alone or the entire L1 ectodomain (Fig 1 a). However, inhibition studies using function-blocking antibodies to either _vß₃ or ß₁ integrin demonstrated a significant disparity in the contribution of these integrins to adhesion on the two substrates (Fig 1 b). Thus, blocking ligation by _vß₃ resulted in a complete abrogation of adhesion to Ig6, but was only partially effective when the entire L1 ectodomain was used as a substrate (Fig 1 b). This disparity can be attributed to supplemental ß₁ integrin recognition of the L1 ectodomain, as adhesion to the L1 ectodomain could only be fully blocked with a combination of antibodies to both _vß₃ and ß₁ integrins (Fig 1 b; right).

View larger version (30K): [in this window] [in a new window]   Figure 1. RGD-independent ß1 integrin binding to the L1 ectodomain involves the third FN-like repeat. (a) Adhesion of M21 cells to immobilized L1 ectodomain or domain Ig6 alone. (b) M21 adhesion to L1 ectodomain or Ig6 in the presence or absence of function-blocking antibody to vß3 (LM609), ß1 integrins (P4C10), v integrins (anti-VNR), or both v and ß1 integrins. (c) M21 adhesion to Ig6 with adjacent FN-like repeats (Ig6-FN1-2 and Ig6-FN1-3) in the presence or absence of function-blocking antibodies. Data shown is the mean of triplicate measurements ± SD.

These findings indicate that recognition of the RGD motif in L1-Ig6 can only partially account for the full measure of integrin binding to the entire L1 ectodomain. One possible explanation for this disparity is the presence of a second non-RGD motif recognized by one or more ß₁ integrins. To identify the location of this motif, we generated multidomain L1 fragments containing the Ig6 domain and adjacent FN-like repeats. While the addition of the first and second proximal FN-like repeats of L1 (Ig6-FN1-2) failed to result in supplemental ß₁ integrin binding (Fig 1 c, left) inclusion of the third FN-like repeat (Ig6-FN1-3) did result in adhesion by both _vß₃ and ß₁ integrin(s) (Fig 1 c, right). These data indicate that recognition of sites within Ig6 (RGD) and the third FN-like repeat (FN3; non-RGD), can fully account for integrin binding to the L1 ectodomain. It should be noted that the Ig-like domains proximal to Ig6 (i.e., Ig5 and Ig4) had no influence on integrin recognition (data not shown).

The Heterodimers ₅ß₁ and ₉ß₁ Are Responsible for ß₁ Recognition of the Third FN-like Repeat of L1
A panel of antiintegrin antibodies was tested to identify the ß₁ integrin(s) responsible for recognition of the L1 ectodomain. These antibodies were first tested in adhesion assays using M21 cells selected for a lack of _v integrin expression (i.e., M21-L cells). Because of the absence of _vß₃ expression, M21-L cell adhesion to L1-Ig6 or L1-Ig6-FN1-2 was minimal (Fig 2 a). Consistent with ß₁ integrin recognition of the third FN-like repeat, a marked increase in adhesion was observed on either Ig6-FN1-3 or on the L1 ectodomain (Fig 2 a). Adhesion of _v(-)M21-L cells to both of these substrates was examined in the presence of function-blocking antibodies to a variety of ß₁ integrins including ₁ß₁, ₂ß₁, ₃ß₁, ₄ß₁, ₅ß₁, ₆ß₁, and ₉ß₁. From these studies, it was confirmed that adhesion could only be abrogated using a combination of mAbs specific for both ₅ß₁ and ₉ß₁ (Fig 2 b). Results obtained with the _v(-)M21-L cells could be reproduced with wild-type M21 cells provided _vß₃ binding by these cells was also blocked (Fig 2 c). It should be noted that whereas wild-type M21 cells were able to utilize ₅ß₁ and ₉ß₁ for adhesion in a physiological cation environment (i.e., 1 mM Ca²⁺, 1 mM Mg²⁺, 0.4 mM Mn²⁺), _v(-)M21-L cells only showed significant adhesion via these integrins in an activating cation environment consisting of 0.4 mM Mn²⁺ alone. This unexpected finding suggests that the selection of M21 cells lacking _v integrin expression has had an unforeseen effect on the activation state of these ß₁ integrins.

View larger version (42K): [in this window] [in a new window]   Figure 2. The third FN-like repeat of L1 is recognized by integrins 5ß1 and 9ß1. (a) Adhesion of v(-)M21-L cells to immobilized L1 ectodomain, the Ig6 domain alone, or Ig6 in conjunction with adjacent FN-like repeats (Ig6-FN1-2 and Ig6-FN1-3). (b and c) Adhesion of v(-)M21-L (b) or M21 cells (c) to L1 ectodomain or Ig6-FN1-3 in the presence or absence of function-blocking antibody to vß3 (LM609), 5ß1 (P1D6), or 9ß1 (Y9A2), alone or in combination. Data shown are the mean of triplicate measurements ± SD.

