(Received for publication, February 6, 1996)
From the
PECAM-1 (CD31) is a 130-kDa member of the immunoglobulin (Ig) gene superfamily that is constitutively expressed at high concentration at endothelial cell intercellular junctions and at moderate density on the surface of circulating leukocytes and platelets. Recent in vitro and in vivo studies have shown that PECAM-1 plays a central role in mediating the extravasation of leukocytes from the vessel wall in response to inflammatory mediators. To study the binding characteristics of PECAM-1, phospholipid vesicles were prepared and examined by flow cytometry and immunofluorescence microscopy for their ability to associate with each other and with cells. Proteoliposomes containing high concentrations of PECAM-1 interacted homophilically with each other, forming large self-aggregates. PECAM-1 proteoliposomes, as well as soluble bivalent PECAM-1 in the form of a PECAM-1/IgG immunoadhesin, associated homophilically with cells expressing human, but not murine, PECAM-1. This binding could be completely inhibited by monoclonal antibody Fab fragments specific for Ig homology Domain 1 or Domains 1 + 2. Binding studies using cells expressing human PECAM-1 deletion mutants and murine/human chimeras confirmed that both Ig Domains 1 and 2 were both necessary and sufficient for homophilic binding. In contrast, engagement of membrane-proximal Domain 6 with monoclonal antibody Fab fragments had the opposite effect and augmented the binding of PECAM-1 proteoliposomes to cells. Thus, PECAM-1, like certain integrins, appears to be capable of antibody-induced conformational changes that alter affinity for its ligand. Similar changes induced by physiologic stimuli could be important in regulating the function of PECAM-1 in vascular cells.
PECAM-1 (CD31) is a 130-kDa member of the immunoglobulin (Ig) gene superfamily (1, 2, 3) that is constitutively expressed at high concentration at endothelial cell intercellular junctions (4) and at moderate density on the surface of circulating leukocytes and platelets (for a recent review, see (5) ). Recent in vitro(6) and in vivo(7, 8) studies have shown that PECAM-1 plays a central role in mediating the extravasation of leukocytes from the vessel wall in response to inflammatory mediators, as transendothelial migration is markedly inhibited in the presence of anti-PECAM-1 antibodies or soluble recombinant PECAM-1. Although PECAM-1 can become phosphorylated on cytoplasmic domain serine residues following cellular activation(9) , the precise way in which circulating leukocytes and the underlying endothelial cell barrier regulate the adhesive state of PECAM-1 to control cell-cell interactions remains poorly understood.
Previous studies suggest that PECAM-1 may be capable of interacting
with multiple cellular targets. The early finding that PECAM-1
localizes to cell-cell borders of monolayer cells in culture only when a PECAM-1-transfected cell is in contact with another cell
expressing PECAM-1 (3) suggested, but did not prove, that
PECAM-1 may be capable of interacting homophilically with itself. Later
studies (10) showed that PECAM-1-transfected L-cells were
capable of interacting with nontransfected L-cells in a
calcium-dependent manner, thus implicating a heterophilic adhesion
mechanism for PECAM-1 as well. Cell surface glycosaminoglycans were
later shown to serve as at least one cellular target for PECAM-1
mediated L-cell aggregation(11) . More recently, the integrin
has been proposed as a candidate
counter-receptor for PECAM-1 heterophilic interactions(12) ,
but these studies await further confirmation.
In the present study, we have used fluorescently labeled proteoliposomes containing highly purified PECAM-1, and a recombinant PECAM-1/IgG chimeric protein (PECAM/Ig) containing the complete extracellular domain of PECAM-1, to further examine the molecular mechanisms involved in PECAM-1-mediated cellular adhesive events. Our results indicate that the primary cellular target for human PECAM-1 is PECAM-1 and that Ig homology Domains 1 and 2 mediate PECAM-1 homophilic adhesion. In addition, Ig Domain 6 may play a heretofore unexpected regulatory role in modulating the adhesive properties of PECAM-1.
