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J. Biol. Chem., Vol. 276, Issue 28, 25783-25790, July 13, 2001
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From the
Received for publication, December 14, 2000, and in revised form, April 12, 2001
Activated leukocyte cell adhesion molecule
(ALCAM/CD166), a member of the immunoglobulin superfamily with five
extracellular immunoglobulin-like domains, facilitates heterophilic
(ALCAM-CD6) and homophilic (ALCAM-ALCAM) cell-cell interactions. While
expressed in a wide variety of tissues and cells, ALCAM is restricted
to subsets of cells usually involved in dynamic growth and/or migration processes. A structure-function analysis, using two monoclonal anti-ALCAM antibodies and a series of amino-terminally deleted ALCAM
constructs, revealed that homophilic cell adhesion depended on ligand
binding mediated by the membrane-distal amino-terminal immunoglobulin
domain and on avidity controlled by ALCAM clustering at the cell
surface involving membrane-proximal immunoglobulin domains.
Co-expression of a transmembrane ALCAM deletion mutant, which lacks the
ligand binding domain, and endogenous wild-type ALCAM inhibited
homophilic cell-cell interactions by interference with ALCAM avidity,
while homophilic, soluble ligand binding remained unaltered. The
extracellular structures of ALCAM thus provide two structurally and
functionally distinguishable modules, one involved in ligand binding
and the other in avidity. Functionality of both modules is required for
stable homophilic ALCAM-ALCAM cell-cell adhesion.
Adhesion molecules play an important role in development,
leukocyte function, and homeostasis in multicellular organisms, which
are mainly governed by inter- and intracellular communication via
cell-cell interactions. Alterations in cellular adhesion and communication can contribute to uncontrolled cell growth (1) and
life-threatening syndromes like leukocyte adhesion deficiency (2).
Activation of adhesion molecules generally involves both modulation of
affinity and avidity. The affinity of adhesion molecules often reflects
a specific conformation of the extracellular ligand-binding domain.
Avidity modulation involves changes in the cell surface distribution of
adhesion molecules (e.g. lateral oligomerization), which
leads to clusters of molecules and thereby specifically increases the
number of available receptors at the site of cell-cell interaction.
Activated leukocyte cell adhesion molecule
(ALCAM/MEMD/CD166)1 is a type
I transmembrane protein and a member of the Ig superfamily. It has over
90% homology with the chicken adhesion molecule BEN/SC1/DM-GRASP (3-5), and it has 30% identity and 50% similarity with the human melanoma cell adhesion molecule Mel-CAM/MUC18/CD146 (6). Furthermore, ALCAM has 93% sequence identity with the candidate liver high density lipoprotein receptor HB2 (7). ALCAM is involved in various
physiological processes including hematopoiesis (8, 9), thymus
development (10), the immune response (11), neurite extension (12),
neural cell migration (13), and osteogenesis (14).
ALCAM has a short cytoplasmic tail and its extracellular part comprises
five Ig domains: two amino-terminal variable (V) type Ig domains
followed by three constant (C) type Ig domains
(V1V2C1C2C3). ALCAM was first identified as a CD6 ligand (15), but it also mediates
homophilic ALCAM-ALCAM interactions. While the heterophilic ALCAM-CD6
interaction is extensively studied and mapped (16-18), little is known
about the molecular basis for the homophilic ALCAM-ALCAM interaction as
observed in human melanoma (19, 20) and hematopoiesis (8, 9).
Previously, we have shown that homophilic ALCAM-mediated cell-cell
adhesion is regulated through actin cytoskeleton-dependent clustering of ALCAM molecules at the cell surface and that this clustering is necessary to obtain stable adhesive interactions (21). In
analogy with other members of the immunoglobulin superfamily like NCAM
(22) and Ng-CAM/L1-CAM (23), it is likely that the formation of ALCAM
cis-homo-oligomers at the cell surface is essential for
strong ligand binding and homophilic interactions.
Here we describe a detailed molecular analysis of the homophilic ALCAM
interaction and the construction of an amino-terminally deleted ALCAM
molecule that inhibited wild-type ALCAM-mediated aggregation in a
dose-dependent manner. These combined data lead us to
propose a model for homophilic ALCAM-mediated cell adhesion.
Cell Lines--
The adherent human melanoma cell lines BLM and
530 (24) were grown as monolayers in Dulbecco's modified Eagle's
medium as described before (19). The erythroleukemia cell line
K562 growing in suspension and ALCAM-transfected K562 cells were
cultured as described previously (21). Regular tests confirmed that
cell lines were free of mycoplasma contamination.
Monoclonal Anti-ALCAM Antibodies--
Anti-ALCAM antibody J4-81
(MA250020, IgG1) was purchased from Antigenix America Inc. (Franklin
Square, NY). Monoclonal anti-ALCAM antibody AZN-L50 (IgG2a) was
generated by immunization of BALB/c mice with K562-ALCAM cells. Four
consecutive days before fusion, mice were boosted intravenously. The
spleen was isolated, and spleen cells were fused with SP2/0 cells using
standard technology. Supernatants of growing hybridomas were tested in
a cell enzyme-linked immunosorbent assay using K562-ALCAM cells and
K562 cells as a positive and negative control, respectively. Positive
hybridomas were recloned several times to obtain true monoclonal
hybridomas. The hybridoma AZN-L50 was selected for its strong and
specific binding to ALCAM and its ability to inhibit the homophilic
ALCAM-ALCAM interaction.
