A Single Immunoglobulin-like Domain of the Human Neural Cell Adhesion Molecule L1 Supports Adhesion by Multiple Vascular and Platelet Integrins

Anti-integrin antibodies used include the following: anti-hamster α₅β₁ mAb PB1, anti-α_v and β₃ integrin polyclonal (anti-vitronetic receptor [VNR]), anti-α_vβ₃ mAb LM609, anti-α_IIbβ₃ mAb LJ-CP8, anti–β₃ integrin mAb 7E3, anti–β₁ integrin mAb P4C10, and anti–α_v integrin mAb 17E6. PB1 was generated and provided by Dr. R. Juliano (University of North Carolina, Chapel Hill, NC), (Brown and Juliano, 1985). Anti–β₁ integrin mAb P4C10 was provided by Dr. E.A. Wayner (University of Minnesota, Twin Cities, MN). The 7E3 antibody was originally generated and characterized by Coller et al. (1986) and the 17E6 antibody (Mitjans et al., 1995) was provided by Dr. S.L. Goodman (Merck KGaA, Darmstadt, Germany). LM609 (Cheresh and Spiro, 1987), anti-VNR, and LJ-CP8 (Niija et al., 1987) were generated within the Scripps Research Institute (La Jolla, CA). The anti–human L1 mAb 5G3 used in this study, was also generated and characterized within the Scripps Research Institute (Mujoo et al., 1986).

L1 peptides were synthesized on a peptide synthesizer (ABI 430A; Applied Biosystems, Inc., Foster City, CA) within the Scripps Research Institute Core Facility. A 15-mer peptide was selected to include the single RGD site in human L1 (i.e., PSITWRGDGRDLQEL). Control peptides were substituted with alanine to give PSITWRADGRDLQEL. For the purpose of immobilization an additional batch of these peptides was made with NH₂-terminal cysteine residues. Peptides were prepared using Rink Amide MBHA or Wang resin (Calbiochem-Novabiochem, La Jolla, CA). After resin deprotection and assembly the peptides were cleaved from the resin with a cleavage cocktail (2.5% ethanedithiol, 5% thioanisole, 5% water, 87.5% trifluoroacetic acid) and subsequently purified by preparative reverse phase HPLC. Peptides were characterized further by analytical HPLC and mass spectroscopy.

The generation and characterization of CHO cells stably transfected to express normal human platelet α_IIbβ₃ (A5 cells) has been described in detail elsewhere (O'Toole et al., 1989, 1990; Frojmovic et al., 1991) and will be described only briefly here. CHO cells were cotransfected with equal amounts of human α_IIb and β₃ expression constructs and a CDM8 vector containing the neomycin resistance gene CDNeo at a ratio of 30:1 (O'Toole et al., 1989). Transfection was performed by the calcium phosphate method followed by glycerol shock. G418-resistant colonies were isolated and positive clones identified by flow cytometry using subunit-specific antibodies. The generation of CHO cells transfected to express the active extracellular domain of α_IIbβ₃ (α_IIbα_LΔβ₃ cells) has been described (O'Toole et al., 1994). Briefly, the cytoplasmic sequence from the α_L integrin subunit was isolated from the appropriate cDNA clone by PCR with the PCR oligonucleotides designed to omit the α_L cytoplasmic sequence VGFFK (i.e., α_LΔ). As previously described, the α_LΔ construct was ligated with a fragment encoding the extracellular and transmembrane domains of the α_IIb integrin subunit. Coexpression of this chimeric internal deletion mutant (α_IIbα_LΔ) with the wild-type β₃ integrin subunit resulted in the stable expression of the α_IIbα_LΔβ₃ heterodimer bearing an active, high affinity extracellular domain of human α_IIbβ₃ (O'Toole et al., 1994). Wild-type CHO-K1 cells and transfected cell lines were maintained in DME supplemented with 10% FCS, 1% glutamine, and 1% non-essential amino acids.

A spontaneously transformed, human umbilical vein endothelial cell line designated ECV304 (Hughes, 1996) was obtained from the American Type Culture Collection (Rockville, MD). A stable α_vβ₃-negative variant of this line was obtained by repeated negative sorting using anti-α_vβ₃ mAb LM609. Sorting of α_vβ₃-negative unstained cells was performed using a FACstar^® flow cytometer (Becton Dickinson, Co., Mountain View, CA). To obtain a stable, α_vβ₃-negative population, the cells were sorted on five consecutive occasions. The ECV304 cells were maintained in M199 medium supplemented with 10% FCS and 1% glutamine.

Blood was collected from the antecubital vein of healthy adult donors through a 19-gauge needle into syringes containing, as anticoagulant the thrombin inhibitor d-phenylalanyl-l-arginine chloromethyl ketone dihydrochloride (PPACK; Bachem Bioscience Inc., Philadelphia, PA) (50 nM final concentration) and supplemented, when indicated, with prostaglandin E₁ (PGE₁; Sigma Chemical Co., St. Louis, MO) (20 nM final concentration) to inhibit platelet activation. None of the donors had taken drugs known to affect platelet function for the preceding 10 d. For the preparation of washed platelets, the blood was supplemented with 5 U/ml of the ADP scavenger apyrase (Sigma Chemical Co.) and centrifuged at 2,500 g for 15 min at room temperature. Plasma was removed and replaced with an equivalent volume of Hepes-Tyrode's buffer, pH 6.5 (10 mM Hepes, 140 mM NaCl, 2.7 mM KCl, 0.4 mM NaH₂PO₄, 10 mM NaHCO₃, and 5 mM dextrose), containing 1 U/ml of apyrase. The resuspended blood cells were centrifuged again at 2,250 g for 10 min. The blood cells were washed twice using Hepes-Tyrode's buffer containing 0.2 U/ml apyrase in the next step and no apyrase in the last step. The final blood cell pellet was reconstituted in Hepes-Tyrode's buffer, pH 7.4, containing 50 mg/ml BSA to adjust the viscosity to that of plasma, and then centrifuged at 700 g for 15 min. The platelet-rich supernatant was collected and supplemented with 1 mM CaCl₂, 1 mM MgCl₂, and 100 μM MnCl₂. The platelet count was adjusted to 100,000 platelets/μl. To analyze the effect of activation on platelet adhesion, the platelets were stimulated with ADP and epinephrine (20 μM final concentration of each) immediately before adding the platelet suspension to the assay plates. Adhesion of non-activated platelets was studied using unstimulated platelets prepared from PGE₁-treated blood.