Figure 1:
Adhesive properties of
PECAM-1 proteoliposomes. A, SDS-PAGE analysis of PECAM-1 and
GPIIb-IIIa incorporated into phospholipid vesicles. PECAM-1 (lane
1) was purified from human platelet detergent lysates by
immunoaffinity chromatography, while GPIIb-IIIa (lane 2) was
prepared using KYGRGDS-Sepharose, as described under
``Experimental Procedures.'' Purified proteins were analyzed
on a silver-stained 4-15% gradient polyacrylamide gel. The
positions of molecular mass standards are shown at left in
kilodaltons. Homophilic aggregation of PECAM-1-containing
proteoliposomes. PECAM-1 (top) and GPIIb-IIIa (bottom) proteoliposomes of varying protein/lipid ratios were
prepared by detergent dialysis and viewed by fluorescence microscopy at
400 magnification. Note that NBD-labeled proteoliposomes having
PECAM-1-to-lipid ratios of greater than 100 µg/mg PC spontaneously
formed large aggregates, while vesicles containing the integrin
GPIIb-IIIa remained evenly dispersed in solution. C,
association of PECAM-1 proteoliposomes with HUVECs. NBD-labeled PECAM-1
proteoliposomes, prepared in the presence of 40 µg of PECAM-1/mg of
PC, were incubated with HUVECs at 22 °C for 90 min and their
association examined by fluorescence (right) and phase
contrast (left) microscopy at
400 magnification.
GPIIb-IIIa proteoliposomes (top panels) were employed as a
control. D, flow cytometric analysis of the association of
PECAM-1 proteoliposomes with HUVECs. NBD-labeled liposomes containing
either no protein (bottom left), GIIb-IIIa (top
right), or PECAM-1 (bottom right) were mixed incubated
with cells as described above and bound fluorescence quantitated by
flow cytometry. Note that the binding of GPIIb-IIIa proteoliposomes was
only slightly greater than that of the no-protein vesicles, while
PECAM-1 proteoliposomes associated significantly with
cells.
Since PECAM-1 vesicles containing only 40 µg of protein/mg of lipid remained largely monomeric in solution (Fig. 1B, middle panel) we used these in all subsequent cell association assays. Such PECAM-1 proteoliposomes bound HUVECS, as determined by both immunofluorescence microscopy (Fig. 1C) and by flow cytometric analysis (Fig. 1D).
To examine potential cellular targets for PECAM-1, murine L-cells and L-cells stably expressing PECAM-1 (Fig. 2A) were also examined for their relative ability to support binding of PECAM-1 proteoliposomes. PECAM-1 proteoliposomes bound to HUVECS and PECAM-1 transfected L-cells (Fig. 2B), but failed to associate with nontransfected L-cells. Additional studies (not shown) revealed that PECAM-1 proteoliposomes associated with other PECAM-1-positive cell lines, such as U937 cells, but not with cells lines (COS, CHO) that did not express PECAM-1. Thus, PECAM-1 proteoliposome binding appears to require cell surface expression of PECAM-1. These findings were confirmed in parallel studies that employed soluble bivalent PECAM-1 in the form of a PECAM-1/IgG construct. As shown in Fig. 2B, PECAM-1/IgG bound well to both HUVECS and PECAM-1-transfected L-cells, but failed to associate with nontransfected L-cells. Interestingly, the binding of both PECAM-1 phospholipid vesicles and PECAM-1/IgG was independent of the presence of divalent cations, as addition of EDTA had no effect on their association. Thus, the mechanism by which purified PECAM-1 associates with cells differs significantly from that implied by the aggregation of PECAM-1-transfected L-cells, in which PECAM-1-transfected L-cells were found to aggregate in a calcium-dependent manner and interacted as readily with nontransfected cells(3, 10) . Furthermore, addition of heparin or heparan sulfate had no effect on the binding of either PECAM-1 proteoliposomes or PECAM-1/IgG to HUVECS or PECAM-1-transfected L-cells (not shown), suggesting that cell surface glycosaminoglycans are incapable of supporting the binding of purified human PECAM-1.