Plate Adhesion Assay--
ALCAM-Fc recombinant protein
consisting of the five extracellular domains of ALCAM fused to the
human IgG1 Fc domain was produced and purified as described earlier
(21). Adhesion of cells to immobilized ALCAM-Fc was tested as described
before (21). Briefly, flat bottom maxisorp 96 wells plates (NUNC,
Roskilde, Denmark) were coated with 4 µg/ml goat
anti-human-Fc-F(ab')2 in TSM (20 mM Tris, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, pH 8.0) for 1 h. Plates were
blocked with 1% (w/v) bovine serum albumin in TSM and subsequently
coated with 250 ng/ml ALCAM-Fc for 1 h. Cells (2 × 104 cells/well) were labeled with Calcein-AM (Molecular
Probes, Inc., Eugene, OR) and preincubated with monoclonal anti-ALCAM
antibody J4-81 (5 µg/ml) and/or AZN-L50 (10 µg/ml) for 5-10 min at
room temperature. Cells were allowed to adhere in triplicate wells of
the coated plates for 20 min (530 cells) or 45 min (KG1, K562-ALCAM) in
culture medium at the indicated temperatures. Nonadherent cells were
removed by washing the wells five times with TSM plus 0.5% bovine
serum albumin at 37 °C. Cells were lysed with lysis buffer (50 mM Tris, 0.1% SDS), and fluorescence was quantified in a
cytofluorometer (PerSeptive Biosystems). Adhesion was expressed as the
percentage (mean ± S.D.) bound cells of the total cells allowed
to adhere in triplicate wells.
Soluble ALCAM-Fc Binding Assay--
For soluble ALCAM-Fc
binding, the indicated concentrations of ALCAM-Fc were added to 5 × 104 cells in culture medium in V-bottom wells in a final
volume of 50 µl. After an incubation of 30 min at 37 °C, cells
were washed once with prewarmed (37 °C) medium and subsequently
incubated with a fluorescein isothiocyanate (FITC)-conjugated secondary goat anti-human Fc antibody (Cappel Inc., West Chester, PA) in medium
for 15 min at 37 °C. After washing with prewarmed medium, cells were
analyzed on a FACScan (Becton Dickinson, Mountain View, CA). The mean
fluorescence intensity is a measure for the amount of ALCAM-Fc
molecules bound to the cells. The percentage of cells that have bound
ligand was determined.
Construction of Amino-terminally Truncated ALCAM
Mutants--
Amino-terminally truncated ALCAM mutants were
generated using the polymerase chain reaction and carefully chosen
restriction sites in pWD201 (2.1-kilobase pair ALCAM cDNA coding
for the leader sequence, the two V-type and three C-type Ig domains,
the transmembrane spanning domain, and the short cytoplasmic tail
(V1V2C1C2C3)
in pZip-neo-(X)-1 containing the neomycin resistance gene (19)). A
schematic representation is shown in Fig.
1. The ALCAM leader sequence was
amplified using a 5' Rev/T3 primer (5'-ATT ACG CCA AGC TCG AA-3') and
3' primers extended with a suitable restriction site. For the
generation of pWD277
(V2C1C2C3-construct),
the Rev/T3 and P9-PstI (5'-AG CAT GCC AGA AGG TAT GAT AAT
GGT ATC TCC ATA T-3') primer pair was used to amplify the leader
sequence. The amplified fragment was subsequently cloned in
SstI/PstI-linearized pWD201. For the generation
of pWD275 (C1C2C3 construct), the
leader was amplified using the Rev/T3 and P8-BalI (5'-CTG
GCC AGA AGG TAT GAT AAT GGT ATC TCC ATA T-3') primer pair and cloned in
SstI/BalI-linearized pWD201. Finally, pWD278
(C2C3 construct) was generated using the Rev/T3
and P7-NheI (5'-GCT AGC AGA TAT TGT GCA AGG TAT GAT AAT GGT
ATC TCC ATA T-3') primer pair. Thereafter, the fragment was cloned in
SstI/NheI-linearized pWD201. The sequences of all
constructs were verified.
Transfection--
FuGENE-6 transfection reagent (Roche Molecular
Biochemicals) was used to transfect the human melanoma cell lines
according to the manufacturer's protocol. In brief, a mixture of
FuGENE-6 and circular DNA (3 µl and 0.5 µg, respectively) was added
dropwise to a 40% confluent monolayer in a six-well plate in medium.
After 48 h, medium was replaced with selection medium (medium plus
1 mg/ml G418 (Life Technologies, Inc.)). Neomycin-resistant colonies were expanded and maintained in medium supplemented with 0.5 mg/ml G418.
