ATVB In Focus
Platelet Activation and the Formation of the Platelet Plug
Series Editor:
Lawrence Brass
Previous Brief Reviews in this Series:
• Tsai H-M. Deficiency of ADAMTS13 causes thrombotic thrombocytopenic purpura. 2003;23:388 –396.
• Quinn MJ, Byzova TV, Qin J, Topol EJ, Plow EF. Integrin ␣IIb3 and its antagonism. 2003;23:945–952.
Signal Transduction Pathways Mediated by PECAM-1
New Roles for an Old Molecule in Platelet and Vascular Cell Biology
Peter J. Newman, Debra K. Newman
Abstract—Recent studies of platelet endothelial cell adhesion molecule-1 (PECAM-1 [CD31])-deficient mice have
revealed that this molecule plays an important role in controlling the activation and survival of cells on which it is
expressed. In this review, we focus on the complex cytoplasmic domain of PECAM-1 and describe what is presently
known about its structure, posttranslational modifications, and binding partners. In addition, we summarize findings that
implicate PECAM-1 as an inhibitor of cellular activation via protein tyrosine kinase– dependent signaling pathways, an
activator of integrins, and a suppressor of cell death via pathways that depend on damage to the mitochondria. The
challenge of future research will be to bridge our understanding of the functional and biochemical properties of
PECAM-1 by establishing mechanistic links between signals transduced by the PECAM-1 cytoplasmic domain and
discrete cellular responses. (Arterioscler Thromb Vasc Biol. 2003;23:953-964.)
Key Words: PECAM-1 䡲 signal transduction 䡲 ITIM 䡲 SHP-2 䡲 alternative splicing
P
latelet endothelial cell adhesion molecule (PECAM)-1 is
a 130-kDa type I transmembrane glycoprotein (GP) that
was originally described as the endothelial cell equivalent of
platelet membrane GPIIa (the integrin 1 subunit),1 a myeloid
differentiation antigen,2,3 and the CD31 antigen present on
the surface of monocytes, granulocytes, platelets, and endothelial cells.4,5 The common identity of these previously
disparate entities as PECAM-1 (CD31) was established in
1990 on its cloning by 3 different groups.6 – 8 Fifteen years
later, more than 1500 articles list PECAM-1 or CD31 in their
title or abstract, although many of these can be attributed to
the pragmatic use of anti–PECAM-1 antibodies to immunochemically identify endothelial cells in histological sections
or to mark angiogenic blood vessels.9 Nonetheless, a great
deal has been learned about the cell and molecular biology of
PECAM-1 in the blood and vascular cells in which it is
expressed, including its chromosomal location, the structure
of its rather complex gene, and its adhesive and signaling
properties. Because all but the latter have been previously
reviewed in detail,10,11 this review will focus on recent studies
that have shed light on the ability of PECAM-1 to transmit
signals that alter cell adhesion, activation, and survival.
Structure of PECAM-1 and Its
Cytoplasmic Domain
Mature PECAM-1 consists of a 574-amino acid extracellular
domain comprised of 6 immunoglobulin (Ig)-like homology
domains, a 19-residue transmembrane domain, and a 118amino acid cytoplasmic tail. Extracellular Ig domain 1
contains specialized sites that mediate trans-homophilic interactions between PECAM-1 molecules on adjacent
cells12–14 and antibodies whose epitope maps to this region
have been shown to be effective in blocking transendothelial
migration of leukocytes15–18 and hematopoietic progenitor
Received February 25, 2003; revision accepted March 24, 2003.
From the Blood Research Institute, The Blood Center of Southeastern Wisconsin (P.J.N., D.K.N.), and the Departments of Pharmacology (P.J.N.),
Cellular Biology (P.J.N.), and Microbiology (D.K.N.) and Cardiovascular Center (P.J.N.), Medical College of Wisconsin, Milwaukee, Wis.