Figure 2:
Homophilic adhesion of PECAM-1. A, cell surface expression of PECAM-1. The indicated cells
were removed from culture flasks with 10 mM EGTA, washed,
stained with PECAM-1.3 monoclonal antibody (40 µg/ml) at 4 °C
for 60 min, followed by FITC-conjugated goat anti-mouse IgG, and
analyzed by flow cytometry. Shown are nontransfected murine L-cells
that do not express PECAM-1, HUVECs, and PECAM-1-transfected L-cells.
Normal mouse IgG (NMIgG, 40 µg/ml) was used as a control. B, homophilic association of PECAM-1-containing
proteoliposomes and PECAM-1/IgG chimeric protein with cells expressing
PECAM-1. For the vesicle binding experiments, cells were incubated with
PECAM-1 proteoliposomes (prepared in the presence of 40 µg of
PECAM-1/mg of PC) and analyzed by flow cytometry as described in the
legend to Fig. 1. GPIIb-IIIa proteoliposomes (40 µg of
GPIIb-IIIa/mg of PC) were used as a control. PECAM-1 proteoliposomes
bound to HUVECs and PECAM-1-transfected L-cells, but not to
nontransfected L-cells. Addition of 2.5 mM CaCl had no beneficial effect on proteoliposome binding. For the
PECAM-1/IgG experiments, cells were incubated with PECAM-1/IgG (20
µg/ml), followed by FITC-conjugated goat anti-human IgG, and
analyzed by flow cytometry. Normal human IgG (NHIgG, 20
µg/ml) was used as the control. Consistent with the cell binding of
PECAM-1 proteoliposomes, PECAM-1/IgG bound HUVECs and PECAM-1
transfected L-cells, but did not associate with nontransfected
L-cells.
Figure 3: PECAM-1 homophilic adhesion is mediated by Ig Domains 1 and 2. The interaction of either PECAM-1 proteoliposomes (A) or PECAM-1/IgG (B) with cells was measured in the presence of the indicated monoclonal antibody fragments (Fabs). Fabs specific for Ig homology Domain 1 (PECAM-1.3) or Domains 1 + 2 (hec7.2) were inhibitory down to background levels, whereas Fabs specific for Ig Domain 5 had no effect. Inhibition by PECAM-1.3 and hec7.2 occurred whether Fabs were added to the liposomes (A, left) or preincubated with cells (A, right). Antibodies bound to Ig Domains 1 or 1 + 2 effectively blocked adhesion of PECAM-1/IgG whether HUVECs (B, left) or PECAM-1-transfected L-cells (B, right) were used as target cells.
To further investigate
the involvement of the amino terminus in mediating PECAM-1 homophilic
interactions, we examined the binding of human PECAM-1/IgG to L-cells
expressing an isoform of PECAM-1 lacking Ig Domain 1 (1 cells). As
shown in Fig. 4, L-cells expressing wild-type full-length
PECAM-1 bound the murine monoclonal antibodies PECAM-1.2 (Domain 6) and
PECAM-1.3 (Domain 1), while the PECAM-1
1 L-cells bound PECAM-1.2,
but failed to react with PECAM-1.3, as would be predicted. PECAM-1
proteoliposomes bound normally to L-cells expressing full-length
PECAM-1, but did not associate with surface-expressed PECAM-1 that
lacked Ig Domain 1 (Fig. 4B). Moreover, PECAM-1/IgG
failed to interact with isoforms of PECAM-1 that lacked either Ig Domains 1 or 2 (Table 2), suggesting that both NH
-terminal Ig homology domains might participate in
the formation of the homophilic contact site.
Figure 4:
PECAM-1 homophilic adhesion requires Ig
Domain 1. A, expression of PECAM-1 on the surface of
transfected L-cell lines. PECAM-1-transfected L-cells expressing either
full-length PECAM-1, or a recombinant isoform missing
NH-terminal Ig homology Domain 1 (PECAM-1
1), were
harvested with 10 mM EGTA in HBSS, washed, and incubated with
either PECAM-1.3 (specific for Ig Domain 1) or PECAM-1.2 (specific for
Ig Domain 6). Normal mouse IgG (NMIgG) was used as the
control. The cells were then washed, stained with FITC-conjugated goat
anti-mouse IgG, and analyzed by flow cytometry. Note the PECAM-1
(
1) L-cells bound PECAM-1.2 normally, but failed to react with
PECAM-1.3. B, PECAM-1 proteoliposomes and PECAM-1 IgG each
bound normally to L-cells expressing full-length PECAM-1, but failed to
bind to to the PECAM-1 isoform lacking Ig Domain 1, demonstrating that
the amino terminus of the molecule is required for PECAM-1 homophilic
interactions.