The Third FN-like Repeat (FN3) Alone Supports Integrin Ligation But Recognition of This Domain Is Regulated by Upstream FN1
To confirm that a second integrin recognition motif is present in the FN3 repeat, we generated a further series of L1 domain fragments consisting of single or multiple FN-like repeats, including FN3 alone as well as FN2-3 and FN1-3. L1 domain fragments consisting of FN1 alone and FN1-2 were generated as controls.

M21 cells failed to adhere to either FN1 or FN1-2, but did display significant dose-dependent adhesion to the FN3 domain alone (Fig 3 a). These findings confirm the importance of FN3 in adhesion and demonstrate that this domain can support adhesion independent of the RGD motif in Ig6. However, it is important to note that adhesion to FN3 was markedly reduced when this domain was offered together with both FN1 and FN2 domains (Fig 3 a, FN1-3). This effect can be attributed to the presence of the first FN repeat (FN1) since adhesion to FN2-3 did not differ markedly from adhesion to FN3 alone (Fig 3 a). Several explanations could account for the disparity in adhesion observed between the FN3 and FN1-3 domain constructs, including unequal adsorption of the GST fusion proteins. To exclude this possibility, we assessed the relative coating efficiency of the FN domain constructs by measuring the amount of immobilized GST present in coated wells. When offered at slightly different concentrations, which resulted in equalization of GST immunoreactivity (Fig 3 b, inset), the significant disparity in adhesion between FN3 and FN1-3 was still observed, even though relatively high amounts of fusion protein were offered (Fig 3 b). It is important to note that cell spreading was also markedly reduced on FN1-3, and that at lower coating concentrations, the disparity in adhesion between FN3 and FN1-3 was even more marked (Fig 3 a). A further explanation for this reduced adhesion associated with the presence of FN1 would be an interaction between this domain and a cellular ligand that negatively regulates integrin ligation. However, it should be noted that FN1 did not impact integrin-mediated adhesion to Ig6. Thus, adhesion to a construct consisting of Ig6 and FN1 was not significantly different from that to Ig6 alone (Fig 3 c).

View larger version (22K): [in this window] [in a new window]   Figure 3. Adhesion to the third FN-like repeat of L1 (FN3) is regulated by the first FN-like repeat (FN1). (a) M21 adhesion to immobilized single domain fragments (FN1 and FN3) and multiple domain fragments (FN1-2, FN1-3, and FN2-3). (b) M21 adhesion to the immobilized FN domain fragments after equalizing for protein adsorption as determined by ELISA detection of GST (b, inset). (c) Adhesion of M21 cells to immobilized Ig6 or Ig6-FN1. Data shown are the mean of triplicate measurements ± SD.

To confirm that the adhesion observed with the FN3 domain alone is due to ligation of ₅ß₁ and ₉ß₁, further inhibition studies were performed with both M21 and _v(-)M21-L cells. As expected, _v(-)M21-L cell adhesion to both FN3 and FN1-3 was reduced by function-blocking antibodies to both ₅ß₁ and ₉ß₁ (Fig 4 a). Wild-type M21 cell adhesion to FN1-3 was similarly abrogated using a combination of antibodies to ₅ß₁ and ₉ß₁ (Fig 4 b, right). Remarkably, and in contrast to adhesion on FN1-3, M21 cell adhesion to FN3 could only be completely abrogated with the further addition of an antibody to _vß₃ (Fig 4 b, left). These findings suggest that part of the antiadhesive activity of FN1 can be attributed to its capacity to significantly limit recognition of FN3 by _vß₃. However, since the adhesion of _v(-)M21-L cells was also reduced in the presence of FN1 (Fig 4 a), it is likely that this domain also limits recognition by ₅ß₁ and ₉ß₁. To further establish that ₉ß₁ can directly support adhesion on FN3, it was determined that CHO cells transfected with the ₉ subunit (Taooka et al. 1999 ) are significantly more adherent on FN3 than mock transfectants, and that the increased adhesion observed can be inhibited with a function-blocking antibody to ₉ß₁ (Fig 4 c).

View larger version (31K): [in this window] [in a new window]   Figure 4. The FN3 domain alone is recognized by vß3 as well as 5ß1 and 9ß1, but this interaction is markedly inhibited by the FN1 domain. (a) Adhesion of v(-)M21-L cells to immobilized FN3 alone or FN3 with the adjacent first and second FN-like domains (FN1-3) in the presence or absence of function-blocking antibodies to 5ß1 (P1D6) or 9ß1 (Y9A2). (b) Adhesion of M21 cells to immobilized FN 3 or FN1-3 in the presence or absence of function blocking antibody to vß3 (LM609), 5ß1 (P1D6), or 9ß1 (Y9A2), alone or in combination. (c) Mock-transfected CHO cells were compared with CHO cells transfected with the human integrin 9 subunit in the presence or absence of function-blocking antibody to 9ß1 (Y9A2). (d) Direct binding of purified vß3 or 5ß1 integrins to immobilized FN 3 or FN1-3 as determined by ELISA. The relative coating efficiency of the constructs was determined by anti-GST ELISA (inset). Data shown are the mean of triplicate measurements ± SD.