Two wild-type and two mutant L1–glutathione-S-transferase (GST) fusion proteins were used in this study. The wild-type fusion proteins consisted of Ig-like domains 4, 5, and 6 (L1-Ig4-6) or the sixth Ig-like domain alone (L1-Ig6). The mutant fusion proteins consist of the sixth Ig-like domain of L1 with the amino acid mutations of Arg-554 and Asp-556 to Lys-554, and Glu-556, respectively (i.e., RGD→ KGE) or the single amino acid mutation Asp-556 to Ala-556 (i.e., RGD→ RGA). Amino acids are numbered as described by Bateman et al. (1996).

The generation and characterization of the L1-Ig4-6 GST fusion protein used in this study has been described in detail elsewhere (Zhao and Siu, 1995). The production of L1-Ig6 GST fusion protein (amino acids 518–614) was as follows. The region of interest was amplified from full-length L1 cDNA, which was provided by Dr. J. Hemperly (Becton Dickinson Research Center, Research Triangle Park, NC). Amplification was performed according to the manufacturer's instructions for the Expand High Fidelity PCR System (Boehringer Mannheim Corp., Indianapolis, IN) using an upstream sense primer specific for nucleotides 1,542–1,562 of the human L1 open reading frame (ORF) and containing an engineered internal EcoRI restriction endonuclease site (5′-AAA GAA TTC ACT CAG ATC ACT-3′) in conjunction with a downstream antisense primer specific for nucleotides 1,829–1,849 of the human L1 ORF, which was also engineered to contain an internal EcoRI site (5′-CGT GAA TTC GGC CCA GGG CTC-3′). The resulting product was digested with EcoRI and subcloned into a pGEX-1λT vector (Pharmacia Biotech Sevrage, Uppsala, Sweden). Competent Escherichia coli strain BL21 cells (Stratagene, La Jolla, CA) were transformed with this construct and resulting colonies were screened by PCR and examined for expression of appropriately sized GST fusion protein by SDS-PAGE, followed by immunoblotting with an anti-GST polyclonal antibody (Upstate Biotechnology, Inc., Lake Placid, NY). The chemiluminescent substrate PS-3 (Lumigen, Inc., Southfield, MI) was used for detection. Dideoxy sequencing of positive clones was performed to verify the integrity of the introduced coding sequence.

The mutant L1-Ig6 RGD→ RGA was generated according to the manufacturer's instructions for the Quickchange Site-Directed Mutagenesis Kit (Stratagene) using the plasmid DNA encoding the L1-Ig6 fusion protein (pGEX-1λT L1-Ig6) as template. Briefly, oligonucleotides corresponding to the sense and antisense sequences of bases 1,654–1,682 of the L1 ORF, which included a change from A to C at base 1,666 (sense: 5′-CCT GGC GTG GGG CCG GTC GAG ACC TCC AG-3′; antisense: 5′-CTG GAG GTC TCG ACC GGC CCC ACG CCA GG-3′) were annealed to a heat-denatured template, and the construct was replicated using Pfu DNA polymerase for 18 cycles. The resulting mixture was digested with the methylation-dependent endonuclease DpnI to degrade the wild-type template. Supercompetent E. coli strain XL-1 blue cells were transformed with this construct by heat shock and resulting colonies were screened and sequenced as described above.

To generate the mutant L1-Ig6 RGD→ KGE the primers for an equivalent L1-Ig6 construct (forward primer: 5′-TGG GAT CCA GAT CAC TCA GGG GC-3′; and the reverse primer: 5′-GCG AAT TCT GGG ATC CCG GCC CAG GGC TCC CCA C-3′) encoding for amino acids 518–614, were used in conjunction with the mutagenic primers 5′-CAT CAC CTG GAA GGG GGA GGG TCG AGA CC-3′ and 5′-GTA GTG GAC CTT CCC CCT CCC AGC TCT GG-3′ in the four primer method (Higuchi, 1990). The amplified product was digested with BamHI and subcloned into this site of pGEX-3X for expression in the E. coli strain JM101. The nucleotide sequence of the insert was confirmed by double-stranded DNA sequencing using the T7 Sequencing™ kit (Pharmacia Biotech Sevrage).

Purification of the recombinant fusion proteins was performed using isopropylthio-β-d-galactoside–induced log-phase cultures essentially as described by the manufacturer for the GST Gene Fusion System (Pharmacia Biotech Sevrage). Briefly, recovered bacteria were lysed by sonication and incubated with detergent before clarification and immobilization of the recombinant protein on a Sepharose 4B–coupled, glutathione affinity matrix (Pharmacia Biotech Sevrage). After extensive washing, the GST fusion proteins were eluted from the matrix with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0, and dialyzed extensively against PBS before use. The fusion proteins were subject to SDS-PAGE to confirm the correct mobility and to confirm purity.

Integrin expression was assessed by FACS^® analysis. Subconfluent cultures were harvested and stained with anti-integrin mAbs at 20 μg/ml or polyclonals diluted 1:40. The cells were then treated with an anti–mouse or anti–rabbit IgG, FITC-conjugated antibody, and were analyzed with a FACScan^® flow cytometer (Becton Dickinson, Co., Mountain View, CA). Control cells were treated with secondary FITC-conjugated antibody only.

Adhesion experiments were performed as detailed by Lagenaur and Lemmon (1987) with some modifications. Purified L1-GST fusion proteins (L1-Ig6 or L1-Ig4-6) dialyzed into PBS were spotted (1-μl spots) and coated onto the bottom of 96-well Titertek plates (ICN Pharmaceuticals, Inc., Costa Mesa, CA) as described (Montgomery et al., 1996). Unless otherwise stated, the fusion proteins were offered at a concentration of 40 μg/ml. Treated and control wells were blocked with 5% BSA for 1–3 h at 37°C. For adhesion studies involving immobilized peptides, wells were precoated overnight with murine IgG2a antibody at 20 μg/ml. Antibody-treated and washed wells were then incubated with the heterobifunctional cross-linker, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), at 30 μg/ml in PBS for 45 min. These wells were then washed and peptides added at 100–200 μg/ml for 2–3 h. Control wells received antibody and SPDP alone. Treated and control wells were blocked with 5% BSA for 1–3 h at 37°C.