To rule out the possibility that deletion of an entire Ig homology domain inadvertently resulted in conformational changes that led to ``loss of function,'' we constructed and expressed in murine L-cells a series of human/murine chimeric proteins that much more closely resembled wild-type PECAM and examined their ability to interact homophilically with human PECAM-1/IgG. As shown in Table 2, human PECAM-1/IgG failed to bind murine PECAM-1 expressed on the surface of L-cells, demonstrating species specificity for PECAM-1 homophilic interactions and permitting us to splice selected regions of human PECAM-1 onto the unreactive murine backbone to search for ``gain of function'' adhesive phenotypes. Substitution of murine Ig Domains 1 + 2, or murine Domain 2 alone, into a human PECAM-1 backbone resulted in loss of human PECAM-1/IgG binding, consistent with a role for both of these domains in mediating homophilic adhesion. Finally, a chimeric construct consisting of human PECAM-1 Ig Domains 1 + 2 fused to a murine PECAM-1 backbone (Domains 3-6) restored homophilic binding. Together with the domain-specific antibody blocking studies shown in Fig. 3, our data strongly implicate Ig homology Domains 1 and 2 as both necessary and sufficient for the formation of the primary homophilic contact site of PECAM-1.
Figure 5:
Endothelial cell
is incapable of supporting the
binding of PECAM-1. A, expression of the integrin
on HUVECs. HUVECs were incubated
with the
complex-specific antibody,
LM609, the
-integrin subunit-specific antibody, AP3,
or PECAM-1.3 at 4 °C for 60 min, followed by FITC-conjugated goat
anti-mouse IgG, and analyzed by flow cytometry. NMIgG at 40 µg/ml
was used as the control. LM609 is a blocking antibody which inhibits
the interaction of
with vitronectin
receptor as described previously(16) . B,
anti-
antibodies do not inhibit the
association of PECAM-1 proteoliposomes with HUVECs. HUVECs were
preincubated with 40 µg/ml of the indicated monoclonal antibodies
at 22 °C for 30 min, washed, and used as the target cells for
PECAM-1 proteoliposome binding, which was performed as described under
``Experimental Procedures.'' NMIgG at 40 µg/ml was used
as a negative control. Note that PECAM-1.3 (specific for Ig homology
Domain 1) completely blocked the association of PECAM-1 proteoliposomes
with HUVECs to background levels, suggesting that cell surface PECAM-1
serves as the sole target for homophilic PECAM-1
interactions.
Like many integrins, PECAM-1 is constitutively expressed on the surface of vascular cells that, under normal conditions, do not interact with each other. To determine whether PECAM-1 might be capable of undergoing conformational changes that result in increased adhesiveness, we attempted to modulate the affinity of PECAM-1 proteoliposomes for cells using murine monoclonal antibodies that bind to domains of PECAM-1 not directly involved in homophilic adhesion. Fab fragments were used to avoid potential activation of Fc receptors. As shown in Fig. 6A, when Fab fragments of PECAM-1.2 and 4G6, each specific for Ig Domain 6, were prebound to PECAM-1 proteoliposomes, binding to HUVECS increased 2-4-fold. The increased association of PECAM-1 proteoliposomes with the cell surface was dependent upon the concentration of ``activating'' Fab used (Fig. 6B) and could be completely inhibited by preincubation of the cells with the Ig Domain 1-specific antibody, PECAM-1.3 (not shown). Not all anti-PECAM-1 antibodies induced enhanced binding, as increasing concentrations of Domain 1-specific Fabs (PECAM-1.3, hec7.2) alone resulted in inhibition of proteoliposome binding down to background fluorescence levels. Thus, PECAM-1, like certain integrins, appears to be capable of undergoing antibody-induced conformational changes that alter affinity for its ligand. For full-length, naturally occurring human PECAM-1 presented in the context of a phospholipid bilayer, that ligand is PECAM-1.