Flow Cytometry: Cell Surface Expression and Aggregation
Assay--
Cells (2 × 105) were incubated with the
indicated mouse monoclonal antibody at 4 µg/ml in PBA
(phosphate-buffered saline containing 1% bovine serum albumin and
0.05% NaN3) for 30 min at 4 °C, washed three times with
PBA, and further incubated with FITC-conjugated goat anti-mouse IgG
antibody (Dako, Glostrup, Denmark) for 30 min at 4 °C. After
washing, positive cells were detected using a FACScan, and the mean
fluorescence intensity was determined.
The aggregation capacity of human melanoma cell lines was measured by a
double colored assay as described previously (19). Briefly, two
separate cell suspensions were labeled fluorescent green or fluorescent
red with 5,6-sulfofluorescein diacetate (50 µg/ml in culture
medium; Molecular Probes) or hydroethidine (40 µg/ml in
culture medium; Plysciences, Warrington, PA), respectively. Following extensive washing, cells were mixed in equal amounts and
allowed to aggregate at 37 °C for 30 min. After incubation, cells
were fixed by adding paraformaldehyde to a final concentration of 0.5%
(w/v) and subsequently analyzed using a FACScan. Aggregation was
expressed as the percentage of double colored events of the total events.
Immunofluorescence--
Immunofluorescence was performed on
methanol/acetone-fixed monolayers of cells grown on a glass surface as
described previously (19). Affinity-purified mouse monoclonal
antibodies were used at 4 µg/ml in phosphate-buffered saline.
Western Blotting--
Cells (7 × 106) were
lysed for 30 min on ice in 1 ml of lysis buffer (25 mM
Tris-HCl, pH 7.5, 1% Nonidet P-40, 100 mM KCl, 10 mM MgCl2, 0.25 mM dithioerythritol,
1 mM phenylmethylsulfonyl fluoride, 1 mM
benzamidine, 2 mM Na2VO4, 10 µg/ml leupeptin, 10 µg/ml aprotinin). Nuclei and the Nonidet P-40
insoluble fraction were spun down at maximum speed in an Eppendorf
centrifuge. Supernatants were stored at
Protein concentration was determined using the Bio-Rad protein assay
and reagents according to the supplier's instructions. Equal amounts
of protein were subjected to 6.5% SDS-polyacrylamide gel
electrophoresis and subsequently transferred to Hybond-C pure membrane
(Amersham Pharmacia Biotech). Membranes were blocked for 1 h at
room temperature in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) and 5% (w/v) low fat milk powder. After washing the membranes
three times with TBS-T, the blots were incubated with AZN-L50 (3 µg/ml in TBS-T supplemented with 3% (w/v) low fat milk powder) for
2 h at room temperature. Following three washes with TBS-T at room
temperature, the membranes were incubated with horseradish
peroxidase-conjugated rabbit anti-mouse IgG (1:2000, Dako, Glostrup,
Denmark) in TBS-T plus 5% milk powder for 1 h at room
temperature. After three washes with TBS-T, proteins were visualized
via the enhanced chemiluminescent reaction (Amersham Pharmacia
Biotech).
Monoclonal Anti-ALCAM Antibodies Have Differential Effects on
ALCAM-mediated Cell Aggregation and Adhesion--
To elucidate the
molecular mechanism underlying the homophilic ALCAM-ALCAM interaction,
the availability of function blocking monoclonal antibodies (mAbs) is
virtually indispensable. The ALCAM-antibody J4-81 has previously been
described to block heterophilic ALCAM-CD6 interactions (25). In
contrast to these findings, we observed that the addition of mAb J4-81
markedly increased homotypic cell clustering of ALCAM-positive but
CD6-negative myelomonocytic KG1 cells (Fig.
2A). Therefore, new mAbs were
generated and selected for the capacity to specifically inhibit
homophilic ALCAM-ALCAM interactions. ALCAM mAb AZN-L50 completely
inhibited the J4-81-induced homotypic cell clustering of KG1 cells to
background levels (Fig. 2A). Ectopic expression of ALCAM in
erythroleukemic K562 cells (K562-ALCAM) resulted in the occurrence of
large ALCAM-mediated cell clusters in suspension that were not observed
in the parental K562 cells. While the addition of mAb J4-81 could not
further enhance cell clustering of K562-ALCAM cells, mAb AZN-L50
completely blocked the spontaneous ALCAM-dependent cell
clustering of these cells (Fig. 2A). Neither mAb J4-81 nor
AZN-L50 had any effect on ALCAM-negative parental K562 cells (Fig.
2A).
Using human melanoma 530 cells, similar activating and inhibiting
effects of the monoclonal antibodies on ALCAM-mediated aggregation were
observed in an aggregation assay analyzed by flow cytometry. While
parental 530 cells do not express ALCAM and do not aggregate, aggregation of these cells is induced by ectopic expression of ALCAM
(530/V1V2C1C2C3)
(19), and this clustering was further increased by 35% using mAb
J4-81, whereas mAb AZN-L50 inhibited the ALCAM-mediated aggregation by
25% as compared with the untreated control (530/ALCAM) (Fig.