CHO cells were harvested using EDTA (0.526 mM) in PBS (versene; Irvine Scientific, Santa Ana, CA) and ECV304 cells with a trypsin-versene mixture (Biowhittaker, Walkerville, MD). All the cells were then given a further wash with the EDTA solution to remove residual cations. The cells were then resuspended in adhesion buffer consisting of HBSS (without calcium and magnesium) supplemented with 10 mM Hepes, and BSA (0.2–1%), with the pH adjusted to 7.4. Divalent cations were added as indicated in the text and included MnCl₂ (0.4 mM), MgCl₂ (1–2 mM), and CaCl₂ (1–2 mM). Platelets were harvested and resuspended in Hepes-Tyrode's buffer as described above. For inhibition studies, the cells and platelets were pretreated with polyclonal antibodies (1:30 dilution), mAbs (80 μg/ml) or peptides (25 μM) for 30 min before the addition of both cells and inhibitors to pretreated wells. Cells were added at 10⁵/well and platelets at 5 × 10⁶/well, and then these plates were spun at 700 rpm to give a continuous monolayer of cells or platelets on the floor of each well. Endothelial cells and CHO cells were allowed to adhere for 30–40 min at 37°C, while the platelets were allowed to adhere for 10 min. At the end of the assay the wells were carefully washed with PBS, and non-adherent cells removed under a constant vacuum. Remaining adherent cells were fixed with 1% paraformaldehyde, and enumerated with the aid of an inverted light microscope. Cells were counted per unit area using a ×15 high powered objective and an ocular grid with a minimum of four areas counted per well. Alternatively, adherent cells or platelets were stained for 20 min with 1% crystal violet in 0.1 M borate, pH 9.0. Dye was eluted with 10% acetic acid and its absorbance determined at 600 nm. All experimental treatments were performed in triplicate.

Frozen sections of normal human skin, squamous cell carcinoma, psoriatic skin, and synovial tissue from the knee joint of patients diagnosed with rheumatoid arthritis were stained for the L1 antigen using mAb 5G3 or for α_vβ₃ expression using mAb LM609. Frozen sections were fixed in cold acetone before removal of endogenous peroxidase with 0.03% H₂O₂. Sections were subsequently blocked with 10% goat serum and 1% BSA in PBS. Primary antibodies were overlaid onto tissue sections at 20 μg/ml. Control sections were treated with isotype-matched murine IgG at 20 μg/ml. Sections were incubated with primary antibodies for 30–60 min at room temperature or overnight at 4°C, and after extensive washing were treated with a secondary anti–mouse IgG biotinylated antibody (LSAB kit; Dako Corp., Carpinteria, CA) for 30 min. After further washing, tissue sections were treated with peroxidase-labeled streptavidin for an additional 30 min. To achieve red or blue-black color the sections were treated with 3-amino-9-ethylcarbazole (AEC) or Vector VIP substrate (Vector Laboratories, Burlingame, CA), respectively. For double staining, the first antigen (α_vβ₃) was revealed using one substrate (AEC), and the sections subsequently restained for the second antigen (L1) using a different substrate (Vector VIP).

Currently, α_vβ₃ is the only integrin that has been shown to interact with human L1 (Ebeling et al., 1996; Montgomery et al., 1996; Duczmal et al., 1997; Pancook et al., 1997). To determine whether L1 is also a ligand for the platelet integrin α_IIbβ₃, we used a CHO cell line (A5) genetically altered to express human platelet α_IIbβ₃ (O'Toole et al., 1989, 1990; Frojmovic et al., 1991). To further determine whether α_IIbβ₃ needs to be in an active state to recognize L1 we used a CHO cell line (α_IIbα_LΔβ₃) transfected to express α_IIbβ₃ in a constitutively active state (O'Toole et al., 1994). Activation was achieved by chimerization of extracellular and transmembrane α_IIb with a cytoplasmic deletion mutant of the α_L integrin subunit (O'Toole et al., 1994). The integrin profile of these transfected cell lines and the CHO wild type was determined by flow cytometry using anti–hamster or anti–human integrin–specific antibodies (Fig. 1 A). A number of salient conclusions can be drawn from this analysis. First, both transfected cell lines have been successfully manipulated to express high levels of human α_IIbβ₃ (LJ-CP8 reactivity). Second, transfection of the human β₃ integrin subunit has also resulted in a pairing with endogenous hamster α_v and consequently expression of chimeric α_vβ₃ (LM609 reactivity). Finally, and of immediate relevance for this study, wild-type and transfected CHO express high levels of endogenous α₅β₁ (PB-1 reactivity) and endogenous α_v integrin(s) (VNR reactivity).

We have previously demonstrated that purified full-length L1 and a recombinant L1 fusion protein (L1-Ig4-6) can support the adhesion of melanoma cells via the integrin α_vβ₃ (Montgomery et al., 1996). We further proposed that it is the sixth Ig-like domain of human L1 that is likely to be relevant for such adhesion by virtue of the presence of a single RGD motif in this domain. Based on this proposition, and for the purposes of this study, we generated a fusion protein consisting of this L1 domain alone (i.e., L1-Ig6). As a first step, this L1-Ig6 recombinant protein was tested for its ability to support adhesion by wild-type and transfected CHO cells.

Importantly, the L1-Ig6 fusion protein supported significant concentration-dependent adhesion by all three CHO cell lines (Fig. 1,B). Furthermore, transfected CHO cells exhibited greater adhesion than the wild-type CHO-K1 cells (Fig. 1,B). These data clearly indicate the importance of the sixth Ig-like domain of L1 for mediating cellular adhesion, and also suggest that transfection and expression of β₃ integrins (α_vβ₃ and/or α_IIbβ₃) can lead to enhanced binding to L1-Ig6. Significantly, CHO cells bearing the active α_IIbβ₃ (α_IIbα_LΔβ₃ cells) showed the greatest level of adhesion, particularly at lower L1-Ig6 concentrations (Fig. 1 B). When offered at saturating concentrations (i.e., >50 μg/ml), L1-Ig6 supported >90% attachment of the α_IIbα_LΔβ₃ cells in contact with the substrate, resulting in a continuous monolayer of spreading cells and these cells were resistant to detachment by the large shear forces generated during the washing of the 96-well plates. When offered at a saturating concentration, vitronectin gave an equivalent response (data not shown).

From the data presented in Fig. 1 B it is evident that the wild-type CHO-K1 cells can interact with the L1-Ig6 fusion protein. To characterize this wild type adhesion we looked for evidence of either β₁ or α_v integrin involvement.

In the presence of Ca²⁺, Mg²⁺, and Mn²⁺, CHO-K1 cell adhesion was completely abrogated by a VNR polyclonal antibody, indicating the involvement of one or more α_v integrins (Fig. 2,A, left). Despite high levels of α₅β₁ expression, a function blocking mAb specific for hamster α₅β₁ (PB1) had no impact on adhesion in this cation environment (Fig. 2,A, left). However, in the presence of Mn²⁺ alone, CHO-K1 adhesion to L1-Ig6 could only be abrogated using a combination of VNR polyclonal antibody and the anti-α₅β₁ mAb PB1 (Fig. 2,A, right). This finding indicates that in the presence of Mn²⁺ alone, α₅β₁ can also recognize human L1-Ig6. However, it is also clear that binding by α₅β₁ must be acutely susceptible to inhibition by either Ca²⁺ or Mg²⁺ since, as stated, we did not observe α₅β₁ involvement when all three cations were present in the adhesion buffer (Fig. 2 A, left).