Correspondence to Peter J. Newman, Blood Research Institute, The Blood Center of Southeastern Wisconsin, PO Box 2178, 638 N. 18th St, Milwaukee,
WI 53201. E-mail pjnewman@bcsew.edu
© 2003 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
DOI: 10.1161/01.ATV.0000071347.69358.D9
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Arterioscler Thromb Vasc Biol.
TABLE 1.
June 2003
Tissue Distribution and Functional Consequences of Alternatively Spliced PECAM-1 Isoforms
Cell Type
Human umbilical vein endothelial cells
Human microvascular endothelium
PECAM-1 Isoforms
Present
Potential Effects on
PECAM-1 Protein Function
Reference
⌬9
Transmembraneless, soluble
23
⌬13
Missing 1st ITIM
24, 30
⌬14
Missing 2nd ITIM
22,30
⌬15
Different C-terminal sequence
30
⌬12
Unknown
30
⌬13
Missing 1st ITIM
30
⌬14
Missing 2nd ITIM
30
⌬15
Different C-terminal sequence
30
Human platelets
⌬15
Different C-terminal sequence
31
Human T lymphocytes
⌬13
Missing 1st ITIM
DK Newman, unpublished
⌬14
Missing 2nd ITIM
DK Newman, unpublished
DK Newman, unpublished
⌬13,14
Missing both ITIMs
Human glioblastomas
⌬14
Missing 2nd ITIM
28
Murine endothelium
⌬14
Missing 2nd ITIM
27
⌬15
Different C-terminal sequence
27
⌬12,15
Different C-terminal sequence
27
⌬12,14
Missing 2nd ITIM
27
⌬14,15*
Different C-terminal sequence, missing ITIM
27
⌬12,14,15
Different C-terminal sequence, missing ITIM
27
⌬14,15
Different C-terminal sequence, missing ITIM
31
⌬14,15
Different C-terminal sequence, missing ITIM
31
⌬14
Missing 2nd ITIM
31
Murine platelets
Strain C129
Strain FVBN
Developing mouse embryos
Murine blastocyst inner cell mass
⌬15
Different C-terminal sequence
31
⌬14,15*
Different C-terminal sequence, missing ITIM
31
⌬12,14,15
Different C-terminal sequence, missing ITIM
31
⌬12
Unknown
26
⌬14
Missing 2nd ITIM
26
⌬15
Different C-terminal sequence
26
⌬12,15
Different C-terminal sequence
25, 26
⌬14,15
Different C-terminal sequence, missing ITIM
26
⌬12,14,15
Different C-terminal sequence, missing ITIM
26
⌬12
Unknown
29
⌬15*
Different C-terminal sequence
29
⌬12,14
Missing ITIM
29
⌬14,15*
Different C-terminal sequence, missing ITIM
29
⌬12,14,15
Different C-terminal sequence, missing ITIM
29
With few exceptions, full-length PECAM-1 is the most abundant species, and the alternatively spliced isoforms indicated are present in only minor
amounts. Highly abundant species, when present, are indicated with an asterisk.
cells,19 malarial parasite binding,20 and angiogenesis in
vivo.21
The cytoplasmic domain of PECAM-1 is complex22 (see
Figure 1) and is encoded by 8 short exons that are differentially
susceptible to alternative splicing, resulting in generation of
mRNA species that encode distinct PECAM-1 isoforms (see
Table 1). These species include a transmembraneless, soluble
form of PECAM-1 lacking exon 9 that is produced by human
umbilical vein endothelial cells and secreted into plasma23 as
well as numerous other variants that lack 1 or more cytoplasmic
domain exons.22,24 –31 Although the existence of few of these
PECAM-1 species have been demonstrated at the protein level,
several of them (indicated with an asterisk in Table 1) represent
relatively abundant mRNA species. Interestingly, whereas exons
1 to 9, 11 to 14, and 16 are all phase 1 exons,22 cytoplasmic
domain– encoding exons 10 and 15 are phase 2 and phase 0,
respectively, and their removal results in PECAM-1 mRNA
species encoding isoforms with a C-terminus that is shorter and
has a different amino acid sequence (䡩, Figure 1). Although no
variants lacking exon 10 have been found to date, there are at
least 3 different PECAM-1 mRNAs missing exon 15, including
2 very abundant species (⌬15 and ⌬14,15) present, for example,
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Newman and Newman
Signal Transduction Pathways Mediated by PECAM-1
955
Figure 1. A, Amino acid sequence of the
full-length human PECAM-1 cytoplasmic
domain (䢇) and of 2 predicted products
of alternatively spliced PECAM-1 mRNA
species in which exons 14 and/or 15 are
deleted and exon 16 is translated in an
alternative reading frame (䡩). Perfectly
conserved tyrosine residues are yellowfilled, perfectly conserved serine residues
are orange-filled, and a free cytoplasmic
cysterine residue near the membrane is
red-filled. B, cDNA and corresponding
predicted amino acid sequences around
the sites at which human PECAM-1
mRNA undergoes alternative splicing,
resulting in removal of exon 14 and/or
15. Dotted lines identify splice junctions,
and stop codons are boxed.