To confirm direct integrin binding to FN3 and to demonstrate regulation of this interaction by FN1, we performed binding assays with purified ₅ß₁ and _vß₃ heterodimers. The binding of these integrins to FN3 or FN1-3 substrates was compared in an ELISA-based assay (Fig 4 d). Significant direct binding between both _vß₃ and ₅ß₁ and FN3 was observed, and both of these interactions were significantly reduced when FN1-3 was used as a substrate (Fig 4 d). This difference was observed despite a slight disparity in coating efficiency, resulting in the availability of more FN1-3 substrate (Fig 4 d, inset).

The findings presented clearly demonstrate that the third FN-like repeat of L1 can be recognized by multiple integrins including _vß₃, ₅ß₁, and ₉ß₁. However, such interactions are markedly reduced in the presence of FN1. This inhibition is most evident in the case of _vß₃ which, when present, appears to have a dominant role in adhesion to FN3, but recognizes FN1-3 poorly.

Sequences within the Putative B-C and C-C' Loop Regions of FN3 Support Integrin Ligation
Integrin binding motifs in the FN- or Ig-like domains of matrix components or cell adhesion molecules are commonly situated on exposed loops or turns between ß-strands. To identify integrin recognition sequences in FN3, we generated a series of peptides corresponding to putative loop regions (Bateman et al. 1996 ). Two active peptide sequences were identified within the putative B-C and C-C' loop regions of FN3.

A peptide based on the putative B-C loop sequence RPVDLAQVKGHLR⁸²⁷ was found to support significant M21 cell attachment (Fig 5 a). Overlapping truncation peptides from this sequence established the minimal active sequence to be QVKGHLR⁸²⁷. Confirming an integrin-dependent interaction, M21 adhesion to this peptide was blocked using a combination of antibodies to both _vß₃ and ₅ß₁ (Fig 5 b). The adhesion of _v(-)M21-L cells to the QVKGHLR⁸²⁷ sequence was blocked by an antibody to ₅ß₁ alone but was unaffected by an mAb to ₉ß₁ (Fig 5 c). Based on amino acid substitution, both lysine⁸²³ and the COOH-terminal arginine⁸²⁷ residues are essential for adhesion to QVKGHLR⁸²⁷ (Fig 5 d). Substitution of the NH₂-terminal glutamine⁸²¹ residue partially reduced adhesion, whereas mutation of histidine⁸²⁵ had no effect. Importantly, the corresponding sequence in the mouse L1 homologue (i.e., QVKGHLK) was also able to support adhesion (Fig 5 d). To confirm direct integrin binding to immobilized QVKGHLR⁸²⁷ peptide, binding assays were performed with purified ₅ß₁ and _vß₃ heterodimers (Fig 5e and Fig f). Significant binding by both _vß₃ and ₅ß₁ was observed and, consistent with results obtained in the adhesion assays, mutation of the lysine⁸²³ residue resulted in the complete loss of binding by both integrins (Fig 5e and Fig f, mutant). Significantly, specific inhibition of integrin binding by the soluble RGD peptide (GRGDSPC) was not detected (Fig 5e and Fig f).

View larger version (34K): [in this window] [in a new window]   Figure 5. A sequence in the putative B-C loop of the FN3 domain supports adhesion via both vß3 and 5ß1. (a) M21 adhesion to an immobilized peptide corresponding to the putative B-C loop of FN3 (RPVDLAQVKGHLR827) or overlapping truncation peptides of this sequence. (b) Adhesion of M21 cells to immobilized peptide QVKGHLR827 in the presence or absence of antibody to vß3 (LM609) or 5ß1 (P1D6), alone or in combination. (c) Adhesion of v(-)M21-L cells to immobilized peptide QVKGHLR827 in the presence or absence of antibodies to 9ß1 (Y9A2) or 5ß1 (P1D6). (d) M21 adhesion to peptides resulting from alanine substitutions within the sequence QVKGHLR827. (e and f) Direct binding of purified vß3 (e) or 5ß1 (f) integrins to immobilized QVKGHLR827 or QVAGHLR827 (mutant) peptide as determined by ELISA. Soluble RGD or RGE peptide (50 µg/ml) was added concurrently with the purified integrins. Mutated residues are underlined, and the data shown are the mean of triplicate measurements ± SD.

