* Roon Research Center for Arteriosclerosis and Thrombosis, Division of Experimental Hemostasis and Thrombosis;
Department of Molecular and Experimental Medicine, Department of Immunology, The Scripps Research Institute, La Jolla,
California 92037; § Banting and Best Department of Medical Research and Department of Biochemistry, University of Toronto,
Ontario, Canada M5G 1L6; and
Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037
The neural cell adhesion molecule L1 has
been shown to function as a homophilic ligand in a variety of dynamic neurological processes. Here we demonstrate that the sixth immunoglobulin-like domain of human L1 (L1-Ig6) can function as a heterophilic ligand for multiple members of the integrin superfamily including v
3,
v
1,
5
1, and
IIb
3. The interaction between L1-Ig6 and
IIb
3 was found to support the rapid
attachment of activated human platelets, whereas a corresponding interaction with
v
3 and
v
1 supported the adhesion of umbilical vein endothelial cells. Mutation of the single Arg-Gly-Asp (RGD) motif in human
L1-Ig6 effectively abrogated binding by the aforementioned integrins. A L1 peptide containing this RGD
motif and corresponding flanking amino acids (PSITWRGDGRDLQEL) effectively blocked L1 integrin interactions and, as an immobilized ligand, supported adhesion via
v
3,
v
1,
5
1, and
IIb
3. Whereas
3
integrin binding to L1-Ig6 was evident in the presence
of either Ca2+, Mg2+, or Mn2+, a corresponding interaction with the
1 integrins was only observed in the
presence of Mn2+. Furthermore, such Mn2+-dependent
binding by
5
1 and
v
1 was significantly inhibited by exogenous Ca2+. Our findings suggest that physiological levels of calcium will impose a hierarchy of integrin binding to L1 such that
v
3 or active
IIb
3 >
v
1 >
5
1. Given that L1 can interact with multiple vascular or platelet integrins it is significant that we also present evidence for de novo L1 expression on blood
vessels associated with certain neoplastic or inflammatory diseases. Together these findings suggest an expanded and novel role for L1 in vascular and thrombogenic processes.
PIONEERING studies on the structure and function of
L1 have established this cell adhesion molecule
(CAM)1 as a member of the immunoglobulin superfamily (IgSF) that plays a quintessential role in neural development (Lindner et al., 1983 Human and mouse L1 and L1-related glycoproteins in
the rat (nerve growth factor-inducible, large external glycoprotein [NILE]), chick (neuron-glial [Ng]CAM, 8D9,
G4), and Drosophila (neuroglia) have been described (Grumet et al., 1984 Reflecting its designation as a neural CAM (NCAM),
L1 is highly expressed on postmitotic neurons of the central and peripheral nervous systems and on pre- or nonmyelinating Schwann cells of the peripheral nervous system
(Lindner et al., 1983 In addition to having a propensity for homophilic binding (Lemmon et al., 1989 The presence of a single Arg-Gly-Asp (RGD) motif in
the sixth Ig-like domain of human L1, and the presence of
two such motifs in the same domain of the murine and rat
L1-homologues prompted early speculation as to whether
L1 might also function as a heterophilic ligand for members of the integrin superfamily. A number of studies have
now reported novel L1-integrin interactions (Ruppert et al., 1995 To date, Antibodies
Anti-integrin antibodies used include the following: anti-hamster Peptides
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.
Cell Lines and Culture
The generation and characterization of CHO cells stably transfected to
express normal human platelet A spontaneously transformed, human umbilical vein endothelial cell
line designated ECV304 (Hughes, 1996 Isolation of Human Platelets
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.
Construction and Expression of L1 Fusion Proteins
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 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 mutant L1-Ig6 RGD To generate the mutant L1-Ig6 RGD Purification of the recombinant fusion proteins was performed using
isopropylthio- Flow Cytometry
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 Assays
Adhesion experiments were performed as detailed by Lagenaur and Lemmon (1987) 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.
Immunohistochemistry
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 Characterization of CHO-K1 Cells and Transfectants
Currently,
Wild-Type and Transfected CHO Cells Display
Concentration-dependent Adhesion to L1-Ig6
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
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 Wild-type CHO-K1 Cells Interact with L1-Ig6
Using 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 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
Since Ca2+ has been shown to inhibit 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.