in the inner cell mass of mouse blastulas.29 As shown, these 2
PECAM-1 isoforms end with a different sequence, E-N-G-R-L-P,
than does full-length PECAM-1. The biologic consequence of this
variation is not known.
Tyrosine Phosphorylation of the PECAM-1
Cytoplasmic Domain
Regulated phosphorylation of both serine and tyrosine residues occurs within the PECAM-1 cytoplasmic domain in
response to numerous forms of cellular stimulation. As is the
case with cytoplasmic domains of other plasma membrane
receptors, PECAM-1 cytoplasmic domain phosphorylation
regulates assembly of signaling complexes and, in some
cases, interactions with various elements of the cytoskeleton.
PECAM-1 can become tyrosine phosphorylated in nearly
all vascular cells in which it is expressed. Although low levels
of constitutive PECAM-1 tyrosine phosphorylation have been
reported in platelets32 and endothelial cells,33 PECAM-1 is
generally not found to be tyrosine phosphorylated in cells
maintained in a resting state. PECAM-1 tyrosine phosphorylation has been detected in stirred platelets,34 in platelets
exposed to pervanadate32 or wheat germ agglutinin,35 and in
platelets induced to aggregate via receptors for collagen34 –36
or thrombin.32,34,35,37,38 Cross-linking of platelet PECAM-1
itself also results in its tyrosine phosphorylation.36,39,40 In
endothelial cells, PECAM-1 tyrosine phosphorylation can be
induced by mechanical force applied directly to PECAM-1,41
on adhesion to immobilized PECAM-1,42 fibronectin,42 or
collagen,33 or on exposure to wheat germ agglutinin,35 fluid
shear stress, or osmotic shock.41,43,44 When it has been
compared, the level of PECAM-1 tyrosine phosphorylation
seems to be decreased in migrating (or nonconfluent) endothelial cells.33,41 PECAM-1 tyrosine phosphorylation has also
been observed in T lymphocytes on cross-linking of PECAM145 or the T cell antigen receptor45,46 and in RBL-2H3
mast-like cells on adherence to fibronectin46 or on crosslinking of the IgE receptor, Fc⑀RI.47 However, not all stimuli
induce tyrosine phosphorylation of PECAM-1, because exposure of endothelial cells to agents that induce membrane
lipid hydrolysis or calcium mobilization does not result in
tyrosine phosphorylation of PECAM-1,43 nor does exposure
of RBL-2H3 cells to the protein kinase C (PKC) agonist
phorbol myristate acetate.47
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Because PECAM-1 does not possess intrinsic kinase activity, the identity of tyrosine kinases able to phosphorylate
PECAM-1 has been the subject of extensive investigation. A
fairly large body of evidence obtained from coprecipitation,34,35 in vitro kinase,48,49 and overexpression50 studies
supports a role for Src family kinases in PECAM-1 tyrosine
phosphorylation. The extent to which individual Src family
kinases are redundant in their ability to phosphorylate
PECAM-1 may, however, depend on the array of Src kinases
that a given cell expresses and the activation conditions to
which that cell is exposed.34,51 Inhibition of PECAM-1
tyrosine phosphorylation by Src family kinase inhibitors is
reported to be more or less complete, suggesting that a
non-Src family tyrosine kinase may also be able to phosphorylate PECAM-1.34,35,41 Candidates include members of the
Csk50 and Syk47 families of protein tyrosine kinases, although
the latter remains controversial.50,51 It remains to be determined whether, under physiological conditions, PECAM-1 is
phosphorylated by both Src and non-Src family tyrosine
kinases, how the cell type and its environment influence the
activation states of enzymes able to phosphorylate
PECAM-1, and whether the tyrosine kinases able to phosphorylate PECAM-1 are dependent on each other’s activity.