Figure 6: Engagement of Ig homology Domain 6 modulates PECAM-1 adhesive interactions. A, monoclonal antibodies specific for Ig Domain 6 increase the association of PECAM-1 proteoliposomes with HUVECs. PECAM-1 proteoliposome binding to HUVECs was performed as described under ``Experimental Procedures,'' with 200 µg/ml of the indicated monoclonal Fabs added to cell/liposome mixture. Protein-free fluorescent PC vesicles, as well as GPIIb-IIIa proteoliposomes, were used as controls for nonspecific binding. 100% binding was taken as the median fluorescence intensity obtained as a result of the association of PECAM-1 proteoliposome with HUVECs in the absence of anti-PECAM-1. Note that the binding of two different monovalent Fabs specific for Ig homology Domain 6 (PECAM-1.2 and 4G6) resulted in increased PECAM-1 proteoliposome binding to HUVECs. B, dose-dependent modulation of PECAM-1 adhesive interactions. HUVECs were incubated with PECAM-1 proteoliposomes in the presence of various concentrations of anti-PECAM-1 monoclonal Fabs. NMIgG Fab fragments were employed as a control. Note that Ig Domain 6 Fabs enhanced binding in a dose-dependent manner, while Ig Domain 1 Fabs inhibited binding down to background fluorescence levels.
In the present study, we have employed a well defined system
using isolated adhesion molecules in the form of proteoliposomes and
IgG chimeric constructs to investigate mechanisms by which human
PECAM-1 mediates cellular interactions. Each of these multivalent forms
offer the potential to achieve affinities hundreds of times greater
than monovalent soluble recombinant receptor
molecules(29, 30) . Our major findings may be
summarized as follows. First, full-length PECAM-1, when present at
sufficient concentration within the plane of the membrane, is capable
of interacting directly with itself in a divalent cation-independent,
homophilic manner (Fig. 1). Second, both phospholipid vesicles
expressing isolated, purified human platelet PECAM-1, and a chimeric
bivalent form of PECAM-1 expressed as an immunoadhesin molecule, bind
specifically to cells that express PECAM-1 (Fig. 2). Third,
these homophilic PECAM-1 interactions are mediated by the first two
NH-terminal Ig homology domains, Ig Domains 1 and 2 ( Fig. 3and Fig. 4). Fourth, other cellular targets,
including cell-surface glycosaminoglycans and surface-expressed
integrins (Fig. 5), are insufficient to support the direct
binding of purified human PECAM-1, even when it is presented in a
high-affinity, multivalent form. Finally, PECAM-1 appears to be the
first homophilic adhesion molecule capable of undergoing affinity
modulation, as engagement of membrane-proximal Ig Domain 6 results in
increased Domains 1 + 2-mediated homophilic binding (Fig. 6).
Cell-cell contacts between adjacent endothelial
cells are mediated by adhesion molecules belonging to several different
gene families, including the integrins and
(31) , at least two
members of the cadherin family(32) , and one member of the
immunoglobulin gene superfamily,
PECAM-1(1, 4, 33) . Previous studies have
shown that PECAM-1 is expressed diffusely on the surface of isolated
endothelial cells or PECAM-1-transfected fibroblasts and that upon
contact with another PECAM-1-expressing cell, the subcellular
distribution of PECAM-1 changes dramatically, with a majority of cell
surface PECAM-1 becoming localized to the intercellular
junction(3) . In contrast, PECAM-1 is not present at the
intercellular junctions formed by PECAM-1-transfected cells in contact
with PECAM-1-negative cells, leading to the hypothesis (3) that
PECAM-1 interacts homophilically with PECAM-1 on an adjacent cell to
effect its concentration at cell-cell borders. The possibility,
however, that an intermediary ligand might link PECAM-1 receptors on
neighboring cells, much the same as activated GPIIb-IIIa receptors on
adjacent platelets are bridged by the symmetrical ligand,
fibrinogen(34, 35) , has never been addressed. Our
finding that purified human PECAM-1 in a phospholipid bilayer interacts
1) directly itself in the absence of added proteins (Fig. 1) and
2) with PECAM-1 expressed on a cell surface ( Fig. 2and Fig. 3) would appear to rule out a requirement for intermediary
co-factors in homophilic PECAM-1 interactions.