CHO Transfectants Use Both Thus far, it has been shown that the CHO transfectants
used in this study have been successfully manipulated to
express significant levels of both In contrast to the situation with the wild-type CHO-K1
cells, cells transfected to express
Platelets Interact with L1-Ig6 Via Activated Having identified activated 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-
Endothelial Cells Interact with L1-Ig6 Using L1 is expressed on a variety of cell types known to interact
with endothelium (Ebeling et al., 1996
Whereas the contribution of Given that both
The Interaction between L1-Ig6 and the 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 Supporting the concept of a RGD-dependent interaction we observed that our L1-RGD peptide, once immobilized, could support significant endothelial cell attachment
via
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
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
L1 Expression Can Be Induced on Endothelial Cells
In Vivo
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
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 The 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 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 As a general rule, all integrins require divalent cations
for ligand recognition (D'Souza et al., 1994 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 The observation that L1 can support an RGD-dependent
interaction with 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 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 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 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
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 ; Moos et al., 1988
). Functions attributed to this neural CAM include such dynamic
processes as cerebellar cell migration (Lindner et al., 1983
)
and neurite fasciculation and outgrowth (Lagenaur and Lemmon, 1987
).
; Bock et al., 1985
; Lemmon and McLoon,
1986
; Mujoo et al., 1986
). These homologues share an extracellular structure consisting of six Ig-like domains and five fibronectin type III-like repeats (Moos et al., 1988
;
Sonderegger and Rathjen, 1992
). These extracellular domains are linked via a single transmembrane sequence to a
short, highly conserved cytoplasmic domain (Reid and
Hemperly, 1992
). Limited structural variation within the
human L1 molecule has been reported and can be attributed to variable glycosylation and two alternatively spliced
mini exons (Reid and Hemperly, 1992
; Jouet et al., 1995
).
; Rathjen and Schachner, 1984
; Martini and Schachner, 1986
). Although classified a neural
recognition molecule, L1 has also been identified on non-neuronal cell types of surprisingly diverse origin. Thus, we
and others, have recently described L1 on human immune
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 both neuroectodermal
and epithelial origin (Mujoo et al., 1986
; Linnemann et al.,
1989
; Reid and Hemperly, 1992
). Apart from such cellular
associations it is apparent that L1 can also be shed and incorporated into the extracellular matrix (Martini and Schachner, 1986
; Poltorak et al., 1990
; Montgomery et al., 1996
).
This consequently implies a dual function for L1 both as a
CAM and a substrate adhesion molecule (SAM).
), L1 has recently emerged as a
ligand that can undergo multiple heterophilic interactions.
Examples include interactions with other members of the
IgSF and even components of the extracellular matrix. Thus,
heterophilic ligands include TAG-1/axonin-1 (Kuhn et al.,
1991
; Felsenfeld et al., 1994
), F3/F11 (Olive et al., 1995
),
laminin (Hall et al., 1997
), and chondroitin sulfate proteoglycans (Grumet et al., 1993
; Friedlander et al., 1994
). Significantly, L1 has also been reported to undergo multiple cis-type interactions with molecules as diverse as NCAM
(Feizi, 1994
), CD9 (Schmidt et al., 1996
), and CD24 (Kadmon et al., 1995
). Interestingly such interactions in the plane
of the cell membrane may serve to modify the specificity
and avidity of L1 binding to ligands associated with the
membranes of juxtaposed cells or the extracellular matrix
(Feizi, 1994
; Kadmon et al., 1995
; Schmidt et al., 1996
).
; Ebeling et al., 1996
; Montgomery et al., 1996
; Duczmal et al., 1997
; Pancook et al., 1997
). In the first of these
studies, Ruppert et al. (1995)
describe an interaction between the integrin
5
1 and murine L1. In a subsequent
study, we report an interaction between human L1 and the
vitronectin receptor
v
3 (Montgomery et al., 1996
); an association that has now been described using a variety of
cell types (Ebeling et al., 1996
; Pancook et al., 1997
; Duczmal et al., 1997
). Whereas an interaction between the integrin
5
1 and murine L1 has been documented, an interaction between this integrin and human L1 has not, despite
the use of cells expressing reasonable levels of this integrin
(Ebeling et al., 1996
). This has prompted some debate
about species-specific recognition perhaps governed by
the presence of an additional RGD site in murine L1 (Ebeling et al., 1996
). Whereas the majority of these studies have
used purified or recombinant L1 as a substrate for integrin-mediated adhesion, it is important that the interaction between integrin
5
1 and murine L1 has also been shown
to mediate cell-cell interaction (Ruppert et al., 1995
).
v
3 is the only member of the integrin superfamily 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
). A primary objective of this
study was to determine whether recognition of human L1
is a singular attribute of
v
3 or an attribute that can be extended to other RGD-dependent integrins. In this regard,
we demonstrate that the sixth Ig-like domain of human L1
can in fact support multiple integrin interactions involving, not just
v
3, but also the integrins
v
1,
IIb
3, and indeed
5
1. A further objective of this study was to address key
structural and regulatory issues related to these L1-integrin interactions, including the central importance of the
single RGD motif and the critical differential effects of specific divalent cations. In this regard, we present evidence
that recognition of L1 by
v
3,
v
1,
IIb
3, and
5
1 is indeed RGD-dependent and that the binding of these different integrins is differentially regulated by physiological levels of calcium. Based on the novel pairing of L1 with
v
1 or
IIb
3, we further demonstrated that this CAM can
support the attachment of both endothelial cells (
v
1)
and activated platelets (
IIb
3). Given the interaction between L1 and the vascular integrins
v
3 and
v
1, it is salient that we also describe de novo L1 expression on blood
vessels associated with certain neoplastic or inflammatory
diseases. Based on these findings we suggest expanded and
novel roles for L1-integrin interactions in vascular and
thrombogenic processes.