A further peptide that was derived from a sequence in the putative C-C' loop region of FN3 (GSQRKHSKRHIHKDHV⁸⁵²) also supported significant cell adhesion (Fig 6 a). Independent alanine substitution of either of the two dibasic RK⁸⁴¹ and KR⁸⁴⁵ sequences within the peptide resulted in a minor loss of cell adhesion, whereas simultaneous mutation of both dibasic sequences abrogated cell adhesion almost completely (Fig 6 a). Alanine replacement of a downstream KD⁸⁵⁰ sequence within the peptide had a negligible effect on cell adhesion, demonstrating the specificity of cell adhesion for the two dibasic sequences. Consistent with these findings, significant cell adhesion was also observed on a 9-mer peptide corresponding to the first half of the entire C-C' loop peptide (GSQRKHSKR⁸⁴⁵), but not a peptide corresponding to the second half of the C-C' loop (HIHKDHV⁸⁵²; Fig 6 a). M21 adhesion to the wild-type C-C' loop peptide GSQRKHSKRHIHKDHV⁸⁵² was partially blocked using a combination of antibodies to ₅ß₁ and _vß₃ (Fig 6 b), whereas the adhesion of _v(-)M21-L cells was also partially blocked by inhibition of ₅ß₁, but was not significantly affected by an antibody to ₉ß₁ (Fig 6 c). It is important to note that a component of M21 cell adhesion to the wild-type GSQRKHSKRHIHKDHV⁸⁵² peptide was found to be integrin-independent, with some degree of adhesion evident even in the presence of EDTA (data not shown).

View larger version (38K): [in this window] [in a new window]   Figure 6. A sequence in the putative C-C' loop of the FN3 domain supports adhesion and is recognized by both vß3 and 5ß1. (a) M21 adhesion to an immobilized peptide derived from a sequence in the putative C-C' loop of FN3 (GSQRKHSKRHIHKDHV852) or peptides resulting from alanine substitution or truncation. (b) Adhesion of M21 cells to immobilized wild-type peptide GSQRKHSKRHIHKDHV852 in the presence or absence of antibodies to vß3 (LM609), 5ß1 (P1D6), or ß1 integrins (P4C10), alone or in combination. (c) Adhesion of v(-)M21-L cells to immobilized wild-type peptide GSQRKHSKRHIHKDHV852 in the presence or absence of antibodies to 9ß1 (Y9A2) or 5ß1 (P1D6). (d and e) ELISA determination of the direct binding of purified vß3 (d) or 5ß1 integrins (e) to immobilized wild-type GSQRKHSKRHIHKDHV852 or mutant peptides derived by alanine substitution or truncation. Mutated residues are underlined, and the data shown are the mean of triplicate measurements ± SD.

To confirm direct integrin binding to the immobilized GSQRKHSKRHIHKDHV⁸⁵² peptide, binding assays were performed with purified ₅ß₁ and _vß₃ heterodimers (Fig 6d and Fig e). Significant binding to the wild-type peptide by both _vß₃ and ₅ß₁ was observed, and independent alanine substitution of either of the two dibasic RK⁸⁴¹ and KR⁸⁴⁵ sequences resulted in some loss of binding by both integrins. Concurrent mutation of both dibasic sequences completely abolished binding by both _vß₃ and ₅ß₁ (Fig 6d and Fig e), which is consistent with results obtained in the adhesion assays. As expected, both integrins bound to the first half of the wild-type peptide exclusively, demonstrating little or no interaction with the second half of the peptide. As with integrin binding to the B-C loop peptide, binding of _vß₃ and ₅ß₁ integrins to the C-C' loop peptide was not specifically inhibited by the soluble RGD peptide (data not shown).

Site-directed mutagenesis of key residues within the putative B-C and C-C' loop regions of FN3 was performed to confirm that they are required for integrin recognition in the context of the whole domain. Specifically, the sequence QVKGHLR⁸²⁷ in the putative B-C loop was mutated to QVAGHLR⁸²⁷, whereas the GSQRKHSKRHIHKDHV⁸⁵² sequence constituting the C-C' loop was mutated to GSQNNHSNNHIHKDHV⁸⁵². Conservative asparagine substitutions were generated within the C-C' loop to minimize unforeseen effects on domain structure, and both of the dibasic RK⁸⁴¹ and KR⁸⁴⁵ sequences were substituted since both were found to contribute to integrin binding (Fig 6). While the wild-type FN3 domain supported both M21 and _v(-)M21-L adhesion at concentrations as low as 50–75 nM, this adhesion was markedly reduced after substitution of the dibasic sets of lysine and arginine residues in the putative C-C' loop (Fig 7, a and b). Mutation of the single lysine⁸²³ residue in the putative B-C loop had a relatively small but significant effect on adhesion, which was principally evident at lower coating concentrations (Fig 7, a and b). To confirm the primary importance of the C-C' loop sequence, direct binding assays were performed with purified ₅ß₁ and _vß₃ integrins. In agreement with the integrin binding results obtained using peptides, ligation of both integrins to the FN3 domain was dose-dependent and saturable, and exhibited significant susceptibility to mutations within the C-C' loop (Fig 7c and Fig d).