In seeming contrast to the homophilic interactions demonstrated in Fig. 1Fig. 2Fig. 3Fig. 4, a number of studies have suggested that PECAM-1 may also be capable of interacting heterophilically with cell surface glycosaminoglycans(11, 36) . These data are derived from experiments in which PECAM-1-transfected murine L-cells were shown to bind, in a divalent cation-dependent manner, as readily to nontransfected L-cells as they do to each other(10, 11) . Aggregation of PECAM-1-transfected cells with nontransfected L-cells was inhibited in the presence of added heparin and other selected glycosaminoglycans, suggesting that glycosaminoglycans might serve as a cellular target for PECAM-1 heterophilic interactions. As shown in the present study, however, when presented in purified form, both human PECAM-1 proteoliposomes and human PECAM-1/IgG interacted with cells efficiently only when PECAM-1 was expressed on the cell surface and did not associate at all with nontransfected murine L-cell fibroblasts (Fig. 2B) or with other PECAM-1-negative cell lines. Binding to cell surface PECAM-1 was unaffected by heparin or RGD peptides and was divalent-cation-independent, providing compelling evidence that the major ligand for purified human PECAM-1 is PECAM-1.
Our findings do not rule out a role for cellular PECAM-1 in mediating heterophilic cellular interactions, especially if one invokes a downstream signaling event that results from either cellular activation or PECAM-1/PECAM-1 engagement. There are several lines of evidence that suggest that PECAM-1-mediated signaling, rather than adhesion per se, could broker heterophilic interactions. First, a number of laboratories have independently shown that engagement of PECAM-1 leads to up-regulation of integrin function(37, 38, 39) , providing evidence for secondary adhesion that, while PECAM-1-dependent, does not involve PECAM-1 adhesion per se. Second, we (9) and others (40) have shown previously that PECAM-1 becomes phosphorylated on its cytoplasmic domain following cellular activation. Either of these two events, shown schematically in Fig. 7, could possibly lead to a change in the adhesive phenotype of the cell, enabling heterophilic adhesion mediated by non-PECAM-1 receptors. The recent findings of DeLisser and colleagues (36, 41) that deletion of portions of the PECAM-1 cytoplasmic domain results in a loss of heterotypic adhesion capability are consistent with a role for the PECAM-1 cytoplasmic domain, particularly exon 14(36, 42) , in mediating this signaling event. Studies are currently in progress to characterize the downstream signaling events that emanate from cell surface engagement of PECAM-1.
Figure 7: Schematic representation of the roles of PECAM-1 in mediating cellular interactions. Two distinct roles for PECAM-1 are depicted, PECAM-1 homophilic adhesion per se, and PECAM-1-dependent signaling leading to heterophilic adhesion mediated by non-PECAM-1 receptors. According to this model, both homophilic as well as heterophilic adhesion are PECAM-1-dependent. Anti-PECAM-1 Fabs binding at or near Ig Domain 1 interfere with homophilic PECAM-1/PECAM-1 interactions, while antibodies bound to Ig Domain 6 propagate long range conformational changes at the amino terminus of the molecule, leading to affinity modulation and tight adhesion. Binding of Domain 6 antibodies may also affect the conformation of the cytoplasmic domain and influence assembly of signaling complexes such that downstream events necessary to effect secondary heterophilic adhesion do not occur.