2B). Neither of the antibodies had any effect on the
ALCAM-negative vector control 530 cells that did not aggregate (Fig.
2B). A similar pattern of activation and blocking of
adhesion by these mAbs was observed when using the human melanoma cell
line BLM expressing endogenous ALCAM (not shown).
In addition to cell-cell aggregation assays, we analyzed the effect of
mAbs J4-81 and AZN-L50 on ALCAM-mediated stable cell adhesion to
immobilized recombinant ALCAM-Fc. KG1 cells were allowed to adhere to
an ALCAM-Fc-coated surface in the presence or absence of ALCAM mAbs
J4-81 and AZN-L50. No ALCAM-mediated adhesion was observed in the
absence of mAbs. The addition of mAb J4-81 induced adhesion of KG1 to
immobilized ALCAM-Fc, which in turn was efficiently inhibited by the
addition of the function blocking mAb AZN-L50 at physiological
temperatures (Fig. 2C). To exclude the possibility that mAb
J4-81 enhanced cellular aggregation and adhesion via cross-linking of
two ALCAM molecules on opposing cells (or immobilized ALCAM-Fc),
adhesion of KG1 to immobilized ALCAM-Fc was performed at lower,
nonphysiological temperatures (Fig. 2C). The strong induction of ALCAM-mediated adhesion of KG1 cells by mAb J4-81 was
temperature-dependent as the observed adhesion was reduced when cell adherence was performed at room temperature or at 4 °C.
Furthermore, the adhesion of KG1 cells induced by mAb J4-81 is
efficiently blocked with mAb AZN-L50 (Fig. 2C). Similar
results were obtained with K562-ALCAM cells (not shown). Thus, a pair of monoclonal antibodies was characterized that either activated (J4-81) or blocked (AZN-L50) the homophilic ALCAM-ALCAM interaction.
Domain Mapping of mAbs J4-81 and AZN-L50 to Specific Ig Domains of
ALCAM--
The results of these functional studies with mAbs are more
informative when the respective antibody epitopes are known. To identify the Ig domains that are involved in the homophilic
ALCAM-mediated interactions, domain mapping was performed for mAbs
J4-81 and AZN-L50. A series of progressively amino-terminally truncated ALCAM mutants was generated and expressed in the ALCAM-negative human
melanoma cell line 530. The extracellular part of wild-type ALCAM
consists of two amino-terminal V-type domains and three membrane-proximal C-type domains (i.e.
V1V2C1C2C3).
Immunofluorescence analysis was performed with the anti-ALCAM
antibodies on the transfected cells to identify the domains required
for antibody recognition (Table I). mAb
J4-81 only recognized wild-type ALCAM, while mAb AZN-L50 recognized all
available ALCAM constructs. Thus, the epitope for anti-ALCAM mAb J4-81
is mapped to domain V1, whereas that for AZN-L50 is located
in the C2C3 module of ALCAM.
The Amino-terminal Ig Domain V1 of ALCAM Is Required
for Ligand Binding and Homophilic Cell-Cell Interactions--
The
adhesion, aggregation, and antibody mapping data obtained from
experiments using mAbs AZN-L50 and J4-81 (Fig. 2 and Table I) suggest
that the membrane-proximal domains C2C3 and the
amino-terminal domain V1 are directly involved in
homophilic ALCAM-ALCAM interactions. To further explore the role of
domain V1 in ALCAM-mediated aggregation, the series of
amino-terminally truncated ALCAM mutants expressed in the
ALCAM-negative human melanoma cell line 530 were subjected to flow
cytometry to determine the cell surface expression of the truncated
ALCAM molecules, using mAb AZN-L50 that recognizes the
membrane-proximal domains C2C3 of ALCAM (Fig.
3A). The ALCAM mutant lacking
domains V1V2C1 did
not localize at the cell surface; therefore, this mutant was not
included in the subsequent aggregation assay. Deletion of domain
V1 reduced the aggregation capacity to the background
levels observed in the control 530 cells transfected with empty vector
(Fig. 3B). In addition, the cell lines expressing truncated
ALCAM did not aggregate with cells expressing wild-type ALCAM (not
shown). From these findings, we conclude that domain V1
is essential in mediating homophilic ALCAM interactions.
Binding of soluble ALCAM-Fc by ALCAM-expressing cells is a sensitive
assay to assess the ALCAM ligand binding capacity, i.e. the
affinity. The ligand binding capacity of the amino-terminally truncated
ALCAM molecules was compared with that of wild-type ALCAM. In line with
the results of the cell aggregation assays, this assay also revealed
that deletion of the amino-terminal domain V1 from the cell
surface-expressed ALCAM
(530/V2C1C2C3)
completely abolished binding of soluble ALCAM-Fc as compared with 530 melanoma cells ectopically expressing wild-type ALCAM
(530/V1V2C1C2C3) (Fig. 3C). Moreover, 530 cells expressing amino-terminally
truncated ALCAM did not adhere to immobilized ALCAM-Fc at all, while
530 cells expressing wild-type ALCAM were readily activated to adhere to ALCAM-Fc by the addition of mAb J4-81 (Fig. 3D).