The sequence of PECAM-1 cDNA has been determined
from 6 species, including human,6 mouse,52 cow,43,53 pig
(Genbank accession No. X98505), rat, and dog (unpublished
data, 1996). Tyrosine residues are perfectly conserved at
positions corresponding to human residues 596, 636, 663, and
686 (yellow-filled circles in Figure 1). Incompletely conserved tyrosine residues include those at positions 638 (histidine in humans, tyrosine in all others), 674 (histidine in all
species except dogs), and 701 (phenylalanine in rats). A
mutant form of human PECAM-1 in which the tyrosine
residues at positions 663 and 686 were replaced with phenylalanine (Y663,686F) failed to become tyrosine phosphorylated in
transfected HEK293 cells exposed to pervanadate, revealing
that, at least under these conditions, Y663 and Y686 are the sole
tyrosine phosphorylation sites in human PECAM-1.54 However, a homologous mutant form of murine PECAM-1 could
become tyrosine phosphorylated on overexpression of the Src
family kinase p56lck in COS-1 cells.50 Although the tyrosine
phosphorylation of Y663,686F PECAM-1 in those cells may
have resulted from overexpression of p56lck, it is also possible
that COS-1, but not HEK293, cells contain a kinase that can
phosphorylate PECAM-1 on tyrosines other than Y663 and
Y686, or that mouse PECAM-1 becomes phosphorylated at a
site (possibly Tyr638) not present in human PECAM-1.50
Interestingly, Y663 is much less efficiently phosphorylated by
either Src or Csk kinases than is Y686,43,50 suggesting that its
phosphorylation may be a rate-limiting step in PECAM-1–
mediated signal transduction. The identity of the kinases or
conditions required for efficient phosphorylation of Y663,
therefore, remain important areas of investigation.
Cytoplasmic Proteins That Bind Tyrosine
Phosphorylated PECAM-1
The best-characterized structural feature of the PECAM-1
cytoplasmic domain is the presence of 2 distinct immunoreceptor tyrosine-based inhibitory motifs (ITIMs) centered
around Y663 and Y686, respectively, and it is the presence of the
paired ITIM within the PECAM-1 cytoplasmic domain that
led several years ago to its assignment to the Ig-ITIM family
of inhibitory receptors.55 Like other members of the Ig-ITIM
family, PECAM-1, when tyrosine phosphorylated, is able to
recruit Src homology 2 (SH2) domain– containing signaling
proteins, which then can initiate signaling pathways, many of
which remain to be defined. Proteins reported to be able to
associate with the PECAM-1 cytoplasmic domain are summarized in Table 2; however, it is important to recognize that
the extent to which these potential binding partners interact
with PECAM-1 is likely to be influenced by their relative
local concentrations, their relative binding affinities, and the
effect of additional posttranslational modifications of the
PECAM-1 cytoplasmic domain.56
The protein most commonly reported to interact with the
PECAM-1 cytoplasmic domain is the SH2 domain– containing protein-tyrosine phosphatase, SHP-2. It is widely accepted that phosphorylation of the PECAM-1 ITIM tyrosine
residues results in both recruitment and activation of SHP2.37,41,45,46,48,50,54,57–59 Interestingly, in a completely unbiased
approach, a bait GST fusion protein containing the tandem
SH2 domains of SHP-2 exclusively bound a peptide comprised of PECAM-1 residues 617 to 711 (ie, containing its
dual ITIM), when tyrosine-phosphorylated, out of an entire