Recently, Piali et al. have reported that
murine lymphokine-activated killer (LAK) cells, which express PECAM-1
on their surface(43) , were able to bind truncated recombinant
forms of murine PECAM-1 that had been immobilized on plastic microtiter
wells(12) . Surprisingly, binding of LAK cells to a PECAM-1
construct consisting only of Ig Domains 1-3 could be inhibited by
preincubating the cells with either RGD peptides or with intact,
bivalent antibodies specific for the integrin
. While only 2-3% of cells
added actually bound their recombinant PECAM-1 construct, even in the
absence of added antibodies, their results nevertheless differ
significantly from our own observations in which not only did
anti-
antibodies have no effect on
full-length human PECAM-1 adhesion, but the binding of both PECAM-1
proteoliposomes or PECAM-1/IgG was completely blocked by pretreating
HUVECS or PECAM-1 transfectants with PECAM-1.3 Fab fragments alone (Fig. 5). Since this Fab fragment is only 50 kDa, it is unlikely
that it would interfere with accessibility of the 100,000
complexes normally found on the
surface of HUVECS(20) . While these discrepancies might be
attributed to species differences (their cells and constructs were of
murine origin), we speculate that the small amount of
-mediated binding observed by Piali
and colleagues may have resulted from secondary signaling events that
may follow PECAM-1/PECAM-1 interactions (Fig. 7). The fact that
neither murine nor human PECAM-1 contains the highly conserved
integrin-binding motif, I(L)-D(E)-S(T)-P (or S)-L, that is present in all integrin-binding Ig superfamily (IgSF) members described
to date, including Ig domains 1 and 4 of VCAM-1 (44, 45, 46, 47) and in Ig Domain 1
of ICAM-1, ICAM-2, ICAM-3, and MadCAM(47, 48) , argues
further that PECAM-1 is not a typical integrin-binding IgSF member.
Our findings that engagement of PECAM-1 Ig Domain 6 may regulate the adhesive properties of the amino terminus represent the first example of affinity modulation of an adhesion molecule outside of the integrin family. Clearly, Domain 6-specific Fab fragments are functioning as a surrogate modulator of PECAM-1 affinity in our system and serve only to demonstrate that PECAM-1 affinity modulation is possible. The actual physiological regulator(s) of PECAM-1 affinity states remain to be identified. However, in light of the fact that long range conformational changes can result in changes in the adhesive phenotype of PECAM-1, the opportunity presents itself to re-examine previous studies of PECAM-1-mediated adhesion in which Ig Domain 6 antibodies were employed and offer alternative interpretations. For example, the recent findings of Liao et al.(49) that antibodies prebound to PECAM-1 Ig Domain 6 prevented release of monocytes from recently transversed endothelium may reflect an antibody-induced increase in the affinity of Ig Domain 1 for its endothelial cell target (i.e. junctional PECAM-1). Likewise, Fawcett et al.(50) found that recombinant PECAM-1 constructs lacking Ig Domain 6, when plated onto microtiter wells, were unable to bind to and capture PECAM-1-positive cells and concluded from their studies that Domain 6 therefore represented a homophilic contact site. In light of the potential ability of Domain 6 to modulate the adhesive phenotype of PECAM-1 (Fig. 6), it is possible that artificial truncated constructs lacking this domain have reduced affinity for cell surface PECAM-1. The fact that truncated constructs extend less far out from the bottom of the microtiter well may also have contributed to their relative inability to bind cells.
In summary, we have used a series of well defined monoclonal antibodies and recombinant PECAM-1 constructs to map the homophilic contact sites involved in PECAM-1 cellular interactions. Both loss of function as well as gain of function mutations were employed and together strongly suggest that Ig homology Domains 1 and 2 are both necessary and sufficient to mediate PECAM-1/PECAM-1 associations. Finally, simulated engagement of Ig Domain 6 resulted in long range propagation of a conformational change at the amino terminus of the molecule, as manifested by a dramatic increase in binding of PECAM-1 proteoliposomes to cells. Future studies aimed at identifying the factors that regulate the adhesive phenotype of PECAM-1, and its subsequent ability to mediate the interactions of leukocytes and platelets with the vessel wall, should have important implications for our understanding of inflammation, angiogenesis, and the immune response.