Furthermore, the function-blocking mAb AZN-L50 (10 µg/ml), which maps
to the membrane-proximal domains C2C3, did not
affect soluble ligand binding affinity by K-ALCAM cells (Fig.
4), whereas it completely inhibited
aggregation of these cells in suspension (Fig. 2A) at the
same concentration. Although the half-maximum ligand-binding value
moderately increased from 1.5 to 3.5 µg/ml in the presence of
AZN-L50, this slight decrease in ligand binding cannot account for the
complete AZN-L50-induced inhibition of cellular aggregation of
K- ALCAM cells in suspension. In conclusion, the membrane-distal domain V1 is critically involved in ligand binding and in
mediating stable ALCAM mediated cell-cell adhesion.
Involvement of the Membrane-proximal Domains
C2C3 of ALCAM in Homophilic Cell-Cell
Interactions--
In addition to the involvement of domain
V1 in ALCAM-mediated aggregation and adhesion, the specific
inhibition of homophilic ALCAM interactions by mAb AZN-L50 suggests
that the membrane-proximal domains C2C3 might
be equally important in the formation of stable cell-cell interactions.
Previously, it was shown that increased clustering of ALCAM molecules
at the cell membrane enhanced avidity, which was required for stable
ALCAM-mediated adhesion (21). To explore the effects of partly deleting
the ligand binding domain in these ALCAM complexes on ALCAM-mediated
cell aggregation, amino-terminally truncated ALCAM
(C1C2C3 = Heterophilic interactions between ALCAM and CD6 have been
extensively studied and mapped (18, 26). In contrast, the molecular basis for homophilic ALCAM-ALCAM interactions has remained largely elusive. Functional homophilic ALCAM-mediated adhesion was demonstrated for melanoma cells (19) and hematopoietic cells (8). Recently, we have
shown that ALCAM-mediated homophilic adhesion is dynamically regulated
through the actin cytoskeleton (21). However, the involvement of
specific domains in ALCAM-ALCAM-mediated adhesion had not been
addressed yet.
Here we have shown that wild-type ALCAM
(V1V2C1C2C3)
is bimodular, consisting of a distinct ligand binding module comprising the membrane-distal domain V1 and an oligomerization module
comprising the membrane-proximal C-type domains
C2C3. Both modules are required for stable cell
adhesion and aggregation. We propose a molecular model that accounts
for the observed properties of homophilic ALCAM interactions (Fig.
6).
Using a series of progressively amino-terminally truncated ALCAM
mutants, we found that the amino-terminal domain V1 is
critically involved in the homophilic interaction. Deletion of domain
V1 not only disrupted the homophilic ALCAM-mediated
cell-cell interaction but also completely prevented binding of soluble
wild-type ALCAM-Fc. These observations indicated that a direct and
exclusive interaction between two opposing domains V1 is
crucial for homophilic ALCAM-mediated adhesion. If multiple Ig domains
were directly involved in the homophilic ALCAM interaction, cells
expressing an ALCAM construct that solely lacks domain V1
would be expected to display a residual capacity to aggregate with
cells expressing wild-type ALCAM. Moreover, truncated ALCAM-expressing
cells would be expected to bind soluble wild-type ALCAM, albeit at
lower affinity, if additional domains were involved in ligand binding.
However, both cell-cell aggregation and soluble ligand binding were
completely abolished upon deletion of the amino-terminal domain
V1. Furthermore, mAb AZN-L50, which maps to the
membrane-proximal domains C2C3, only slightly
decreased the soluble ligand binding affinity, whereas it completely
inhibited aggregation of these cells in suspension at the same
concentration (10 µg/ml). It is therefore unlikely that the slight
decrease in soluble ligand binding (i.e. increase in
half-maximal ligand-binding value from 1.5 to 3.5 µg/ml in the
presence of AZN-L50) accounted for the total inhibition of
ALCAM-mediated cellular aggregation via AZN-L50. Thus, these data
indicate that ligand binding in itself is independent of the C-type Ig
domains and that homophilic ALCAM-ALCAM trans-interactions
are exclusively mediated by binding of opposing amino-terminal
V1 domains.
mAb J4-81, which specifically recognizes domain V1,
enhanced homophilic ALCAM-mediated cell adhesion and aggregation,
possibly by inducing a conformational change that promotes ligand
binding. KG1 cells with abundant ALCAM cell surface expression were
only able to form homotypic cell clusters after activation by mAb
J4-81. In contrast, spontaneous cell clustering of K562 cells with
ectopic ALCAM expression could not be further enhanced by J4-81
treatment. These findings suggest differential activation of ALCAM in
these two cell lines. We could exclude the possibility that mAb
J4-81-enhanced aggregation and adhesion to immobilized ALCAM-Fc is
caused by cross-linking ALCAM molecules of opposing cells, because the
stimulatory effect of mAb J4-81 was temperature-dependent
and efficiently inhibited by the blocking anti- ALCAM mAb AZN-L50.