View larger version (41K): [in this window] [in a new window]   Figure 7. Substitution of the dibasic sequences in the putative C-C' loop of the FN3 domain suppresses adhesion and direct integrin binding. (a and b) Adhesion of M21 (a) or v(-)M21-L cells (b) to wild-type FN3 domain or mutant FN3 domains containing conservative asparagine substitution of the two dibasic sequences within the C-C' loop (RK841 and KR845), or a single lysine823 to alanine823 substitution within the B-C loop. (c and d) ELISA determination of the direct binding of purified vß3 (c) or 5ß1 integrins (d) to immobilized wild-type FN3 domain or mutant FN3 containing asparagine substitutions within the C-C' loop. Data shown are the mean of triplicate measurements ± SD.

Based on our findings, we propose that the FN3 domain of L1 contains two novel integrin binding motifs that account for RGD-independent recognition by ₅ß₁ and _vß₃. Substitution studies demonstrate that the dibasic RK⁸⁴¹ and KR⁸⁴⁵ sequences present in the putative C-C' loop of FN3 are of primary importance for integrin recognition. A second motif in the putative B-C loop of the FN3 domain (QVKGHLR/K⁸²⁷) also contributes to ₅ß₁ and _vß₃ binding, albeit to a lesser degree. It is important to note that both QVKGHLR⁸²⁷ and GSQRKHSKRHIHKDHV⁸⁵² peptides were ineffective as soluble antagonists in as much as they failed to inhibit adhesion to themselves. Indeed, a paradoxical increase in adhesion was often observed when these peptides were offered during the adhesion assay (data not shown). One explanation for this effect would be that the soluble peptides can self-associate with the immobilized peptide, thereby resulting in multimerized peptide which is then recognized by integrin _vß₃ and/or ₅ß₁. While we have obtained clear evidence that ₉ß₁ can support adhesion to FN3, we failed to identify the binding motif. It may prove that recognition by this integrin is subject to conformational constraints that are violated by the use of linear peptides.

Integrins Recognize Multimerized FN3 and Homoaggregation of This Domain Is Regulated by the Dibasic Sequences in the Putative C-C' Loop and by the Presence of FN1
Purified FN3 and FN2-3 fusion proteins (GST or His) were both observed to precipitate at high protein concentrations, suggesting a propensity for homoaggregation. In contrast, such precipitation was not observed with either Ig6-FN1-3 or FN1-3 fusion proteins. These observations, coupled with the prior observation that the presence of FN1 is inhibitory to cell adhesion on FN3, raised the possibility that integrins preferentially recognize the FN3 domain as a homomultimer and that inhibition of integrin recognition by FN1 is related to the ability of this domain to inhibit such multimerization. It was also observed that precipitation of the FN3 domain was markedly reduced after substitution of the dibasic RK⁸⁴¹ and KR⁸⁴⁵ sequences present in the putative C-C' loop sequence of FN3. This raised the further possibility that the peptide sequence GSQRKHSKRHIHKDHV⁸⁵², which is of primary importance for integrin recognition, is also important for FN3 homomultimerization. Indeed, a propensity for self-association by the GSQRKHSKRHIHKDHV⁸⁵² peptide could explain the paradoxical finding that this peptide can function as a soluble agonist in adhesion assays. Confirmation of this hypothesis would require that several stipulations be fulfilled: (1) FN3 is multimerized at the time of integrin recognition; (2) the presence of FN1 limits FN3-mediated multimerization; and (3) the GSQRKHSKRHIHKDHV⁸⁵² peptide self-associates and mutation of the C-C' loop sequence, which results in a loss of integrin recognition, also prevents multimerization.