Materials and Methods
5
1
mAb PB1, anti-
v and
3 integrin polyclonal (anti-vitronetic receptor
[VNR]), anti-
v
3 mAb LM609, anti-
IIb
3 mAb LJ-CP8, anti-
3 integrin
mAb 7E3, anti-
1 integrin mAb P4C10, and anti-
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-
1 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
).
IIb
3 (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
IIb and
3 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
IIb
3 (
IIb
L
3 cells) has been described (O'Toole et al.,
1994
). Briefly, the cytoplasmic sequence from the
L integrin subunit was
isolated from the appropriate cDNA clone by PCR with the PCR oligonucleotides designed to omit the
L cytoplasmic sequence VGFFK (i.e.,
L
). As previously described, the
L
construct was ligated with a fragment encoding the extracellular and transmembrane domains of the
IIb
integrin subunit. Coexpression of this chimeric internal deletion mutant
(
IIb
L
) with the wild-type
3 integrin subunit resulted in the stable expression of the
IIb
L
3 heterodimer bearing an active, high affinity extracellular domain of human
IIb
3 (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.
) was obtained from the American
Type Culture Collection (Rockville, MD). A stable
v
3-negative variant
of this line was obtained by repeated negative sorting using anti-
v
3 mAb
LM609. Sorting of
v
3-negative unstained cells was performed using a
FACstar® flow cytometer (Becton Dickinson, Co., Mountain View, CA).
To obtain a stable,
v
3-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.
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 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-1
T 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.
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-1
T 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.
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 SequencingTM kit (Pharmacia Biotech Sevrage).
-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.
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.
v
3 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
(
v
3) was revealed using one substrate (AEC), and the sections subsequently restained for the second antigen (L1) using a different substrate
(Vector VIP).
Results
v
3 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
IIb
3, we used a CHO cell line (A5) genetically altered to express human platelet
IIb
3 (O'Toole et al.,
1989
, 1990
; Frojmovic et al., 1991
). To further determine
whether
IIb
3 needs to be in an active state to recognize
L1 we used a CHO cell line (
IIb
L
3) transfected to express
IIb
3 in a constitutively active state (O'Toole et al.,
1994
). Activation was achieved by chimerization of extracellular and transmembrane
IIb with a cytoplasmic deletion mutant of the
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
IIb
3 (LJ-CP8 reactivity). Second, transfection
of the human
3 integrin subunit has also resulted in a
pairing with endogenous hamster
v and consequently expression of chimeric
v
3 (LM609 reactivity). Finally, and
of immediate relevance for this study, wild-type and transfected CHO express high levels of endogenous
5
1 (PB-1
reactivity) and endogenous
v integrin(s) (VNR reactivity).
Fig. 1.
(A) Integrin profiles of wild-type CHO-K1 cells, of
CHO cells transfected with IIb and
3 subunits (A5) and of CHO
cells transfected to express active
IIb
3 (
IIb
L
3 cells). (B) Concentration-dependent adhesion of CHO-K1, A5, and
IIb
L
3
cells to L1-Ig6. (A) Integrin expression is represented by FACS®
histograms. Cells were treated with antibodies to hamster
5
1 (monoclonal PB1), to human
v
3 (mAb LM609), to human
IIb
3 (mAb LJ-CP8), to hamster or human
v or
3 integrin subunits (polyclonal VNR), or to human
3 integrins (mAb 7E3).
These cells were subsequently stained with fluorescein-conjugated goat anti-mouse or goat anti-rabbit antibodies and were
analyzed using a FACScan® flow cytometer. Control cells were
treated with secondary fluorescein-conjugated antibody only. (B)
Wild-type or transfected cells were allowed to adhere to immobilized L1-Ig6 fusion protein offered at concentrations ranging
from 1 to 100 µg/ml. After 30 min non-adherent cells were removed by washing and the remaining adherent cells counted per unit area with a ×15 high powered objective. Experimental treatments were performed in triplicate with a minimum of four areas
counted per well. No significant adhesion was observed on GST
alone and any minimal residual adhesion to BSA-blocked plastic
alone has been subtracted from the cell counts shown. Error bars
represent ±1 SD.