The observed enhancement of homophilic ALCAM-mediated adhesion and
aggregation by mAb J4-81 is in contrast with the inhibitory effect of
this antibody on the heterophilic ALCAM-CD6 interaction (25). This indicates that different mechanisms are regulating the homophilic and
heterophilic ALCAM ligand binding interactions.
The inhibitory effect of AZN-L50, which is mapped to the
membrane-proximal domains C2C3, indicated that
these domains are also essential for the homophilic ALCAM-mediated
interaction. In analogy with other Ig superfamily molecules like IgG,
major histocompatibility complex class II, NCAM (27), and Ng-CAM/L1-CAM (23), these C-type Ig domains are probably involved in the formation of
cis-homo-oligomers at the cell surface via lateral
oligomerization. Aruffo and co-workers (28) have speculated that ALCAM
oligomerization might be essential for the heterophilic ALCAM-CD6
interaction. Recently, we have shown that homophilic ALCAM-mediated
cell adhesion is regulated through actin
cytoskeleton-dependent clustering of ALCAM molecules at the
cell surface. The resulting increased avidity of ALCAM clusters is
essential to obtain stable adhesive interactions (21). Here we show
that expression of Since the epitope for ALCAM-blocking mAb AZN-L50 is localized in
domains C2C3, it is tempting to speculate that
the inhibitory effect of this mAb is a result of prohibited lateral
oligomerization of ALCAM molecules by steric hindrance. This notion is
supported by the observation that mAb AZN-L50 does not affect soluble
ligand binding (Fig. 4) and therefore only modulates ALCAM avidity.
Although inhibition of cell aggregation via In conclusion, we have demonstrated that ALCAM-mediated homophilic
interactions most likely require lateral homo-oligomerization through
the membrane-proximal C-type Ig domains, while the membrane-distal Ig
domain V1 is exclusively involved in ligand binding.
Coordinate oligomerization and ligand binding leads to the formation of
a tight, bilayered ALCAM network, enabling stable adhesive interactions.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
These two authors contributed equally to this work.
**
To whom correspondence should be addressed. Tel.: 31-24-3616619;
Fax: 31-24-3540525; E-mail: G.Swart@bioch.kun.nl.
Published, JBC Papers in Press, April 16, 2001, DOI 10.1074/jbc.M011272200
The abbreviations used are:
ALCAM, activated
leukocyte cell adhesion molecule;
FITC, fluorescein isothiocyanate;
mAb, monoclonal antibody;
NCAM, neural cell adhesion molecule.
Molecular Basis for the Homophilic Activated Leukocyte
Cell Adhesion Molecule (ALCAM)-ALCAM Interaction*
§,,
,,**
University of Nijmegen, Department of
Biochemistry, P. O. Box 9101, NL-6500 HB Nijmegen, The Netherlands,
the ¶ Department of Tumor Immunology, University Medical Center
St. Radboud, Nijmegen, The Netherlands, and Roche Diagnostics
GmbH, Nonnenwald 2, D-82372 Penzberg, Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Construction of NH2-terminally
truncated ALCAM mutants. Using the polymerase chain reaction and
standard cloning procedures, deletion mutants of ALCAM were made as
described under "Materials and Methods." Primers P9, P8, and P7
contained a PstI, BalI, and NheI
restriction site respectively, which facilitated cloning of the
generated fragment into double-digested ALCAM cDNA (pWD201) at the
SstI and the respective restriction enzyme site.
V1 and V2, V-type domains;
C1, C2, and
C3, C-type domains; TM, transmembrane
domain; cyto, cytoplasmic tail; P7,
P8, and P9, primers 7-9; Rev/T3,
reverse T3 primer.
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Monoclonal anti-ALCAM antibodies have
differential effects on ALCAM-mediated cell aggregation and
adhesion. A, phase-contrast microscopy. Ectopic ALCAM
expression in erythroleukemic K562 cells (K562-ALCAM) induced cell
aggregation in culture, whereas vector control K562 cells did not
aggregate. mAb J4-81(5 µg/ml) could not further enhance this
spontaneous aggregation, but the ALCAM-mediated aggregation was
efficiently inhibited by the mAb AZN-L50 (10 µg/ml). Both antibodies
had no effect on vector control K562 cells. Aggregation of wild-type
ALCAM-expressing myelomonocytic KG1 cells significantly increased upon
treatment with mAb J4-81. This J4-81-induced cell aggregation was
completely inhibited with mAb AZN-L50. B, cell-cell
aggregation in the absence or presence of an anti-ALCAM mAb was
quantified by two-color flow cytometry. mAb J4-81 (5 µg/ml) increased
the cell aggregation of the ectopically ALCAM-expressing human melanoma
cell line 530/ALCAM with 35% as compared with the untreated cells,
while mAb AZN-L50 (5 µg/ml) reduced this aggregation with 25% with
respect to the untreated control. Neither of the antibodies had any
effect on the ALCAM-negative vector control cell line. Increasing
concentrations of mAb AZN-L50 up to 10 µg/ml did not further reduce
the cellular aggregation (not shown). C, myelomonocytic KG1
cells were allowed to adhere to immobilized recombinant human ALCAM-Fc,
in the presence or absence of the ALCAM mAb J4-81 (5 µg/ml) or
AZN-L50 (10 µg/ml) at the indicated temperatures, and the percentage
of adhering cells was determined. ALCAM-mediated cell adhesion was
strongly induced by mAb J4-81. This J4-81-induced adhesion was
efficiently inhibited when tumor cells were incubated with mAb AZN-L50
(10 µg/ml) prior to adhesion. mAb-induced adhesion required
physiological temperature, since it was clearly inhibited at lower
temperatures, indicating that J4-81 induction of adhesion is not due to
cross-linking of opposing ALCAM molecules.