Since Ca²⁺ has been shown to inhibit β₁ integrin ligation (Kirchhofer, 1991; Mould et al., 1995) we determined whether this cation was responsible for a lack of α₅β₁ involvement in a mixed cation environment (Fig. 2,A, left). To this end, CHO-K1 cells were allowed to adhere to L1-Ig6 in the presence of Mn²⁺ and increasing concentrations of exogenous Ca²⁺. To discriminate between α₅β₁ and α_v integrin–dependent binding, the cells were treated with either PB1 or anti-VNR antibody. Using this approach, a significant inverse correlation was observed between the concentration of exogenous Ca²⁺ and α₅β₁-dependent binding (Fig. 2,B) such that the addition of 2 mM calcium reduced the level of α₅β₁-dependent binding by >80% (Fig. 2 B). In contrast, the same concentration of calcium reduced the α_v integrin(s)–dependent binding by only 20%.

It is important to note that when the divalent cations were added to the adhesion buffer individually only Mn²⁺ could support significant wild-type CHO-K1 adhesion (Fig. 2 C). Together these findings indicate, not only an absolute requirement for Mn²⁺, but also a pivotal role for Ca²⁺ in the differential regulation of integrin binding to L1.

Thus far, it has been shown that the CHO transfectants used in this study have been successfully manipulated to express significant levels of both α_vβ₃ and α_IIbβ₃ (Fig. 1,A) and that these same cells have an increased ability to bind to the sixth Ig-like domain of L1 (Fig. 1 B). A further series of experiments was performed to determine whether these β₃ integrins can indeed recognize L1 and, if so, how the binding of these integrins is regulated.

In contrast to the situation with the wild-type CHO-K1 cells, cells transfected to express α_vβ₃ and α_IIbβ₃ (A5 and α_IIbα_LΔβ₃ cells) were able to recognize L1 in the presence of either Ca²⁺ alone or Mg²⁺ alone (Fig. 3 vs. Fig. 2,C). Most importantly, this de novo adhesion could be attributed to both the chimeric α_vβ₃ and active α_IIbβ₃. Thus, CHO cells engineered to express active α_IIbβ₃ (α_IIbα_LΔβ₃ cells) showed significant adhesion to L1-Ig6 in the presence of Ca²⁺ or Mg²⁺, and this adhesion could only be significantly reduced using a combination of antibodies to both α_vβ₃ and α_IIbβ₃ (Fig. 3, bottom). It is important to note, that in the presence of Ca²⁺ alone, α_IIbβ₃-dependent binding to L1-Ig6 was only evident in the CHO cells engineered to express active α_IIbβ₃ (α_IIbα_LΔβ₃ cells). Thus, in A5 cells bearing α_IIbβ₃ in its resting state, adhesion to L1-Ig6 in the presence of Ca²⁺ was fully inhibited by mAb LM609 (i.e., α_vβ₃ dependent) with no evident contribution by α_IIbβ₃ (Fig. 3, top left). This finding implies that α_IIbβ₃-dependent recognition of L1 requires this integrin to be in its activated state. However, it is interesting to note that Mg²⁺ alone is sufficient to activate α_IIbβ₃ binding by the A5 cells. Thus, the adhesion of these cells, in the presence of Mg²⁺ alone, could only be abrogated using antibodies to both α_IIbβ₃ and α_vβ₃ (Fig. 3, top right). Because of endogenous α_v integrin and α₅β₁ binding, adhesion in the presence of Mn²⁺ could only be abrogated using a combination of polyclonal antibodies to both α₅β₁ and β₃ integrins (VNR and PB1, data not shown). The ability of the different cations to support adhesion of the transfected cell lines was in the order Mn²⁺ > Mg²⁺ > Ca²⁺ (Fig. 3; Mn²⁺ not shown).

Having identified activated α_IIbβ₃ as a heterophilic ligand for L1-Ig6 in our CHO model we wished to determine whether this integrin will also mediate platelet attachment. An interaction between L1- and platelet α_IIbβ₃ would then suggest a novel physiological function in thrombogenic processes. In this regard, L1 could contribute either as a cellular ligand expressed on myelomonocytic cells (Pancook et al., 1997), metastatic neuroectodemal tumors (Linnemann et al., 1989), or endothelial cells, or as a shed ligand in solution or associated with subcellular matrix (Martini and Schachner, 1986; Poltorak et al., 1990; Montgomery et al., 1996).

In adhesion assays comparable to those performed with the CHO cells we observed that L1-Ig4-6, L1-Ig6, and the immobilized L1-derived peptide (C)PSITWRGDGRDLQEL could all support the rapid and significant attachment of activated platelets (Fig. 4, A–C). This adhesion was not affected by anti-α_vβ₃ mAb LM609 alone, but was abrogated by the mAb 7E3, which is a potent function-blocking antibody, specific for both α_vβ₃ and α_IIbβ₃ (Fig. 4, A–C). The affinity of activated platelets for immobilized L1-Ig6, and the strong inhibitory effect of mAb 7E3, is also evident from the photomicrographs presented in Fig. 4, D and E. Together these findings are consistent with a role for platelet α_IIbβ₃. The contribution of α_IIbβ₃ was also confirmed using the specific anti-α_IIbβ₃ mAb LJ-CP8, but this mAb was generally less effective than 7E3 (data not shown). It is important to note that these results were only obtained using platelets bearing active α_IIbβ₃ as a result of stimulation with ADP and epinephrine. Thus, unstimulated platelets, prepared from PGE₁-treated blood, showed only minimal adhesion to L1-Ig4-6 (Fig. 4,A). These data, like the data obtained using the CHO cells, clearly indicate that α_IIbβ₃ needs to be in an active conformation to bind L1. However, as described for other peptides, recognition of the L1-derived peptide was not fully dependent on platelet activation (Fig. 4,C). When offered at saturating concentrations, the L1 fusion proteins supported the rapid attachment of a uniform monolayer of platelets such that the majority of platelets offered and in contact with the substrate appeared to attach (Fig. 4 D). When used as a positive control, vitronectin supported comparable levels of platelet attachment (data not shown). However, as with L1-Ig4-6 significant attachment to vitronectin required platelet activation.