[View Larger Version of this Image (22K GIF file)]
v
3 (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.
3 integrins (
v
3 and/or
IIb
3) can lead to enhanced
binding to L1-Ig6. Significantly, CHO cells bearing the active
IIb
3 (
IIb
L
3 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
IIb
L
3
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).
5
1 and an
v Integrin(s) and These
Heterophilic Interactions Are Differentially Regulated
by Divalent Cations
1 or
v integrin involvement.
v integrins (Fig. 2 A, left). Despite high levels of
5
1 expression, a function blocking mAb specific for hamster
5
1
(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-
5
1 mAb PB1 (Fig. 2 A, right). This finding
indicates that in the presence of Mn2+ alone,
5
1 can also
recognize human L1-Ig6. However, it is also clear that
binding by
5
1 must be acutely susceptible to inhibition by either Ca2+ or Mg2+ since, as stated, we did not observe
5
1 involvement when all three cations were present in
the adhesion buffer (Fig. 2 A, left).
Fig. 2.
Wild-type CHO-K1 cells adhere to L1-Ig6 using an v
integrin(s) and
5
1, and these heterophilic interactions are critically and differentially regulated by exogenous Ca2+. (A) Wild-type cells were allowed to adhere to immobilized L1-Ig6 fusion
protein in the presence of Ca2+ (2 mM), Mg2+ (2 mM), and Mn2+
(0.4 mM), or in the presence of Mn2+ alone (0.4 mM). Some cells
were pretreated with antibody to human
v
3 (mAb LM609; 80 µg/ml), to hamster
5
1 (PB1 ascites 1:30) or to VNR (polyclonal
anti-VNR; 1:30), or a combination of antibodies. The level of
control adhesion achieved in the absence of inhibitors is taken as
100%. In the presence of mixed cations this was equivalent to 82 adherent cells per field and in the presence of Mn2+ alone this
was equivalent to 255 adherent cells per field. Both, a combination of the two preceding antibodies. (B) Wild-type cells were allowed to adhere to immobilized L1-Ig6 fusion protein in the presence of Mn2+ (0.4 mM) alone or in the presence of Mn2+ (0.4 mM)
and increasing concentrations of Ca2+ (0.5-2 mM). Cells were
pretreated with antibody to hamster
5
1 (PB1 ascites 1:30) or to
VNR (polyclonal anti-VNR; 1:30). (C) Wild-type cells were allowed to adhere to immobilized L1-Ig6 fusion protein in the presence of Ca2+ (1 mM) alone, Mg2+ (1 mM) alone or Mn2+ (0.4 mM)
alone. The L1-Ig6 fusion protein was offered at a concentration of 40 µg/ml. Cells were allowed to adhere for 30-40 min in the presence or absence of the antibodies, and adherent cells counted per unit area with a ×15 high powered objective. Experimental treatments were performed in triplicate with a minimum of four areas counted per well. Error bars represent ±1 SD.
[View Larger Version of this Image (36K GIF file)]
1 integrin ligation
(Kirchhofer, 1991; Mould et al., 1995
) we determined
whether this cation was responsible for a lack of
5
1 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
5
1 and
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
5
1-dependent
binding (Fig. 2 B) such that the addition of 2 mM calcium
reduced the level of
5
1-dependent binding by >80%
(Fig. 2 B). In contrast, the same concentration of calcium reduced the
v integrin(s)-dependent binding by only 20%.
v
3 and Activated
IIb
3
to Interact with L1-Ig6
v
3 and
IIb
3 (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
3 integrins can indeed recognize L1 and, if so, how
the binding of these integrins is regulated.
v
3 and
IIb
3 (A5 and
IIb
L
3 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
v
3 and active
IIb
3. Thus,
CHO cells engineered to express active
IIb
3 (
IIb
L
3 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
v
3 and
IIb
3 (Fig. 3, bottom). It is important to
note, that in the presence of Ca2+ alone,
IIb
3-dependent
binding to L1-Ig6 was only evident in the CHO cells engineered to express active
IIb
3 (
IIb
L
3 cells). Thus, in
A5 cells bearing
IIb
3 in its resting state, adhesion to L1-Ig6 in the presence of Ca2+ was fully inhibited by mAb
LM609 (i.e.,
v
3 dependent) with no evident contribution
by
IIb
3 (Fig. 3, top left). This finding implies that
IIb
3-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
IIb
3 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
IIb
3 and
v
3 (Fig. 3, top right). Because of endogenous
v integrin and
5
1 binding, adhesion in the presence of Mn2+ could only be abrogated using a combination
of polyclonal antibodies to both
5
1 and
3 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).
Fig. 3.