Immunofluorescence-based epitope mapping of two monoclonal
anti-ALCAM antibodies
View larger version (33K):
[in a new window]
Fig. 3.
The amino-terminal V-type Ig domain
(V1) is essential for ligand binding and ALCAM-mediated
cell aggregation. A, cell surface expression levels of
wild-type ALCAM and truncated ALCAM mutants in the ALCAM-negative human
melanoma cell line 530 were determined by flow cytometry employing the
mAb AZN-L50 (4 µg/ml), which recognizes all mutants, since its
epitope resides in the membrane-proximal domains
C2C3. Open histograms
represent the isotype control staining, and the gray
histograms represent the AZN-L50 staining of wild type and
truncated ALCAM. The constructs lacking the first or both V-type Ig
domains, V1C1C2C3 or
C1C2C3 respectively, were expressed
at the cell surface. The construct C2C3 lacking
both V-type Ig domains and the first C-type Ig domain did not localize
to the cell surface. B, cell lines expressing wild-type or
mutant ALCAM were subjected to an aggregation assay and quantified by
flow cytometry. Wild-type or NH2-terminally truncated ALCAM
molecules were expressed in the ALCAM-negative melanoma cell line 530. Cells expressing wild type ALCAM aggregated strongly, while truncation
of the first amino-terminal domain V1 already completely
abrogated cell aggregation. Subsequent deletion of more domains did not
have an additional effect on inhibition of adhesion. C,
cells
(530/V1V2C1C2C3,
530/truncated ALCAM) were incubated with soluble ALCAM-Fc at 37 °C,
and bound ALCAM-Fc was detected with a FITC-conjugated anti-human-Fc
antibody. The percentage of cells that had bound ligand was determined
by flow cytometry. The ectopically ALCAM-expressing cell line
530/V1V2C1C2C3
could efficiently bind soluble ALCAM-Fc in a
concentration-dependent manner. In contrast, ALCAM-negative
530 vector control cells (530) and 530 cells expressing truncated ALCAM
(530/V2C1C2C3 and
530/C1C2C3) did not bind soluble
ALCAM-Fc. D, 530 cells, or 530 cells expressing wild-type or
truncated ALCAM mutants were allowed to adhere to immobilized
recombinant human ALCAM-Fc, in the presence or absence of the ALCAM mAb
J4-81 (5 µg/ml) or AZN-L50 (10 µg/ml), and the percentage of
adhering cells was determined. Wild-type ALCAM-expressing 530 cells
were readily activated to adhere to ALCAM-Fc by the addition of mAb
J4-81, and J4-81-induced adhesion was inhibited by the addition of
AZN-L50. ALCAM mutants lacking the membrane-distal domain
V1 or control-transfected 530 control cells did not adhere
to immobilized ALCAM-Fc, and adhesion could not be induced by the
addition of J4-81.
View larger version (24K):
[in a new window]
Fig. 4.
Aggregation blocking mAb AZN-L50 slightly
reduced the soluble ALCAM-Fc ligand-binding capacity of K562-ALCAM
cells. Cells (K562-ALCAM) were incubated with soluble ALCAM-Fc in
the presence or absence of the aggregation-blocking mAb AZN-L50 (10 µg/ml). The percentage of cells that have bound ALCAM-Fc was
determined by flow cytometry using a FITC-conjugated anti-human-Fc
antibody. mAb AZN-L50 moderately increased the half-maximum
ligand-binding capacity from 1.5 to 3.5 µg/ml, indicative of a
marginally reduced ligand binding capacity.
N-ALCAM) was
introduced into BLM melanoma cells expressing wild-type ALCAM by stable
transfection. Three independently isolated transfected cell clones
(BLM/N-ALCAM-1, BLM/N-ALCAM-2, and BLM/N-ALCAM-3) with
different N-ALCAM expression levels were selected for further
analysis. Overexpression of N-ALCAM led to a decreased aggregation
capacity of the BLM cells in a dose-dependent manner (Fig.
5, A and B),
despite unaltered expression levels of wild-type ALCAM molecules (Fig.