phage display cDNA library.60 The PECAM-1/SHP-2 interaction in cells requires PECAM-1 tyrosine phosphorylation at
both positions 663 and 686.50,54 However, in vitro binding
studies using the individual SH2 domains of SHP-2 have
shown that the N-terminal SH2 domain interacts with high
affinity with the N-terminal ITIM of PECAM-1,54,58 whereas
the C-terminal SH2 domain of SHP-2 preferentially interacts,
albeit with lower affinity, with pY686-containing PECAM-1
phosphopeptide.54 It is possible that the high affinity interaction between the N-terminal SH2 domain of SHP-2 and the
pY663-containing ITIM of PECAM-1 may compensate for the
inefficiency with which Y663 is phosphorylated under physiological conditions (see above). Furthermore, the level of
PECAM-1 tyrosine phosphorylation seems to be enhanced
when PECAM-1 is expressed along with a dominant-negative
form of SHP-2, suggesting that PECAM-1 is an SHP-2
substrate and that the extent of PECAM-1/SHP-2 complex
formation may be regulated by PECAM-1 dephosphorylation
mediated by bound SHP-2.50
In contrast with numerous reports documenting PECAM1/SHP-2 interactions, there are conflicting data concerning
interactions between PECAM-1 and the SHP-2–related phosphatase, SHP-1, which has also been shown to associate with
PECAM-1, albeit in much lesser amounts than does SHP2.46,50,57–59 As with SHP-2, both PECAM-1 ITIMs seem to be
required to support SHP-1 binding50; however, unlike SHP-2,
the affinity between SHP-1 and pY663 is identical to that of
pY686, and the affinities of both interactions are significantly
lower than those reported for SHP-2.57 In fact, one study was
unable to detect an interaction of the individual SH2 domains
of SHP-1 with either PECAM-1 phosphopeptide.58 Thus, the
functional relevance of the PECAM-1/SHP-1 interaction
remains controversial.
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Signal Transduction Pathways Mediated by PECAM-1
TABLE 2. Cells, Stimulation Conditions, and Assays Used to Identify Proteins That Form Complexes With PECAM-1 or
Its Cytoplasmic Domain
Protein
SHP-2
Cells
Platelets
BAECs
HUVECs, BAECs
Thrombin-induced aggregation
Anti-PECAM-1 coprecipitation
37, 38
Y
Osmotic stress
GFP-SHP-2 border localization
41
Y
Anti-SHP-2 coprecipitation
41, 48
Y
Y
Anti-PECAM-1 coprecipitation
42, 48, 79
Anti-PECAM-1 coprecipitation
45, 46
Y
B cells
Receptor cross-linking
Anti-PECAM-1 chimera coprecipitation
59
Y
Fc⑀RI cross-linking
Anti-SHP-2 coprecipitation
46
Y
Src or Csk family kinase transfection
Anti-SHP-2 coprecipitation
50
Y
Pervanadate
Anti-PECAM-1 coprecipitation
54
Y
THP-1 cells
Pervanadate
GST-SHP-2-SH2-SH2 far Western blot
58
Y
RBL-2H3 cells
P ervanadate
GST-SHP-1 coprecipitation
46
Y
Src or Csk family kinase transfection
Anti-SHP-1 coprecipitation
50
Y
TRAP-induced aggregation
Anti-PECAM-1 coprecipitation
57
Y
Pervanadate
GST-SHP-1-SH2-SH2 far Western blot
58
Y
Receptor cross-linking
Anti-PECAM-1 chimera coprecipitation
59
Y
COS-1 transfectants
Platelets
THP-1 cells
B cells
RBL-2H3 cells
Fc⑀RI cross-linking
Anti-SHP-1 coprecipitation
46
N
T cells
Pervanadate
Anti-PECAM-1 coprecipitation
46
N
BAECs
Src transfection
Fyn
BAECs
PLC␥1
THP-1 cells
BAECs
PI3K
Y/N*
Osmotic, fluid shear stress
HEK293 cells
SHIP
References
Pervanadate or receptor cross-linking
COS-1 transfectants
Src
Assay Type
T cells
RBL-2H3 cells
SHP-1
Conditions
THP-1 cells