Both v
3 and activated
IIb
3 can support attachment
to L1-Ig6 and such ligation can occur in the presence of Ca2+
or Mg2+ alone. CHO cells transfected with the human
IIb and
3
subunits (A5 cells) or transfected to express active
IIb
3 (
IIb
L
3 cells) were allowed to adhere to immobilized L1-Ig6 fusion protein in the presence of Ca2+ (2 mM), or Mg2+ (2 mM) alone.
Some cells were pretreated with antibody to human
v
3 (mAb
LM609; 80 µg/ml), or to human
IIb
3 (mAb LJ-CP8; 80 µg/ml)
or with a combination of antibodies. The L1-Ig6 was offered at 40 µg/ml. Cells were allowed to adhere for 40 min in the presence or
absence of the antibodies, and adherent cells counted per unit area with a ×15 high powered objective. Both, a combination of the two preceding antibodies. Experimental treatments were performed in triplicate with a minimum of four areas counted per
well. Error bars represent ±1 SD.
[View Larger Version of this Image (48K GIF file)]
IIb
3
IIb
3 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
IIb
3 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
).
v
3 mAb LM609 alone, but was
abrogated by the mAb 7E3, which is a potent function-blocking antibody, specific for both
v
3 and
IIb
3 (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
IIb
3. The contribution of
IIb
3 was also
confirmed using the specific anti-
IIb
3 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
IIb
3 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
IIb
3 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.
Fig. 4.
Activated IIb
3 can mediate the attachment of platelets to immobilized L1 fusion proteins and the L1-derived peptide (C)PSITWRGDGRDLQEL. (A-C) Stimulated or unstimulated human platelets were allowed to adhere to immobilized L1
fusion proteins (L1-Ig4-6 or L1-Ig6; A and B) or to immobilized,
L1-derived RGD peptide (C). Some of the platelets were pretreated with antibody to
v
3 (mAb LM609; 80 µg/ml), or to
v
3
and
IIb
3 (mAb 7E3; 80 µg/ml). The platelets were allowed to
adhere for 10 min in the presence or absence of the antibodies
and the adherent platelets subsequently stained with crystal violet for quantification. Experimental treatments were performed
in triplicate. Error bars represent ±1 SD. (D and E) Photomicrographs of stimulated platelets adhering to L1-Ig6 in the absence
(D) or presence of mAb 7E3 (E). Adherent platelets were
stained with crystal violet and are confined to the circular area
coated with the fusion protein. Individual platelets cannot be discriminated because of their small size and some aggregation. The
platelets were allowed to adhere for 10 min in the presence or absence of 7E3. Any minimal residual adhesion to BSA-blocked plastic alone has been subtracted from the OD values shown. Bar, 300 µm.
[View Larger Versions of these Images (46 + 74K GIF file)]
v
3
and
v
1 and These Heterophilic Interactions Are
Differentially Regulated by Divalent Cations
; 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
v
3 and a high level of
1 integrins (Fig. 5 A, left column). We also detected expression of
v
5 (mAb P1F6) at a median fluorescence comparable
to that found for
v
3 (not shown). Consistent with our
findings using the transfected CHO cells, we observed that
endothelial
v
3 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-
1 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).
Fig. 5.
(A) Integrin profiles of wild-type ECV304 human umbilical vein endothelial cells or ECV304 cells repeatedly sorted
for a lack of v
3 expression. (B and C)
v
3- and
v
1-dependent
adhesion of wild-type or sorted ECV304 endothelial cells to L1-Ig6. (A) Integrin expression is represented by FACS® histograms.
Cells were treated with antibodies to
v integrins (mAb 17E6), to
v
3 (mAb LM609), to
v or
3 integrin subunits (polyclonal VNR),
or to
1 integrins (mAb P4C10). These cells were subsequently
stained with fluorescein-conjugated goat anti-mouse or goat anti-
rabbit antibodies and were analyzed using a FACScan® flow cytometer. Control cells were treated with secondary fluorescein-conjugated antibody only. (B and C) Wild-type or sorted cells
were allowed to adhere to immobilized L1-Ig6 fusion protein offered at 40 µg/ml. Adhesion was performed in the presence of
Ca2+ (2 mM), Mg2+ (2 mM), and Mn2+ (0.4 mM). Some cells were
pretreated with antibody to
v
3 (mAb LM609; 80 µg/ml), to
1
integrins (mAb P4C10; 80 µg/ml), to
v integrins (MAb 17E6; 80 µg/ml), or with a combination of antibodies. After 40 min non-
adherent cells were removed by washing and the remaining
adherent cells counted per unit area with a ×15 high powered objective. Both, a combination of the two preceding antibodies. Experimental treatments were performed in triplicate with a minimum of four areas counted per well. Error bars represent ±1 SD.