L1 is expressed on a variety of cell types known to interact with endothelium (Ebeling et al., 1996; Pancook et al., 1997); and shed L1 may also associate with components of the subendothelial matrix (Hall et al., 1997). This distribution raises the question of if and how endothelial cells interact with L1. To address this issue we used a transformed cell line (ECV304) derived from human umbilical vein endothelial cells (Hughes et al., 1996). These wild-type cells were found to express both α_vβ₃ and a high level of β₁ integrins (Fig. 5,A, left column). We also detected expression of α_vβ₅ (mAb P1F6) at a median fluorescence comparable to that found for α_vβ₃ (not shown). Consistent with our findings using the transfected CHO cells, we observed that endothelial α_vβ₃ can also promote adhesion to L1-Ig6 and this adhesion is evident in the presence of calcium (Fig. 5,B). Adhesion by these wild-type endothelial cells was only marginally inhibited by the anti–β₁ integrin mAb P4C10 (Fig. 5 B). A similar pattern of adhesion was obtained using primary human dermal microvascular endothelial cells (Clonetics, San Diego, CA) (data not shown).

Whereas the contribution of α_vβ₃ to a variety of vascular processes is well documented it is also clear that this integrin is either absent or only marginally expressed by quiescent endothelial cells (Brooks et al., 1994). Accordingly, we wished to determine whether L1 could also be recognized by α_vβ₃-negative endothelial cells. To address this we exploited ECV304 endothelial cells that had been repeatedly FACS^® sorted for a lack of α_vβ₃ expression. This approach proved successful, resulting in the generation of a stable population of α_vβ₃-negative cells (Fig. 5,A, right column). The levels of α_vβ₅ and β₁ integrin expression in these cells remained unchanged (not shown; and Fig. 5,A, right column). Remarkably, these α_vβ₃-negative cells also showed significant adhesion to L1-Ig6 (Fig. 5,C). However, in contrast to the wild-type cells, adhesion was unaffected by an antibody to α_vβ₃ (LM609) but was fully inhibited by a mAb to β₁ integrins (i.e., P4C10) (Fig. 5,C). Furthermore, such adhesion was also completely abrogated with an antibody specific for α_v integrins (Fig. 5 C). Together these results are consistent with an interaction between L1 and endothelial α_vβ₁.

Given that both α_vβ₃ and α_vβ₁ can interact with L1-Ig6, we wished to determine why α_vβ₃ is the dominant integrin in wild-type ECV304 cell adhesion (Fig. 5,B). In this regard, we tested the hypothesis that exogenous calcium may favor the use of α_vβ₃ rather than α_vβ₁. We observed a number of facts supporting this hypothesis. First, significant α_vβ₁ binding could be induced in the wild-type cells, but only in the presence of Mn²⁺ alone (Fig. 6,A). Thus, whereas wild-type ECV304 cell adhesion in the presence of Ca²⁺ could be abrogated with anti-α_vβ₃ mAb LM609 (Fig. 5,B), the adhesion of these cells in the presence of Mn²⁺ alone could only be blocked using a combination of antibodies reactive with both α_vβ₃ and α_vβ₁ (i.e., LM609 and P4C10), or with a mAb reactive with both α_v integrins (i.e., 17E6) (Fig. 6,A, right). Second, minimal wild-type adhesion was observed in the presence of Ca²⁺ or Mg²⁺ alone and this appeared to be fully dependent upon α_vβ₃ (Fig. 6,A). Finally, in an experiment analogous to that performed with the wild-type CHO cells (Fig. 2,B), we observed that the α_vβ₁-mediated (Mn²⁺-dependent) adhesion of the sorted, α_vβ₃-negative endothelial cells was significantly more susceptible to inhibition by exogenous Ca²⁺ than the wild-type adhesion (Fig. 6,B). Together these data indicate that in the absence of α_vβ₃ expression, quiescent endothelial cells may use α_vβ₁ to bind to L1, however with the induction of α_vβ₃ expression, physiological levels of calcium are likely to favor the use of this integrin. It is also clear that whereas α_vβ₁-mediated adhesion is susceptible to inhibition by exogenous Ca²⁺, this inhibition is less pronounced than that seen when adhesion is mediated via α₅β₁ (Fig. 2 B).

Thus far we have demonstrated that a single Ig-like domain of L1 can support multiple integrin interactions. This same domain contains an RGD integrin recognition motif that may provide the binding site for all the aforementioned integrins. However it is also clear that a given RGD site may or may not support adhesion depending on flanking sequences, conformational restraints and accessibility (D'Souza et al., 1991; Haas and Plow, 1994). Furthermore, although α_vβ₃, α₅β₁, α_vβ₁, and α_IIbβ₃ have all been reported to interact with RGD motifs, α₅β₁ and the β₃ integrins have also been shown to interact with non-RGD sequences (Koivunen et al., 1994). To help address this issue we generated a 15-mer peptide based on a sequence in the sixth Ig-like domain of L1 and inclusive of the RGD recognition motif (i.e., PSITWRGDGRDQEL). This peptide was tested for its efficacy both as an immobilized ligand and as soluble inhibitor of L1 integrin binding.

Supporting the concept of a RGD-dependent interaction we observed that our L1-RGD peptide, once immobilized, could support significant endothelial cell attachment via α_vβ₃, and α_vβ₁ (Fig. 7, Endothelial) and CHO cell attachment via endogenous α₅β₁ and transfected α_IIbβ₃ (Fig. 7, CHO). Specific integrin–peptide interactions were confirmed using the function blocking antibodies indicated (Fig. 7). When offered at the same concentration, a control peptide (C)PSITWRADGRDQEL was ineffective as an adhesive ligand. However, it should be noted that because of the conservative glycine to alanine mutation (i.e., RGD→ RAD) this control peptide could also support some attachment but only at significantly higher concentrations (not shown).

Further support for RGD-dependent interaction with L1 was obtained using the same L1-RGD peptide as a soluble inhibitor. At a concentration of 25 μM, the peptide effectively abrogated endothelial cell adhesion to L1-Ig6 in the presence of Mn²⁺ alone (Fig. 8, left). We have previously demonstrated that adhesion by these cells in the presence of this cation involves both α_vβ₃ and α_vβ₁ (Fig. 6,A, right). Likewise, we also observed a complete inhibition of wild-type CHO-K1 adhesion in the presence of Mn²⁺ (Fig. 8, middle). According to our previous data, this is consistent with an inhibition of endogenous α₅β₁ and the α_v integrin(s) (Fig. 2,A, right). Adhesion by the CHO cells transfected to express active α_IIbβ₃ was less sensitive to inhibition via the RGD peptide when used at 25 μM (Fig. 8, right), but could be fully inhibited at higher peptide concentrations (not shown). Used at an equivalent concentration our control peptide was ineffective at preventing adhesion. But again because of the conservative mutation of this peptide (i.e., RGD→ RAD) this peptide could also inhibit attachment at high concentrations (e.g., 250 μM).