GST-Src-SH2 coprecipitation
49
Y
GST-PECAM-1-cyt coprecipitation
43, 49
Y
Osmotic stress
GST-Fyn-SH2 coprecipitation
48
Y
Pervanadate
GST-PLC␥1-SH2-SH2 far Western blot
58
Y
Osmotic stress
GST-PLC␥1-SH2-SH2-SH3 coprecipitation
48
N
Pervanadate
GST-SHIP-SH2 far Western blot
58
Y
B cells
Receptor cross-linking
Anti-PECAM-1 chimera coprecipitation
59
Y
PMNs
None
Anti-PECAM-1 capture, anti-PI3K ELISA
64
Y
BAECs
Osmotic stress
GST-PI3K-SH2 coprecipitation
48
N
Grb2
RBL-2H3 cells
Pervanadate
Anti-SHP-2 coprecipitation
46
Y
Gab1
BAECs
Shear stress
STAT3, 5
Anti-Gab1 border localization
41
Y
Anti-PECAM-1, anti-Gab1 coprecipitation
41
N
Lck transfection
Anti-PECAM-1 coprecipitation
63
Y
None
Anti-PECAM-1, anti-STAT coprecipitation
63
Y
Days 7.5–9.5 of gestation
Anti-PECAM-1 coprecipitation
73
Y
None
Anti-PECAM-1 coprecipitation
73
Y
EOMA cells
PKC inhibition
Anti-PECAM-1 coprecipitation
73
Y
SW480 cells
PECAM-1 expression
Anti-␥-catenin border localization
73
Y
None
Anti-PECAM-1 coprecipitation
76, 79
Y
PECAM-1 expression
Anti--catenin border localization
73
Y
HEK293 cells
HUVECs
␥-catenin
Murine embryos
HUVECs
-catenin
Endothelial cells
SW480 cells
BAECs indicates bovine aortic endothelial cells; HUVECs, human umbilical vein endothelial cells; EOMA, hemagioendothelioma.
*Y refers to a study in which an association was found. N refers to a study in which an association was actively looked for but not found.
Several other SH2 domain– containing proteins have been
evaluated for their ability to associate with PECAM-1,
including selected members of the Src family,43,48,49 the
5⬘-inositol phosphatase, SHIP,58 and PLC␥1,58,59 although the
latter association is controversial.48 Interestingly, the SH2
domain of SHIP is similar to that present in an adaptor protein
known as SAP (SLAM-associated protein), both of which
seem to interact preferentially with a unique type of ITIM that
has been termed an ITSM.61 SAP binding sites are characterized by the presence of a T or S residue at position ⫺2
relative to the ITIM phosphotyrosine.62 Because the
C-terminal ITIM of PECAM-1 contains a T at the ⫺2
position of Y686 (Figure 1), it may be able to bind SAP (Kim
Nichols, University of Pennsylvania, unpublished observations, 2002), although the biological conditions under which
PECAM-1 and SAP might interact in intact cells are presently
not known.
PECAM-1 immunoprecipitates have been shown to contain several phosphoproteins of unknown identity,34,42,49,54,57
and it is likely that some of these associate with the
PECAM-1 cytoplasmic domain indirectly or in an ITIMindependent manner. Signal transducers and activators of
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Arterioscler Thromb Vasc Biol.
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transcription 3 and 5 likely represent the best known examples of ITIM-independent binding, because their interaction
with PECAM-1 seems to require PECAM-1 Y701 but not
ITIM residues Y663 or Y686.63 Phosphatidylinositol-3 kinase,
on the other hand, may be an example of a signaling molecule
that interacts indirectly with PECAM-1, because it could be
detected in anti–PECAM-1 mAb-coated microwells incubated with whole neutrophil lysates64 but was not found
associated with PECAM-1 in direct binding studies.48,58 The
adaptor molecules Grb2 and Gab1 also may interact with
tyrosine-phosphorylated PECAM-1 in an indirect manner.
