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.
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Abstract |
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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 RK
HSK
RH846. The integrin
9ß1, which also recognizes the FN3 domain, colocalizes with L1 in a manner restricted to sites of cellcell contact. We propose that distal receptor ligation events at the cellcell 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
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Introduction |
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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 (
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 (
Structurefunction studies have defined multiple interactive moieties within L1 that facilitate either homophilic or heterophilic interactions (vß3 and
5ß1 (
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 (
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 cellcell 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 cellcell interactions.
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Materials and Methods |
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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 antihuman L1 mAb 5G3 (
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-integrindeficient 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 (
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 (
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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 (
Adhesion Assays
Adhesion assays were performed essentially as described previously (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 1540 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 antiL1-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 antirabbit 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 (1040 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 antimouse 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 antirabbit 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 antiL1-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-100containing 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 GSepharose (Pierce Chemical Co.) before overnight incubation with 5 µg anti-
9ß1 mAb Y9A2. Antibodyantigen complexes were precipitated with protein GSepharose, washed with lysis buffer, and boiled in reducing SDS-PAGE sample buffer before being subjected to SDS-PAGE and immunoblotting with an antiL1-ECD pAb and a combination of antiß1 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 ASepharose (Sigma Chemical Co.) before overnight incubation with 15 µg of either anti-5G3Ag pAb or a control anti-GST pAb. Antibodyantigen complexes were precipitated with protein ASepharose 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 12 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 antirabbit Ig (Southern Biotechnology) or donkey antimouse 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.
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Results |
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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 (vß3 (
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).
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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.
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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).
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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 (
9ß1 (Fig 4 c).
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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 (
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).
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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).
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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 5075 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).
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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).
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Upon SDS-PAGE resolution under nondenaturing conditions (i.e., without boiling), most of the FN3His 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 (HIHKDHV852;
HSK
PHIHKDHV852;
Treatment of FN3His 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 proteasemediated cleavage within the FN3 domain is a potentially important mechanism for regulating its functional activity.
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A Paradigm for L1Integrin 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 L1L1 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.
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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 L1L1 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 L1L1 ligation by binding to an NH2-terminal Ig-like domain (
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 (
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 cellcell 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 cellcell 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 cellcell 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 cellcell 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). Colocalization between
9ß1 and L1 on individual cells that were separated from cell aggregates supports the concept of a cis-interaction between these two ligands.
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As a further test of a direct association between 9ß1 and L1, coimmunoprecipitation studies were performed using the anti-
9ß1 mAb Y9A2. Cell lysates were made from
v(-)M21-L cells maintained as aggregates or as subconfluent monolayers. These lysates were treated with mAb Y9A2 and Western blot analysis was performed to detect the presence of coprecipitated L1. Consistent with an interaction at sites of cellcell contact, L1 was readily detected in Y9A2 immunoprecipitates derived from lysates of aggregated
v(-)M21-L cells (Fig 11 k, Agg.). In contrast, only minimal reactivity was evident in the lysate derived from monolayer cultures (Fig 11 k, Mono.). As further confirmation of L1integrin association, reverse immunoprecipitation was performed using an anti-L1 polyclonal antibody (anti-5G3 Ag-pAb). In these studies, Western blot analysis was performed to detect the presence of coprecipitated ß1,
5, ß3, and ß5 integrin subunits. Providing further evidence for a possible association between L1 and
9ß1, Western blot analysis confirmed the presence of the ß1 integrin subunit in immunoprecipitates derived from aggregated
v(-)M21-L cells (Fig 11 l). Because of a lack of antibodies suitable for the detection of the
9 subunit by Western blotting, we cannot definitively claim that the presence of the ß1 integrin subunit is due to the coprecipitation of
9ß1. However, given that
9ß1 both colocalizes with and coprecipitates L1, it is likely that at least a component of the ß1 present is due to its association with
9. Consistent with reports that have indicated an association between L1 and
5ß1 and
vß3, both ß3 and
5 integrin subunits were also identified in immunoprecipitates of aggregated M21 or M21-L cells (Fig 11 l). In contrast, despite the presence of significant amounts of
vß5 in M21 cell lysates, no significant coprecipitation of the ß5 subunit was detected (Fig 11 l). Importantly, none of the integrin subunits were detected after immunoprecipitation using a control antibody to GST (Fig 11 l).