Aforementioned work with the L1-RGD peptide supports the concept of a RGD-dependent interaction between integrins and L1. However, to address this issue definitively, we sought to demonstrate that mutation of the RGD site in L1-Ig6 is sufficient to abrogate integrin binding. To this end, we generated additional L1-Ig6 fusion proteins containing the mutations RGD→ KGE or RGD→ RGA. Importantly, the conservative RGD→ KGE mutation completely abrogated α_vβ₃- and α_vβ₁-dependent binding by the endothelial cells (Fig. 9, Endothelial). This same mutation and the RGD→ RGA mutation also significantly abrogated α₅β₁-dependent binding by wild-type CHO cells (Fig. 9, CHO, wild-type). Some residual low level binding to both mutations was still observed, perhaps indicating that the mutated sequences can still be recognized to some degree by α₅β₁. Alternatively, a second α₅β₁-binding motif may exist within the L1-Ig6 domain. Interestingly the RGD→ KGE mutation could still be recognized to a significant level by α_IIbβ₃ (Fig. 9, CHO, Active α_IIbβ₃). However, the alanine substitution mutant (RGA) was sufficient to fully abrogate α_IIbβ₃-mediated binding.

We have identified a variety of endothelial and platelet integrins that can interact with the sixth Ig-like domain of L1. Such heterophilic interactions prompted us to determine whether L1 can be expressed on endothelial cells. This expression would suggest the potential for L1 integrin interactions in vascular processes such as angiogenesis and thrombosis; these are processes that require homotypic or heterotypic (platelet) interactions involving endothelial cells.

To address this issue, we looked for L1 expression on normal or quiescent blood vessels and on activated or angiogenic vessels associated with neoplastic or inflammatory diseases. In normal human skin, L1 was absent or minimally expressed by the dermal vessels (Fig. 10,G). However, significant expression of L1 was observed on vessels proximal to a squamous cell carcinoma (Fig. 10, A and B). These proximal vessels also expressed high levels of α_vβ₃ (Fig. 10,C); a documented marker of angiogenic blood vessels (Brooks et al., 1994). Importantly, α_vβ₃ and its heterophilic ligand L1, could be colocalized on these angiogenic blood vessels (Fig. 10, D and E). It is important to note, that L1 was not detected on all the angiogenic vessels identified by α_vβ₃ (Fig. 10, D and E); and that whereas α_vβ₃ expression was evident on intra-tumor vessels, L1 was only detected on angiogenic vessels in normal skin tissue peripheral to the tumor (Fig. 10 F). This pattern of expression may indicate the induction of L1 expression during a limited phase of blood vessel maturation. In a preliminary analysis, we did not detect L1 on vessels associated with either breast or lung tumors suggesting that induction of vascular L1 may be tissue or organ specific.

Interestingly, expression of vascular L1 was also observed in synovial tissues obtained from three out of five patients diagnosed with rheumatoid arthritis (Fig. 10 H). In a preliminary study, L1 was also detected on vessels in psoriatic skin (not shown). Furthermore, whereas we detected little or no L1 expression on cultured human dermal microvascular endothelial cells (Clonetics) we did detect significant L1 levels on the surface of the ECV304 endothelial cell line (data not shown). Together these findings may indicate that de novo L1 expression can be induced on endothelial cells as a result of stimulation by specific, tumor-associated or inflammatory cytokines. Such vascular L1 may then function as a receptor either for itself or for the vascular and platelet integrins identified in this study.

In this work we have detailed the interaction between a single Ig-like domain within L1 and multiple integrins including α_vβ₃, α_vβ₁, α₅β₁, and α_IIbβ₃. To our knowledge this is the first observation that both α_vβ₁ and α_IIbβ₃ can interact with a member of the IgSF. Indeed, L1 may be unique within this family in its capacity to interact with multiple RGD-dependent integrins. Key structural and regulatory issues have also been addressed including the central importance of a single RGD motif and the critical regulatory effect of physiological levels of calcium. Based on these novel integrin–CAM interactions, and the novel observation that L1 can be expressed on blood vessels under various pathogenic conditions, we propose that L1 may have an expanded and unexpected role in various vascular and thrombogenic processes.

The β₃ and β₁ integrins identified in this study as heterophilic receptors for the sixth Ig-like domain of L1 share a collective ability to recognize RGD motifs within their respective ligands (D'Souza et al., 1991; Haas and Plow, 1994; Marshall et al., 1995). In this regard, the single RGD sequence present in L1-Ig6 (Reid and Hemperly, 1992) would appear to be a legitimate putative recognition motif. However, it is also apparent that the conformational and sequential environment of a given RGD site and its accessibility will ultimately dictate whether it can truly function as a recognition motif for a given integrin. Furthermore, it is now widely documented that non-RGD motifs can also be recognized by α_vβ₃, α₅β₁, and α_IIbβ₃ (Koivunen et al., 1994). To demonstrate definitively that the single RGD sequence present in human L1 is indeed critical for binding by α_vβ₃, α_vβ₁, α₅β₁, and α_IIbβ₃ we demonstrate that two mutations of this site in L1-Ig6 reduce or abrogate binding by all four of these integrins. Furthermore we demonstrate that a L1-RGD peptide with the relevant flanking sequences (i.e., PSITWRGDGRDLQEL) is effective both as an immobilized substrate and as a soluble inhibitor for all of the integrins identified.

As stated, the sequence environment of a given RGD site is important in determining the strength and specificity of integrin interactions (Kunicki et al., 1997). In the case of L1, the RGD site is flanked by both a tryptophan and a glycine (i.e., WRGDG). In this regard, phage display libraries have identified numerous α_vβ₃ and α₅β₁ peptide ligands that include a glycine residue COOH-terminal to the RGD (Healy et al., 1995; and Koivunen et al., 1994). Interestingly, peptides with a strong affinity for α_IIbβ₃ generally have a large hydrophobic residue COOH-terminal to the RGD (O'Neil et al., 1992). This preference may explain why at low concentrations our L1 peptide was more efficient at supporting attachment via α_vβ₃ than via α_IIbβ₃ (data not shown). Integrin-binding peptides containing a tryptophan residue NH₂-terminal to the RGD have rarely been identified by phage display libraries, but one has been reported with specificity for α_vβ₃ (Healy et al., 1995). It is interesting to note, that although we detected significant α_vβ₅ expression on the ECV304 endothelial cells used in this study (data not shown), we found no evidence for an interaction between L1-Ig6 (or the L1-RGD peptide) and this RGD-dependent integrin (data not shown). This would suggest that the sequence environment of the RGD site in L1 is not suitable for recognition by α_vβ₅.