As a first step, we looked for evidence of FN3 multimerization by both column chromatography and SDS-PAGE. To avoid potential complexities associated with the presence of GST as a fusion partner, these studies were performed with an FN3-His construct. After gel filtration on a Sephracryl S-200 column, the FN3 domain was observed to elute as a series of high molecular mass complexes (Fig 8 a, top). Based on size relative to molecular mass standards, the smallest complexes were trimers (3x) and hexamers (6x), with relatively little monomer (1x) evident. The relative proportion of monomer present in different preparations varied with some preparations having little or no evidence of any monomer whatsoever. It should be noted that any precipitate present in the FN3 preparations was removed by centrifugation before fractionation by column chromatography. In support of these findings with the FN3-His protein, separation of the FN3-GST preparation also revealed the presence of trimeric and higher order complexes (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 8. Homomultimerization of the FN3 domain is mediated by dibasic sequences in the putative C-C' loop and regulated by the presence of FN1. (a) Sephacryl S-200 gel filtration elution profiles of wild-type FN3 domain (top) or mutant FN3 containing conservative asparagine substitution of the two dibasic sequences (RK841 and KR845) within the C-C' loop (bottom). Elution points of monomer (1x), trimer (3x), and hexamer (6x) in relation to molecular mass standards are denoted. (b) SDS-PAGE analysis of wild-type FN3 (lane 1), FN3 in which the two dibasic pairs within the C-C' loop are replaced with asparagines (lane 2), or FN3 in which lysine823 within the B-C loop is replaced with alanine (lane 3). Separated proteins were detected by immunoblotting with the anti–L1 ECD pAb. (c) SDS-PAGE and immunoblot comparison of FN3 in conjunction with adjacent FN-like domains (FN2-3 or FN1-3). The relative migration of monomeric (1x), trimeric (3x), and hexameric species (6x) are indicated. (d) SDS-PAGE comparison of wild-type GSQRKHSKRHIHKDHV852 peptide (lane 2), with single dibasic alanine mutant peptides (GSQAAHSKRHIHKDHV852, lane 3; GSQRKHSAAHIHKDHV852, lane 4) and the double dibasic alanine mutant peptide (GSQAAHSAAHIHKDHV852, lane 5). A peptide derived from the Ig6 domain of L1 (PSITWRGDGRDLQEL544) is shown for comparison (lane 1). The relative migration of the peptides in relation to molecular mass standards is shown at the left.

Upon SDS-PAGE resolution under nondenaturing conditions (i.e., without boiling), most of the FN3–His complexes were resolved into the monomer (16 kD, 1x; Fig 8 b, lane 1). However, even in the presence of SDS, a significant amount of trimeric FN3 (48 kD, 3x) was detected. Depending upon the amount of material loaded, small amounts of hexameric FN3 (96 kD, 6x) and sometimes even higher order species could also be detected (Fig 8 b, lane 1). Taken together, these data demonstrate that, under native conditions, the FN3 domain self-associates to form large multimeric complexes and that the most stable multimeric configuration appears to be a trimer (SDS-PAGE), which can further self-associate to form higher order complexes (gel filtration).

Importantly, substitution of the dibasic RK⁸⁴¹ and KR⁸⁴⁵ sequences present in the putative C-C' loop sequence of FN3, which effectively abrogated integrin binding, was also found to limit FN3 multimerization. This is demonstrated by the large amount of monomeric FN3 evident on fractionation (Fig 8 a, bottom) and by an almost complete absence of trimeric FN3 (3x) evident on SDS-PAGE (Fig 8 b, lane 2). The small amount of complexed FN3 remaining despite mutation of the dibasic sequences likely reflects the conservative substitution of the arginine and lysine residues with asparagines. In contrast, alanine mutation of the lysine⁸²³ residue in the putative B-C loop, which only marginally effected adhesion, did not obviously effect the multimerization of FN3 (Fig 8 b, lane 3). This lack of effect on domain multimerization was also observed upon fractionation of the lysine⁸²³ mutant by gel filtration (data not shown). Together, these data suggest that integrin binding to FN3 primarily involves recognition of multimers.

Confirming the hypothesis proposed above, a comparison of complex formation by FN1-3 versus FN3 demonstrates that the presence of FN1 does indeed limit multimerization mediated by the FN3 domain. Thus, the proportion of trimeric FN1-3 (140 kD, 3x) evident on SDS-PAGE was found to be markedly lower than that observed with both FN3 (Fig 8 b, lane 1) and FN2-3 (Fig 8 c). That the FN2-3 construct forms complexes as efficiently as FN3 is consistent with the ability of this two-domain construct to support adhesion at levels equivalent to FN3 alone (Fig 3 a). The observation that FN1-3 is still capable of limited multimerization may explain why FN1-3 can still be recognized by integrins at high coating concentrations.

A final requirement of the hypothesis proposed above is that the GSQRKHSKRHIHKDHV⁸⁵² peptide derived from the C-C' loop of FN3 can self-associate and, as a result, support integrin recognition as a peptide complex. This would support the concept that the C-C' loop of FN3 promotes both multimerization and integrin binding. On SDS-PAGE the 16-mer GSQRKHSKRHIHKDHV⁸⁵² peptide, which has a predicted molecular mass of 2 kD, was found to migrate as a primary species of ~6 kD, indicative of an SDS-stable tripeptide complex (Fig 8 d, lane 2). A 15-mer peptide of 1.95 kD derived from the Ig6 domain of L1 (PSITWRGDGRDLQEL⁵⁴⁴) is shown for comparison (Fig 8 d, lane 1). Interestingly, separate alanine substitution of either of the two dibasic RK⁸⁴¹ or KR⁸⁴⁵ sequences in the GSQRKHSKRHIHKDHV⁸⁵² peptide resulted in a reduction in molecular mass, which is consistent with the formation of di- rather than tripeptide complexes (Fig 8 d, lanes 3 and 4). Finally, simultaneous alanine replacement of both sets of dibasic residues resulted in a peptide that resolved as a monomeric species (Fig 8 d, lane 5). Importantly, these same alanine substitutions also abrogated cell adhesion and integrin binding (Fig 6). Taken together, these data indicate that optimal integrin binding to the GSQRKHSKRHIHKDHV⁸⁵² peptide under native conditions involves recognition of tripeptide or higher order peptide complexes and confirms a role for the dibasic RK⁸⁴¹ and KR⁸⁴⁵ sequences in domain and peptide multimerization.