Based on the observation that L1integrin colocalization is primarily restricted to sites of cellcell contact, it is to be expected that only a small fraction of the available integrin pool will be directly associated with L1. In our system, despite attempts to maximize aggregation, we still observed that many cells remained single and, even in aggregates, we often observed variable recruitment into cellcell contact sites. It also needs to be recognized that some integrins are sequestered in large intracellular pools. In the case of the ß1 integrin subunit, the amount specifically coprecipitated with L1 appears to be <1% of the total available cellular ß1. However, since the L1-reactive ß1 integrins (i.e., 5ß1 and
9ß1) constitute only a small fraction of the total ß1 integrin expression by M21-L cells (<10%) this is not unexpected. Consistent with this, we estimate that significantly more of the available
5ß1 integrin pool coprecipitated with L1 (~5%). However, such estimates may be underestimates since it is not clear how stable L1integrin complexes are under the detergent conditions used during the immunoprecipitation.
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Discussion |
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In this study, we have shown that integrin binding to intact L1 cannot be solely attributed to the RGD motif present in the sixth Ig-like domain, but rather involves additional recognition of sequences in the third FN-like domain (FN3). As such, we describe two novel integrin-binding motifs located within the putative B-C and C-C' loops of the FN3 domain (QVKGHLR827 and GSQRKHSKR845). Site-directed mutagenesis of key residues within these motifs confirmed the primary importance of the sequence GSQRKHSKR845 for both cell adhesion and direct integrin binding to the FN3 domain. Remarkably, the same dibasic sequences responsible for supporting integrin binding to this motif (RK841 and KR845) are also shown to promote the multimerization of the FN3 domain. In this regard, multimerization of the GSQRKHSKR845 sequence precedes, and may be required for, interaction with both vß3 and
5ß1 integrins. Importantly, both homomultimerization of, and integrin binding to FN3 is shown to be regulated both by conformational constraints imposed by other FN-like domains (FN1) and by plasmin-mediated cleavage within the sequence RK
HSK
RH846. Based on our findings, we propose that L1 may have an expanded role in the regulation of integrin function, especially at the cellcell interface, where bridging events appear to allow L1integrin (
9ß1) colocalization.
The large multidomain organization of L1 has permitted the evolution of domains and domain regions with distinct functional attributes. A recent model based on the crystal structure of hemolin suggests that an antiparallel alignment involving the first four Ig-like domains of L1 is responsible for homophilic L1L1 binding (
In this study, we propose that the functional activity of the FN-like domains of L1 may also be regulated by open (extended) and closed (globular) conformations. In this regard, a recent structural analysis of L1 by rotary shadowing and transmission electron microscopy confirmed that the FN-like repeats do indeed form a tight globular structure (
While we suggest that homophilic ligation can alter the conformation of the FN-like repeats, thereby exposing the FN3 domain and promoting integrin recruitment, other heterophilic interactions may also have a role. In this regard, it is of particular interest that TAG-1/axonin-1, a neuron-specific CAM, has been shown to promote neurite extension via a mechanism that requires both an L1-like molecule and ß1 integrins (
The concept that the FN3 domain is involved in self-association is supported by earlier work, which showed that beads coated with FN-like domains 35 of L1 had a strong tendency to self-associate (
Based on our findings, we propose that the FN3 domain is of essential importance for the biological activity of L1. As a consequence, it is expected that the activity of this domain will be tightly regulated. While we propose that conformational constraints are a primary mechanism for regulating the activity of FN3, we also detail a second mechanism based on proteolytic cleavage. An important and unifying property of L1-like proteins is a susceptibility to posttranslational cleavage, thus, human and mouse L1, as well as related molecules in the rat and chick, are sensitive to cleavage within the FN3 domain, yielding fragments that have been detected in neural tissue in vivo (HSK
RH (
The extent to which the FN3 domain promotes either cis- or trans-integrin interactions remains to be resolved. Multimerization of L1 in the plane of the cell membrane via a domain (i.e., FN3) that is in close proximity to the membrane may favor the recruitment of integrins in a cis-type of interaction. The demonstration of colocalization between 9ß1 and L1 on individual cells separated from cell aggregates supports the concept of a cis-interaction. In this regard, cis-interactions between integrins and other receptors, including members of the IgSF, are well documented and are believed to be an important mechanism for both regulating the activational status of the integrin, and for the formation of signaling complexes at the cellcell or cellsubstrate interface (
5ß1 and
9ß1 to the apparent exclusion of other ß1 integrins does, however, suggest the involvement of the
-subunit.