Although evidence for an association between PECAM-1 and
Grb2 is conflicting,46,58 Gab1 has been shown to colocalize
with PECAM-1 and SHP-2 at endothelial cell borders after
exposure to fluid shear stress.41 Immunoprecipitation experiments, however, were unable to demonstrate direct binding
of Gab1 to PECAM-1.41 Because both Grb265,66 and Gab167
can bind directly to tyrosine-phosphorylated SHP-2, it is
possible that PECAM-1/SHP-2/Gab1 and PECAM-1/SHP-2/
Grb2 ternary signaling complexes may form in response to
certain forms of cellular stimulation. The relevance of these
interactions in regulating PECAM-1–mediated cell adhesion,
activation, and survival represent an important area of future
investigation.
Serine Phosphorylation of the PECAM-1
Cytoplasmic Domain
PECAM-1 serine, but not threonine, residues have been
shown to be phosphorylated in resting platelets and endothelial cells, and the level of serine phosphorylation increases 2to 3-fold on cellular activation.32,68 –72 PKC is thought to play
a primary role in PECAM-1 serine phosphorylation68,71,73;
however, a role for other kinases has been proposed because
of the observation that the level of platelet PECAM-1 serine
phosphorylation induced by phorbol myristate acetate, which
activates only PKC, is lower than that induced by thrombin,
which activates PKC and possibly other kinases as well.69
Proposed serine phosphorylation sites in PECAM-1 include
S673,73 S620, S670, and S687.43 Although 3 of these (S620, S670, and
S687; see orange-filled circles in Figure 1) are well conserved,
the sites and consequences of serine phosphorylation within
the PECAM-1 cytoplasmic domain remain to be determined.
PECAM-1/Cytoskeletal Interactions
There is growing evidence that PECAM-1 associates both
physically and functionally with the underlying cytoskeleton.
Using the operational definition of detergent insolubility as a
measure of cytoskeletal connectedness, it was found more
than 10 years ago that, in resting platelets, only ⬇10% of
PECAM-1 partitions with the Triton-insoluble cytoskeleton,
which is largely composed of F-actin, filamen (ABP-280),
and spectrin.69 During thrombin-induced platelet aggregation,
however, more than 60% of total cellular PECAM-1 becomes
detergent insoluble, indicating that the degree of PECAM-1
association with the Triton-insoluble cytoskeleton depends on
the activation state of the cell.69 In support of this notion,
⬇20% to 30% of PECAM-1 was found associated with the
cytoskeleton in confluent endothelial cells,73,74 increasing to
⬇65% during cell migration.73
Poggi et al,75 using an NK cell model system, were the first
to demonstrate a functional relationship between PECAM-1
and the cytoskeleton when they showed that mAb-induced
cross-linking of PECAM-1 on the cell surface could induce
cell spreading and cytoskeletal rearrangement. Shortly thereafter, Matsumura et al76 showed that addition to endothelial
cells of a combination of VE-cadherin–specific and PECAM1–specific antibodies resulted in reorganization of F-actin
filaments into discrete foci. Although less specific than using
PECAM-1–specific reagents, addition of wheat germ agglutinin, a multivalent lectin that binds to and cross-links
PECAM-1 (as well as a host of other cell surface receptors),
has also been shown to increase actin assembly at the cell
periphery.35 Taken together, it would seem that PECAM-1 is
able to bind to and coordinate the assembly of F-actin
filaments, especially in association with changes in cell shape
or during cell migration.
Although the molecules linking PECAM-1 and the actin
cytoskeleton have not been established with certainty,
-catenin and ␥-catenin (also known as plakoglobin) seem to
be likely candidates. The catenins are scaffolding proteins
normally involved in anchoring a class of adherens junctional
proteins known as cadherins to the cortical actin cytoskeleton.