Plasmin Regulates FN3 Multimerization and Integrin Binding
Human L1, as well as related molecules in the mouse, rat (NILE), and chick (Ng-CAM and Nr-CAM) have been shown to be sensitive to posttranslational cleavage within the FN3 domain (Faissner et al. 1985 ; Sadoul et al. 1988 ; Nybroe et al. 1990 ; Burgoon et al. 1995 ). Importantly, this cleavage has been shown to involve the same dibasic sequences that we have identified as important for integrin binding and multimerization. Thus, trypsin has been shown to cleave after the second dibasic sequence (GSQRKHSKRHIHKDHV⁸⁵²; Faissner et al. 1985 ; Sadoul et al. 1988 ), whereas we have recently demonstrated that plasmin cleaves both dibasic sequences (GSQRKHSKPHIHKDHV⁸⁵²; Nayeem et al. 1999 ). Based on this information, we questioned whether posttranslational cleavage of the FN3 domain represents a mechanism for regulating both multimerization and integrin binding.

Treatment of FN3–His complexes with plasmin resulted in a dose-dependent dissolution of the trimeric FN3 complexes evident on SDS-PAGE (Fig 9 a). A loss of trimeric FN3 complexes was evident at plasmin concentrations as low as 0.01 U/ml. Consistent with this finding, treatment of immobilized FN3 substrate with plasmin at a concentration of 0.01 U/ml also resulted in a >60% inhibition of adhesion by both M21 and _v(-)M21-L cells (Fig 9 b, right). At the same concentration, plasmin had no effect on the RGD-dependent adhesion of M21 cells to the Ig6 domain of L1 (Fig 9 b, left). Interestingly, plasmin treatment of the L1 ectodomain also resulted in a marked inhibition of adhesion by _v(-)M21-L cells, but only marginally decreased adhesion by M21 cells (Fig 9 b, middle). This result is consistent with the finding that _v(-)M21-L adhesion to the L1 ectodomain is highly dependent on interactions with the FN3 domain, while M21 cells can still adhere via interaction with the RGD motif in the Ig6 domain. Together, these data support the concept that serine protease–mediated cleavage within the FN3 domain is a potentially important mechanism for regulating its functional activity.

View larger version (42K): [in this window] [in a new window]   Figure 9. Plasmin regulates FN3 multimerization and integrin binding. (a) SDS-PAGE and immunoblotting analysis of complexed wild-type FN3 treated with plasmin. FN3 in solution was treated for 90 min with plasmin before SDS-PAGE and immunoblotting with the anti–L1-ECD pAb. The FN3 trimer band (48 kD) was analyzed by scanning densitometry and graphed in arbitrary density units. (b) Adhesion of M21 or v(-)M21-L cells to Ig6, FN3, or L1 ectodomain treated with plasmin. Immobilized proteins were treated with or without 0.01 U/ml plasmin for 90 min, washed, and blocked before the addition of cells. Data shown are the mean of triplicate measurements ± SD.

A Paradigm for L1–Integrin Interactions
Based on our findings, it is evident that FN1 limits homomultimerization via the FN3 domain. One possible explanation for this would be steric hindrance in which the FN1 and FN3 domains are folded back upon one another in a closed globular conformation. Homomultimerization via the FN3 domain and concomitant integrin recruitment may only occur after a change in conformation that promotes a more extended open configuration. Homophilic L1–L1 ligation via the Ig-like domains and/or integrin interaction with the RGD motif in Ig6 may be mechanisms for inducing such a change in conformation. A schematic representation of such a model is shown in Fig 10 a.