Changes in L1 conformation and concomitant L1 clustering may also be important for the recruitment of other heterophilic ligands reported to undergo cis-interactions with L1. Candidate ligands include the following: CD9 (
The integrin 9ß1 identified as an L1 binding partner in this study has a limited tissue distribution that includes epithelia, muscle, and cells of the myelomonocytic series (
9ß1 integrin is also present on melanoma cells (
9ß1 is expected since L1 also has been described on a variety of epithelial structures (
9ß1 can promote neutrophil transendothelial cell migration via an interaction with VCAM-1 (
9ß1 interactions may also modulate transendothelial cell migration either directly by promoting adhesion to the endothelium or indirectly by modulating the activity of
9ß1 and its ability to bind to VCAM-1. Similarly, the ß1-mediated adhesion of eosinophils to endothelial cells has also been shown to be modulated by ligation of PECAM-1 on endothelial cells (
The observation that the integrins identified in this study are interacting with multimeric FN3 is consistent with the concept that L1 clustering is important for the recruitment of integrins, and would also explain why 9ß1 can only be colocalized with L1 at cellcell interfaces, where classical L1L1 homophilic interactions and consequent clustering would be expected to occur. The concept that ligand polymerization can potentiate integrin binding is supported by recent work that demonstrates preferential integrin-mediated adhesion to multivalent, rather than monovalent extracellular matrix components in lymphoid cells (
An important implication of the work presented here is that cellcell contact and concomitant L1 clustering can potentiate integrin activation and/or signaling. As a consequence, L1 may directly modulate those processes normally associated with integrin-binding to the extracellular matrix, including cell motility, survival, and proliferation. The fact that vß3 can associate with L1 via interaction with its RGD motif (
vß3 is essential for the survival of melanoma cells in the dermal microenvironment, however, detailed examination of these cells indicates that they are surviving as cell clusters (
vß3 ligation may represent an important survival mechanism. The ability of L1 to induce signaling via its interaction with integrins requires confirmation, however, the early steps of neurite extension on L1 are dependent on protein phosphorylation events involving the nonreceptor tyrosine kinase pp60c-src, a known mediator of integrin signaling (
The findings reported here significantly extend our current knowledge of L1integrin interactions. We have documented two novel integrin-binding sequences within putative loops in the third FN-like domain of L1. One of these sequences is shown to self-associate, resulting in both domain multimerization and integrin ligation. To our knowledge, this is the first report of a spontaneously self-associating peptide sequence that can also support direct integrin binding. Dual regulation of FN3-integrin binding is proposed based on conformational constraints and sensitivity to proteolysis. Based on our observations, and those of others, we propose a model in which distal trans-ligation of L1 at the cellcell interface induces a conformation change within the L1 ectodomain that culminates in receptor multimerization and integrin recruitment via the FN3 domain. The findings presented in this study further suggest an intimate association between integrins and L1, an association that may have important implications for downstream signaling events.
Note Added in Proof. While this manucript was being reviewed, it was reported that the third fibronectin repeat of L1 is required for L1 to serve as an optimal substratum for neurite extension. Stallcup, W.B. 2000. J. Neurosci. Res. 61:3343.
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Footnotes |
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1 Abbreviations used in this paper: CAM, cell adhesion molecule; ECD, ectodomain; FN, fibronectin; pAb, polyclonal antibody; RGD, arginine-glycine-aspartate.
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Acknowledgements |
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This study was supported by the National Cancer Institute (NCI) RO1 grant CA69112-01 and by National Heart, Lung and Blood Institute (NHLB) RO1 grant HL62477-01 (to A.M.P. Montgomery) and by the NCI research fellowship (S. Silletti) 1F32 CA72192-01. This is Scripps manuscript #12868-IMM.
Submitted: 13 December 1999
Revised: 1 May 2000
Accepted: 23 May 2000
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References |
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