[View Larger Version of this Image (31K GIF file)]
v
3 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
v
3-negative endothelial cells. To address this
we exploited ECV304 endothelial cells that had been repeatedly FACS® sorted for a lack of
v
3 expression. This
approach proved successful, resulting in the generation of
a stable population of
v
3-negative cells (Fig. 5 A, right
column). The levels of
v
5 and
1 integrin expression in
these cells remained unchanged (not shown; and Fig. 5 A,
right column). Remarkably, these
v
3-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
v
3 (LM609) but was fully inhibited by a mAb to
1 integrins (i.e., P4C10) (Fig. 5 C).
Furthermore, such adhesion was also completely abrogated with an antibody specific for
v integrins (Fig. 5 C).
Together these results are consistent with an interaction
between L1 and endothelial
v
1.
v
3 and
v
1 can interact with L1-Ig6,
we wished to determine why
v
3 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
v
3 rather than
v
1. We observed a
number of facts supporting this hypothesis. First, significant
v
1 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-
v
3 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
v
3 and
v
1 (i.e., LM609
and P4C10), or with a mAb reactive with both
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
v
3
(Fig. 6 A). Finally, in an experiment analogous to that performed with the wild-type CHO cells (Fig. 2 B), we observed that the
v
1-mediated (Mn2+-dependent) adhesion of the sorted,
v
3-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
v
3 expression,
quiescent endothelial cells may use
v
1 to bind to L1,
however with the induction of
v
3 expression, physiological levels of calcium are likely to favor the use of this integrin. It is also clear that whereas
v
1-mediated adhesion
is susceptible to inhibition by exogenous Ca2+, this inhibition is less pronounced than that seen when adhesion is
mediated via
5
1 (Fig. 2 B).
Fig. 6.
Usage of v
1 by wild-type ECV304 human umbilical
vein endothelial cells is dictated by a requirement for Mn2+ and
by inhibition by Ca2+. (A) Wild-type ECV304 endothelial cells
were allowed to adhere to immobilized L1-Ig6 in the presence of
Ca2+ alone (1 mM), Mg2+ alone (1 mM), or Mn2+ alone (0.4 mM).
Some cells were pretreated with antibody to
v
3 (mAb LM609;
80 µg/ml), to
1 integrins (mAb P4C10; 80 µg/ml), to
v integrins
(mAb 17E6; 80 µg/ml), or with a combination of antibodies. (B)
Wild-type or sorted endothelial cells were allowed to adhere to immobilized L1-Ig6 fusion protein in the presence of Mn2+ (0.4 mM)
alone or in combination with increasing concentrations of Ca2+
(0.5-2 mM). After 40 min non-adherent cells were removed by
washing and the remaining adherent cells counted per unit area
with a ×15 high powered objective. Both, refers to a combination
of the two preceding antibodies. Experimental treatments were
performed in triplicate with a minimum of four areas counted per
well. Error bars represent ±1 SD.
[View Larger Version of this Image (31K GIF file)]
3 or
1
Integrins Is RGD-dependent
; Haas and Plow, 1994
). Furthermore, although
v
3,
5
1,
v
1, and
IIb
3 have all been reported to interact with RGD motifs,
5
1 and the
3 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.
v
3, and
v
1 (Fig. 7, Endothelial) and CHO cell attachment via endogenous
5
1 and transfected
IIb
3 (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).
Fig. 7.
All of the integrins characterized can
recognize the L1-derived peptide (C)PSITWRGDGRDLQEL. Wild-type or sorted ECV304
endothelial cells and wild-type or transfected CHO
cells (IIb
L
3 cells) were allowed to adhere to
immobilized 16-mer peptide (C)PSITWRGDGRDLQEL or to control peptide (C)PSITWRADGRDLQEL. Adhesion was performed in the
presence of Ca2+ (2 mM), Mg2+ (2 mM), and
Mn2+ (0.4 mM) or in the presence of Mg2+ (1 mM) alone, or Mn2+ alone (0.4 mM). Some cells
were pretreated with antibody to human
IIb
3
(mAb LJ-CP8; 80 µg/ml), to
v
3 (mAb LM609;
80 µg/ml), to
1 integrins (mAb P4C10; 80 µg/
ml), to
v integrins (mAb 17E6; 80 µg/ml), to
hamster
5
1 (PB1 ascites 1:30) or to VNR (polyclonal anti-VNR; 1:30), or with a combination of
antibodies. After 40 min, non-adherent cells were
removed by washing and the remaining adherent
cells stained with crystal violet. Both, a combination of the two preceding antibodies. Experimental treatments were performed in triplicate with a
minimum of four areas counted per well. Error
bars represent ±1 SD.
[View Larger Version of this Image (38K GIF file)]
v
3 and
v
1 (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
5
1 and the
v integrin(s) (Fig. 2 A, right). Adhesion by the CHO cells
transfected to express active
IIb
3 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).