5A). Therefore, elimination of domains
V1V2 of ALCAM generates a dominant negative
ALCAM molecule with respect to ALCAM-mediated homophilic cell
aggregation when introduced in cells expressing wild-type ALCAM. To
analyze whether the introduction of N-ALCAM influenced the ligand
binding affinity of wild-type ALCAM, the soluble ligand binding
capacity of wild-type ALCAM-expressing BLM cells co-expressing
N-ALCAM was compared with parental BLM cells. The BLM cell lines
expressing N-ALCAM did not show a reduction in their capacity to
bind soluble ALCAM-Fc compared with the control BLM cells transfected
with the empty expression vector (BLM) (Fig. 5C). Control
cells (BLM) and cells transfected with truncated ALCAM (BLM/N-ALCAM)
displayed similar apparent half-maximal ligand-binding values (1.6 and
1.3 µg/ml, respectively). This strongly indicates that the decreased
aggregation capacity of the N-ALCAM-expressing cells is due to a
decrease in wild-type ALCAM avidity rather than a decrease in the
ligand binding capacity.
View larger version (19K):
[in a new window]
Fig. 5.
N-ALCAM
(C1C2C3), expressed in the
wild-type ALCAM-expressing cell line BLM, reduced cell aggregation
in a dose-dependent fashion without affecting soluble
ligand binding affinity. Cell clones overexpressing
amino-terminally truncated ALCAM (N-ALCAM) were generated from the
parental cell line BLM with endogenous ALCAM expression by stable
transfection. Three independently isolated cell clones
(BLM/N-ALCAM-1, -2, and-3) expressing different levels of N-ALCAM
were selected for further analysis. A, expression levels of
the truncated molecule were determined by Western blotting using mAb
AZN-L50 (3 µg/ml) that recognizes the membrane-proximal Ig domains
C2C3. The three established cell lines showed
different expression levels of the truncated ALCAM molecule, while
expression of the wild-type ALCAM molecule remained unaltered.
B, the established cell lines expressing truncated ALCAM
(BLM/N-ALCAM -1, -2, and -3) were subjected to an in
vitro aggregation assay and analyzed by two-color flow cytometry.
Expression of truncated ALCAM reduced the aggregation capacity of these
cells as compared with the vector control cell line (BLM/CTRL) in a
dose-dependent manner, and thus truncated ALCAM
(C1C2C3) functions as a dominant
negative molecule with respect to wild-type ALCAM mediated aggregation.
C, cells (BLM, BLM/N-ALCAM-1, -2, and -3) were incubated
with soluble ALCAM-Fc. The percentage of cells that have bound
ALCAM-Fc was determined by flow cytometry using a FITC-conjugated
anti-human Fc antibody. Ectopic N-ALCAM expression in BLM cells with
wild-type ALCAM did not affect the capacity to bind soluble ALCAM-Fc
ligand, demonstrating that ectopic expression of N- ALCAM does not
change the affinity of endogenously expressed ALCAM for soluble
ALCAM-Fc.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (39K):
[in a new window]
Fig. 6.
Model for the oligomeric homophilic
ALCAM-ALCAM interaction. A, the membrane-proximal
C-type Ig domains are involved in lateral oligomerization of ALCAM,
which is inhibited with the anti-C2C3 mAb
AZN-L50, while the NH2-terminal domain V1
mediates the actual homophilic interaction. Deletion of domain
V1 resulted in a molecule that cannot bind soluble ALCAM
and, therefore, has lost the ability to mediate cell aggregation. For
reasons of simplicity, the formation of a dimer is shown, but the
actual mechanism could involve the formation of higher oligomers of
multiple ALCAM molecules, creating a two-dimensional lattice.
B, a model for the dominant negative effect of truncated
ALCAM. Overexpression of C1C2C3
ALCAM in cells already expressing wild type ALCAM results in the
formation of hetero-oligomers of wild-type and truncated ALCAM
molecules and subsequently interferes in the tight ALCAM network
formation because only isolated ligand binding domains V1
are exposed instead of oligomers. Expression of the truncated molecule
will reduce but not completely inhibit the ALCAM-mediated aggregation
via the Ig domain V1, because ligand-binding modules are
still provided by the endogenously expressed wild-type ALCAM.
N-ALCAM, which lacks the ligand binding domain,
in the wild-type ALCAM-expressing BLM cells reduced cell aggregation
but not soluble ligand binding. This indicates that N-ALCAM
expression changes wild-type ALCAM avidity rather than affinity,
suggesting a direct interaction of N-ALCAM with wild-type ALCAM
without disturbing soluble ligand binding. In the model proposed in
Fig. 6A, ALCAM monomers form lateral oligomers via their
membrane-proximal C-type domains, whereas the amino-terminal domain
V1 mediates the actual ligand binding. Triggering of
lateral oligomerization might be a ligand-induced event, which possibly
occurs through a conformational change of ALCAM. This concept is
supported by previous findings that in response to ligand binding, the
linkage of ALCAM to the actin cytoskeleton is strengthened (21), which
can stabilize oligomers at the cell surface. Similar to ligand-induced
clustering, mAb J4-81 might induce a conformational change that
triggers oligomerization.
N-ALCAM was dependent on
its expression level, aggregation could not completely be reduced to
background levels. This residual cell-cell aggregation capacity is
consistent with the proposed model because some homophilic ALCAM-mediated cell-cell interactions are maintained (Fig.
6B).
FOOTNOTES
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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