The conformational or stereochemical presentation of the RGD site is also a key element in dictating receptor recognition and affinity. Secondary structural analysis of RGD recognition motifs in fibronectin (FNIII₁₀) and Foot and Mouth Disease Virus support an emerging model of the RGD being presented at the apex of a flexible loop that extends outwards from the protein core. The side chains of Arg and Asp are purported to face away from each other and are flexible enough to adopt the proper conformation for high affinity integrin binding (Haas and Plow, 1994). By using a combination of electron microscopic analysis and computer-assisted modeling, Drescher et al. (1996) conclude that the RGD sites of murine L1, and the single conserved RGD motif of human L1, are exposed at the molecular surface in a loop or turn between two β-strands. Furthermore they suggest that in the context of the whole molecule, the sixth Ig-like domain of L1 possesses greater surface hydrophobicity than the other Ig-like domains suggesting that it, and contained RGD site(s), can participate in intermolecular interactions.

As a general rule, all integrins require divalent cations for ligand recognition (D'Souza et al., 1994) and two potential mechanisms have been proposed for this dependence. First, specific cations may induce a conformational change in the integrin that favors ligand binding (Mould et al., 1995). Second, the cation may be required to form a ternary complex with the ligand (e.g., RGD) and the integrin; this complex is envisaged to be an unstable but requisite intermediate with the cation eventually being displaced as the ligand–receptor complex stabilizes (D'Souza et al., 1994). Irrespective of the mechanism, it is now well documented that different divalent cations can dramatically and differentially influence integrin-binding affinity and selection. The findings of this study provide a case in point. Thus, whereas we observed that Ca²⁺ is able to support a limited interaction between L1 and α_vβ₃ or α_IIbβ₃, this same cation profoundly suppressed a Mn²⁺-dependent interaction with α₅β₁ or α_vβ₁. It is important to note, that such a dichotomy between these β₁ integrins and the β₃ integrins has been documented for other ligands. Thus, Ca²⁺ has been shown to support both α_vβ₃ or α_IIbβ₃ attachment to RGD peptides or vitronectin (Kirchhofer, 1991; Suehiro et al., 1996), whereas this same cation suppresses the attachment of α₅β₁ and α_vβ₁ to fibronectin and an RGD peptide, respectively (Kirchhofer, 1991; Mould et al., 1995). Recently, Suehiro et al. (1996) described a novel classification for defining β₃ ligands depending upon their ability to support the attachment of α_vβ₃ and/or α_IIbβ₃ in the presence of different cations. Thus, class I β₃ ligands should be able to support attachment of both integrins either in the presence of Ca²⁺ or Mn²⁺. A class II ligand will also support attachment of α_IIbβ₃ in the presence of either Ca²⁺ or Mn²⁺, but α_vβ₃ attachment is suppressed by Ca²⁺. Finally, class III ligands bind exclusively to α_IIbβ₃ under all cation conditions, whereas class IV ligands bind exclusively to α_vβ₃. According to this classification scheme we propose that L1 is a novel class I ligand for β₃ integrins, together with vitronectin, RGD peptides and disintegrin group A (Suehiro et al., 1996).

From the findings presented it is probable that Ca²⁺ will act as a potent physiological regulator of L1–integrin interactions. Thus, given the presence or absence of a given integrin, physiological calcium concentrations are likely to favor a hierarchy of L1–integrin interaction such that α_vβ₃ or activated α_IIbβ₃ > α_vβ₁ > α₅β₁. This hierarchy is likely to be further compounded by transnegative dominance, a recently described phenomenon in which the ligation of a β₃ integrin has been shown to suppress the function of a β₁ integrin (Diaz-Gonzalez et al., 1996). It could legitimately be argued from our data that physiological levels of calcium would effectively preclude an interaction between α₅β₁ and L1-Ig6. However, it is important to note that an interaction between α₅β₁ and murine L1 has been observed in the presence of calcium but only after an undefined activational event after ligation of CD24 (heat stable antigen) (Kadmon et al., 1995). Thus, α₅β₁ may indeed have a physiological role in L1 binding, but it is likely to be regulated by a requirement for additional activational signals.

The observation that L1 can support an RGD-dependent interaction with α_vβ₃ confirms our earlier study that demonstrated that melanoma cells can interact with either full-length L1 or an L1 fusion protein (L1-Ig4-6) via α_vβ₃ (Montgomery et al., 1996). The interaction between human L1 and α_vβ₃ has also been confirmed using lymphocytic cell lines (Ebeling et al., 1996). The finding that human L1-Ig6 can also interact with α_IIbβ₃, α_vβ₁, and α₅β₁ has not (to our knowledge) been reported, and significantly expands the potential repertoire of heterophilic L1 interactions that this CAM can support and the range of cell types that may be involved. It is important to note that, contrary to our findings, a previous study did not detect any significant interaction between human L1 and α₅β₁ (Ebeling et al., 1996). The most obvious explanation for this is the strict cation requirement of this interaction and perhaps transnegative dominance by α_vβ₃. It should also be noted that our CHO cells express very high levels of α₅β₁. The observation that human L1 can interact with α₅β₁ is, however, in agreement with a report demonstrating an interaction between this integrin and murine L1 (Ruppert et al., 1995). In this regard, it is noteworthy that the sixth Ig-like domain of murine L1 contains an additional RGD sequence (LGD in human L1) and that this sequence, like the human motif, may well be available for interaction on an exposed loop (Drescher et al., 1996). It is conceivable that the absence of this second RGD sequence in humans may make it a less favorable ligand for α₅β₁. However, it is also of interest that human L1 retains some additional non-RGD tripeptide sequences such as NGR (fibronectin [FN]-like domain 3), STF (FN-like domain 2), and ETA (Ig-like domain 4), which if exposed in the right stereochemical configuration, could augment the interaction between L1 and α₅β₁. Thus, all three of these tripeptide sequences have been identified as important for the interaction of non-RGD, 7-mer peptides with α₅β₁ (Koivunen et al., 1993).