Fig. 8.
Integrin-dependent adhesion to L1-Ig6 can be abrogated or reduced using soluble L1-derived peptide PSITWRGDGRDLQEL. Wild-type ECV304 endothelial cells and wild-type or transfected CHO cells (IIb
L
3 cells) were allowed to
adhere to immobilized L1-Ig6. Some cells were pretreated and
subsequently adhered in the presence of the soluble peptide
PSITWRGDGRDLQEL (25 µM) or the control peptide
PSITWRADGRDLQEL (25 µM). Some CHO cells were pretreated with antibody to human
IIb
3 (mAb LJ-CP8; 80 µg/ml).
Adhesion was performed in the presence of Mg2+ (1 mM) alone,
or Mn2+ alone (0.4 mM). After 40 min, non-adherent cells were
removed by washing and the remaining adherent cells counted
per unit area with a ×15 high powered objective. The level of
control adhesion achieved in the absence of peptide inhibitors
was taken as 100%. In the case of wild-type endothelial and CHO
cells this was equivalent to 188 and 160 adherent cells per field,
respectively; in the case of the CHO cells bearing active
IIb
3,
this was equivalent to 232 adherent cells per field. Experimental
treatments were performed in triplicate with a minimum of four
areas counted per well. Results are expressed as a percent of the
adhesion observed in the absence of peptide. Error bars represent ±1 SD.
[View Larger Version of this Image (49K GIF file)]
KGE or RGD
RGA. Importantly, the conservative RGD
KGE mutation completely abrogated
v
3- and
v
1-dependent binding by the endothelial cells (Fig. 9, Endothelial). This same
mutation and the RGD
RGA mutation also significantly
abrogated
5
1-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
5
1. Alternatively, a second
5
1-binding motif
may exist within the L1-Ig6 domain. Interestingly the
RGD
KGE mutation could still be recognized to a significant level by
IIb
3 (Fig. 9, CHO, Active
IIb
3). However,
the alanine substitution mutant (RGA) was sufficient to
fully abrogate
IIb
3-mediated binding.
Fig. 9.
Mutation of the RGD sequence in the
sixth Ig-like domain of L1 abrogates binding by
v
3,
v
1, and
IIb
3 and reduces binding mediated by
5
1. Wild-type or sorted ECV304 endothelial cells and wild-type or transfected CHO
cells (
IIb
L
3 cells), were allowed to adhere to
immobilized L1-Ig6 or to L1-Ig6 containing the
mutated sequences RGD
KGE or RGD
RGA.
Adhesion was performed in the presence of Ca2+
(2 mM), Mg2+ (2 mM), and Mn2+ (0.4 mM), or in
the presence of Mg2+ (1 mM) alone, or Mn2+
alone (0.4 mM). Some cells were pretreated with
antibody to human
IIb
3 (mAb LJ-CP8; 80 µg/
ml). After 40 min, non-adherent cells were removed by washing and the remaining adherent
cells counted per unit area with a ×15 high powered objective. The level of control adhesion
achieved on the wild-type fusion protein was taken as 100%. In the case of wild-type and
sorted endothelial cells this was equivalent to 168 and 127 adherent cells per field, respectively, in the case of the wild-type CHO cells
this was equivalent to 82 (mixed cations) and 203 (Mn2+ alone) adherent cells per field and in the case of CHO cells bearing active
IIb
3
this was equivalent to 224 cells per field. Experimental treatments were performed in triplicate with a minimum of four areas counted
per well. Results are expressed as a percent of the adhesion observed on wild-type L1-Ig6. Error bars represent ±1 SD.
[View Larger Version of this Image (34K GIF file)]
v
3 (Fig. 10 C); a documented marker of angiogenic
blood vessels (Brooks et al., 1994
). Importantly,
v
3 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
v
3 (Fig. 10, D and E); and that whereas
v
3 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.
Fig. 10.
L1 expression can be induced on
blood vessels. L1 expression associated with
vessels proximal to a squamous cell carcinoma (A and B). L1 staining was performed
using the anti-L1 mAb 5G3 and the substrate
AEC to give a red stain. L1 is evident on the small vessels in the dermis (arrows). Vessels
proximal to the tumor also expressed v
3: a
marker of angiogenic blood vessels (C).