View larger version (44K): [in this window] [in a new window]   Figure 10. L1 conformation and L1–L1 homophilic interaction may regulate integrin recruitment via the FN3 domain. (a) A potential model for the interaction between integrins and the FN3 domain of L1. The Ig- and FN-like domains of L1 are assumed to form a closed globular conformation according to the findings of Su et al. 1998 and Drescher et al. 1996 . Distal ligation events involving the Ig-like domains of L1 (L1–L1 or L1–integrin) are postulated to cause a conformational change, resulting in a permissive open conformation that can support L1 clustering via FN3 and subsequent integrin recruitment. Potential implications of this multimerization and integrin recruitment include L1-integrin–dependent signal transduction. (b) Anti-Ig6–specific mAb (LP1B9) recognition of its epitope in the Ig6 domain alone, or Ig6 with adjacent FN-like domains (Ig6-FN1-2 and Ig6-FN1-3) as determined by ELISA. Immobilized proteins were incubated with the anti–L1-Ig6 mAb LP1B9, washed, and were detected colorimetrically with HRP-conjugated secondary antibody and OPD. (c) Adhesion of M21 or v(-)M21-L cells to the L1 ectodomain (L1-ECD) or FN3 after pretreatment of cells with the function-blocking anti-L1 mAb 5G3. Data shown are the mean of triplicate measurements ± SD.

Certain predictions can be made based on the model proposed. First, it should be possible to show that, as a result of folding between FN1 and FN3, accessibility to certain domain regions will be limited. In this regard, an mAb specific for an epitope in the Ig6 domain of L1 (mAb LP1B9) was able to recognize Ig6-FN1 and Ig6-FN1-2, but was very limited in its ability to recognize Ig6-FN1-3 (Fig 10 b). This result is hard to reconcile if Ig6-FN1-3 simply forms a linear structure, but can be explained readily if there is a folding event that juxtaposes FN3 with FN1 and Ig6.

A further prediction of the model presented is that inhibition of upstream L1 ligation (e.g., homophilic L1–L1 ligation) will also inhibit integrin-dependent adhesion via FN3 because the FN-like repeats will remain in a closed conformation. In this regard, we observed that integrin-dependent adhesion of _v(-)M21-L cells to the L1 ectodomain could be significantly reduced by an antibody (5G3) that blocks homophilic L1–L1 ligation by binding to an NH₂-terminal Ig-like domain (Montgomery et al. 1996 ; Nayeem et al. 1999 ; Fig 10 c, left). It is unlikely that this antibody is inhibiting _v(-)M21-L adhesion by directly blocking integrin recognition of the FN3 domain since it recognizes a distal epitope and fails to prevent integrin binding to the Ig6 domain, which is located closer to the antibody binding site. However, since _v(-)M21-L cells express high levels of L1, a homophilic interaction with the immobilized L1 could induce the conformation change required for binding of these cells to FN3. The 5G3 antibody was markedly less effective at preventing M21 cell adhesion to the L1 ectodomain (Fig 10 c, right), presumably because these cells are still able to recognize the RGD motif in the Ig6 domain via _vß₃. It is interesting to note that the 5G3 antibody also had some minimal inhibitory activity when the FN3 domain alone was offered as a substrate (Fig 10 c). This inhibition may indicate a limited but direct interaction between cellular L1 and the immobilized FN3 domain. In this regard, it has been shown that FN constructs containing the FN3 domain can interact with other Ig-like domains present in the L1 ectodomain (Holm et al. 1995 ).

Based on the premise that distal L1 ligation events are required to promote FN3 multimerization and integrin recruitment, it is also to be expected that L1 and integrins will only colocalize at the cell–cell interface, where such distal ligation events are expected to occur. Double immunofluorescence was performed to test this prediction. The ₉ß₁ integrin was selected for analysis since previous studies with the _v(-)M21-L cells demonstrated that this integrin is primarily involved in adhesion to the FN3 domain rather than the RGD motif in Ig6. Aggregates of _v(-)M21-L cells were analyzed after simultaneous staining for ₉ß₁ and L1. Both L1 and ₉ß₁ were observed to be recruited to sites of cell–cell contact (Fig 11, a and b) and significant colocalization is evident at these sites (Fig 11c and Fig d). However, it is also important to note that these ligands do not appear to colocalize unless recruited to the cell–cell interface as demonstrated by confocal microscopic analysis (Fig 11 d). Since the juxtaposition of two cell membranes could give the illusion of colocalization, it was further determined whether such colocalization is still evident on single cells obtained after the gentle disruption of stained cell aggregates. The disruption of cell aggregates was performed with simultaneous fixation. Using this approach, we observed significant modulation of both L1 and ₉ß₁ on some of the single cells obtained (Fig 11e and Fig f). Based on the absence of such obvious modulation in the absence of prior aggregation, it is likely that areas of modulation are a result of prior cell–cell contact. Importantly, even under these conditions, we still observed significant colocalization of L1 and ₉ß₁ (Fig 11 g). Significant colocalization was not observed on those single cells that failed to display evidence of modulation (Fig 11 g, asterisk). Adopting the same experimental approach, but with all steps performed at 37°C in the absence of sodium azide, we observed further marked modulation of L1 and ₉ß₁ expression (Fig 11h and Fig i), and again significant colocalization was observed (Fig 11 j). Colocalizatio