L1 and the integrin counter receptors identified in this study are expressed on multiple cell types of diverse histological origin. This suggests the potential for a plethora of interactions and functions that have yet to be described. This is especially true given the observation that L1 expression is not strictly confined to cells of the nervous system. Thus, we and others have recently described L1 expression on human cells of both myelomonocytic and lymphoid origin (Ebeling et al., 1996; Pancook et al., 1997). L1 has also been described on epithelial cells of the intestine and urogenital tract (Thor et al., 1987; Kowitz et al., 1992; Kujat et al., 1995) and on transformed cells of diverse histological origin, including melanoma, neuroblastoma, embryonal carcinoma, osteogenic sarcoma, squamous lung carcinoma, squamous skin carcinoma, pheochromocytoma, rhabdomyosarcoma and retinoblastoma cell lines (Mujoo et al., 1986; Linnemann et al., 1989; Reid and Hemperly, 1992). Finally, in this study we have also shown that L1 expression can be induced on certain endothelial cells. This said, however, the extent to which L1-Ig6–integrin pairing contributes to either homotypic or heterotypic cell–cell interaction among these diverse cell types remains to be determined. It is important to note that Ruppert et al. (1995) have demonstrated that the interaction between murine L1 and α₅β₁ can promote significant homotypic cell aggregation. It is also of interest that L1–integrin pairing may be modulated by other molecules that associate with L1 in the plane of the membrane such as CD24 (Kadmon et al., 1995; Ruppert et al., 1995). Conceivably, different cis interactions involving L1 may sterically or conformationally alter the accessibility of the RGD site thereby regulating L1–integrin interactions.

In the context of L1–integrin interactions, it is important to note, that L1 may be equally or more relevant as a substrate adhesion molecule. Thus, a number of studies have described incorporation of shed L1 into the occluding extracellular matrix (ECM). In the adrenal medulla, for example, L1 immunoreactivity has been found in the ECM adjacent to chromaffin cells (Poltorak et al., 1990). Martini and Schachner (1986) have reported L1 expression in basement membranes associated with murine Schwann cells and in association with collagen fibrils of the endoneurium, and we have previously reported L1 expression associated with strands of intra-tumor laminin (Montgomery et al., 1996). Given these reports and our current findings, it is likely that deposition of this CAM will significantly alter the microenvironment of a cell to favor the kind of integrin interactions described in this study.

As stated, the capacity of L1 to interact with multiple integrins on cells of diverse histological origin may lead to a plethora of interactions that will add to the functional significance of this CAM. Two examples of this are provided in this study. First, we have demonstrated that L1-Ig6 can support significant platelet adhesion via α_IIbβ₃. Given the expression of L1 on endothelial cells, and myelomonocytic cells (Ebeling et al., 1996; Pancook et al., 1997), and the capacity of L1 to associate with basement membrane components (e.g., Laminin; Hall et al., 1997), one could predict a role for L1 in thrombogenic processes that involve these cell types or structures. Since L1 is also highly expressed on many neuroectodermal tumors, it may be that an interaction between L1 and active α_IIbβ₃ will contribute to tumor-associated thrombosis: a process thought to be important for the lodgment step of hematogenous metastasis (Felding-Habermann et al., 1996). Second, we have also demonstrated a novel interaction between L1-Ig6 and endothelial cells via either vascular α_vβ₃ or α_vβ₁. It is conceivable that these interactions will contribute to the rolling, arrest, and/or attachment of L1-expressing cells on, or to, endothelium. This possibility is particularly intriguing given the expression of L1 on trafficking immune cells and metastatic tumor cell lines (Linnemann et al., 1989; Reid and Hemperly, 1992; Ebeling et al., 1996; Pancook et al., 1997). Our observation that L1 expression can be induced on blood vessels associated with certain neoplastic or inflammatory diseases may indicate a role for L1–integrin or L1–L1 interactions in the maturation of new blood vessels and/or reflect an induction of de novo L1 expression by inflammatory or tumor-associated cytokines.

Whereas the focus of this study has been on non-neural cell types, the data presented may have some interesting implications for neuronal processes. For example, both α_vβ₃ and α_vβ₁ have been implicated in avian neural crest cell adhesion and migration (Delannet et al., 1994). In a recent study, Milner et al. (1996) demonstrated the importance of α_vβ₁ for migration by oligodendrocyte precursors. Interestingly, the authors speculate that, among other ligands, L1 present within axonal tracts might serve as a potential α_vβ₁ ligand, providing a mechanism for guiding migrating oligodendrocytes. Whereas this is merely speculation, our data clearly support the potential for L1–α_vβ₁ interactions during neural development. At this stage, no evidence yet exists to show that L1–integrin interactions promote neurite extension or other neuronal processes involving L1; these processes appear to be primarily dependent upon a homophilic interaction. However, consideration needs to be given to the integrin repertoire of the cell being tested. For example, α_vβ₁ is expressed on oligodendrocyte precursors but is lost on differentiation. Similarly, attention needs to be given to the appropriate cation environment. Thus, it is evident from this study that expression of α_vβ₁ adhesion is dependent upon the presence of Mn²⁺ but is inhibited by Ca²⁺.

The findings of this study extend the range and significance of L1–integrin interactions and add to our understanding of how these heterophilic interactions are regulated. In addition to the documented interaction between human L1 and α_vβ₃, we have demonstrated that this CAM is a relatively promiscuous ligand, supporting further novel heterophilic interactions with α_vβ₁, α₅β₁, and α_IIbβ₃. It is further shown that these integrins share a collective ability to recognize a single RGD motif in the sixth Ig-like domain of human L1 and that the binding of these integrins to this motif is critically and differentially regulated by physiological levels of calcium. Based on the novel interactions involving α_vβ₁ and α_IIbβ₃ we have shown that the sixth Ig-like domain of human L1 can support significant endothelial cell and platelet attachment. Based on these findings, and the observation that L1 expression can be induced on endothelial cells, we propose an expanded role for this CAM in vascular and thrombogenic processes.

The authors wish to thank Dr. R.A. Reisfeld of the Scripps Research Institute for his support and encouragement. This is Scripps manuscript No. 10821-IMM.

This study was supported by National Institutes of Health RO1 Grant CA69112-01 (A.M.P. Montgomery), by National Institutes of Health R29 Grant CA67988 (B. Felding-Habermann), and by the Medical Research Council of Canada (C-H. Siu). P. Yip is supported by an Ontario Graduate Studentship (Canada), and S. Silletti by a National Cancer Institute research fellowship (1/F32/CA72192-01).

CAM

cell adhesion molecule

GST

glutathione-S-transferase

IgSF

immunoglobulin super family

L1-Ig6

sixth immunoglobulin-like domain of human L1

NCAM

neural cell adhesion molecule

NILE

nerve growth factor–inducible, large external protein

NgCAM

neuron–glial CAM

ORF

open reading frame

PGE₁

prostaglandin E₁

RGD

Arg-Gly-Asp

VNR

vitronectic receptor