Staining for
v
3 was performed using
LM609 and the substrate AEC to give a red
stain. Vascular L1 and
v
3 expression could be colocalized on a subset of vessels (D and
E). Vessels staining for
v
3 are red (AEC),
whereas vessels also costaining with L1 (arrow) are darker brown to blue-black (AEC
and VIP). Angiogenic vessels expressing L1
were detected in normal dermis between the tumor mass (left arrow) and the epidermis
(right arrow) (F). The tumor and epidermis
are stained for cytokeratin (F). Vessels in the
dermis of normal human skin were found to
be negative or very weakly positive for L1
(G). Vessels in synovial tissue derived from
the joint of a patient with rheumatoid arthritis also showed evidence of L1 expression
(H). L1 staining was performed using the
anti-L1 antibody 5G3 and the substrate AEC
to give a red stain. Bars: (A) 100 µm; (B) 50 µm; (C and D) 20 µm; (E) 25 µm; (F) 200 µm; (G and H) 100 µm.
[View Larger Version of this Image (121K GIF file)]
Discussion
v
3,
v
1,
5
1, and
IIb
3. To our knowledge this
is the first observation that both
v
1 and
IIb
3 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.
3 and
1 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
v
3,
5
1, and
IIb
3 (Koivunen et al.,
1994
). To demonstrate definitively that the single RGD sequence present in human L1 is indeed critical for binding
by
v
3,
v
1,
5
1, and
IIb
3 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.
). 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
v
3 and
5
1 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
IIb
3 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
v
3 than via
IIb
3
(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
v
3 (Healy et al., 1995
). It is
interesting to note, that although we detected significant
v
5 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
v
5.
). 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
-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.
) 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
v
3 or
IIb
3,
this same cation profoundly suppressed a Mn2+-dependent
interaction with
5
1 or
v
1. It is important to note, that
such a dichotomy between these
1 integrins and the
3 integrins has been documented for other ligands. Thus, Ca2+
has been shown to support both
v
3 or
IIb
3 attachment
to RGD peptides or vitronectin (Kirchhofer, 1991; Suehiro et al., 1996
), whereas this same cation suppresses the
attachment of
5
1 and
v
1 to fibronectin and an RGD
peptide, respectively (Kirchhofer, 1991; Mould et al., 1995
).
Recently, Suehiro et al. (1996)
described a novel classification for defining
3 ligands depending upon their ability to
support the attachment of
v
3 and/or
IIb
3 in the presence of different cations. Thus, class I
3 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
IIb
3 in the presence of either Ca2+ or
Mn2+, but
v
3 attachment is suppressed by Ca2+. Finally,
class III ligands bind exclusively to
IIb
3 under all cation
conditions, whereas class IV ligands bind exclusively to
v
3. According to this classification scheme we propose
that L1 is a novel class I ligand for
3 integrins, together
with vitronectin, RGD peptides and disintegrin group A
(Suehiro et al., 1996
).
v
3
or activated
IIb
3 >
v
1 >
5
1. This hierarchy is likely
to be further compounded by transnegative dominance, a
recently described phenomenon in which the ligation of a
3 integrin has been shown to suppress the function of a
1
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
5
1 and L1-Ig6. However, it is important to note that an
interaction between
5
1 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,
5
1 may indeed have a physiological role in L1 binding, but it is likely to be regulated
by a requirement for additional activational signals.
v
3 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
v
3 (Montgomery et al., 1996
). The interaction between human L1
and
v
3 has also been confirmed using lymphocytic cell
lines (Ebeling et al., 1996
). The finding that human L1-Ig6
can also interact with
IIb
3,
v
1, and
5
1 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
5
1 (Ebeling et al., 1996
).
The most obvious explanation for this is the strict cation
requirement of this interaction and perhaps transnegative
dominance by
v
3. It should also be noted that our CHO
cells express very high levels of
5
1. The observation that
human L1 can interact with
5
1 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
5
1. 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
5
1. Thus, all three of these tripeptide sequences
have been identified as important for the interaction of
non-RGD, 7-mer peptides with
5
1 (Koivunen et al., 1993
).
; 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
5
1 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.
). 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.
IIb
3. 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
IIb
3 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
v
3 or
v
1. 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.
v
3 and
v
1 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
v
1 for migration by oligodendrocyte precursors. Interestingly, the authors speculate that, among other
ligands, L1 present within axonal tracts might serve as a
potential
v
1 ligand, providing a mechanism for guiding
migrating oligodendrocytes. Whereas this is merely speculation, our data clearly support the potential for L1-
v
1
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,
v
1 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
v
1 adhesion is dependent upon the presence
of Mn2+ but is inhibited by Ca2+.
v
3, we have demonstrated that this CAM
is a relatively promiscuous ligand, supporting further novel
heterophilic interactions with
v
1,
5
1, and
IIb
3. 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
v
1 and
IIb
3 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.
Received for publication 2 May 1997 and in revised form 22 August 1997.
Address all correspondence to Anthony Montgomery, Department of Immunology, R218, The Scripps Research Institute, La Jolla, CA 92037. Tel.: (619) 784-8109. Fax: (619) 784-2708. E-mail: Montg{at}scripps.eduThe 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.
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