Although early studies did not find an association between ␣-,
-, or ␥-catenin and PECAM-1,77 Matsumura et al76 found
that  -catenin could be coimmunoprecipitated with
PECAM-1 using special cell homogenization conditions that
improved the solubility of the underlying membrane skeleton.
The authors proposed that a functional adherens junctional
complex of PECAM-1/-catenin/F-actin, perhaps in conjunction with VE-cadherin, might regulate processes such as
endothelial cell tube formation. The finding that anti–
PECAM-1 antibodies markedly inhibit the ability of endothelial cells to organize and form 3-dimensional networks in
Matrigel78 also support this concept. Ilan et al79 went on to
characterize the molecular requirements for PECAM-1/catenin complex formation, which include tyrosine phosphorylation of -catenin. This interaction seems to be ITIMindependent, because tyrosine-phosphorylated -catenin
bound similarly to wild-type, Y663F, and Y686F forms of
PECAM-1. Because SHP-2 and -catenin seemed to bind
different sites on the PECAM-1 cytoplasmic domain, the
authors suggested that SHP-2, when recruited to PECAM-1/
-catenin complexes, regulates the tyrosine phosphorylation
state of -catenin. The physiological importance of -catenin
tyrosine phosphorylation, however, remains to be established.
In addition to -catenin, PECAM-1 also has been reported to
associate with ␥-catenin.73 The molecular requirements for
␥-catenin association with PECAM-1 differ from those of
-catenin, however, because ␥-catenin does not need to be
tyrosine phosphorylated to bind. Interestingly, PKC-mediated
serine phosphorylation of the PECAM-1 cytoplasmic domain,
as might occur early in the platelet activation process or
during endothelial cell migration, was found to inhibit
PECAM-1/␥-catenin interactions. Taken together, these data
suggest that dynamic interactions between PECAM-1 and the
catenins may be responsible for anchoring PECAM-1 to the
cytoskeleton. The relationship between such cytoskeletal
connections and the ability of PECAM-1 to regulate integrin
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Newman and Newman
Signal Transduction Pathways Mediated by PECAM-1
activation, endothelial responses to fluid shear stress, protection from phagocytosis, or cell survival (all discussed below)
are important issues that remain to be addressed.
ITIM-Mediated Inhibitory Function
By virtue of their ability to recruit and activate proteintyrosine or inositol phosphatases, ITIM-containing inhibitory
receptors are thought to function primarily by counteracting
signal transduction pathways initiated by activating receptors
that recruit, via their immunoreceptor tyrosine-based activating motifs (ITAMs), protein tyrosine kinases.80 Inhibitory
receptors, therefore, raise the threshold for cellular activation
and regulate ITAM receptor–mediated cellular activation
events. PECAM-1 is the only known ITIM-containing receptor on human or murine platelets, and, because the most
prominent PECAM-1– binding protein, SHP-2, is capable of
transmitting both stimulatory and inhibitory signals into the
cell,81 determining whether PECAM-1 exerts positive or
negative regulatory effects has been the subject of much
investigation.
The first evidence that PECAM-1 could function as an
inhibitory receptor came from studies in which PECAM-1
was artificially forced into proximity with selected activating
receptors. This was done by cross-linking antibodies bound to
PECAM-1 with antibodies bound to ITAM-containing antigen receptors on lymphocytes. On coligation with the T cell45
or B cell51,59 receptor, PECAM-1 was found to attenuate
antigen receptor–induced signaling and cellular responses in
a manner that depended on the integrity of the PECAM-1
ITIMs and that either required SHP-251 or preferred SHP-2
over SHP-1.59
Evidence, albeit less direct, for the inhibitory function of
PECAM-1 has also been obtained by examining the effects of
PECAM-1 cross-linking on collagen-induced platelet activation. Collagen binds to platelets via the integrin ␣21, but
subsequent signal transduction occurs primarily via GPVI,
which exists noncovalently associated within the plane of the
plasma membrane with the ITAM-bearing FcR␥-chain.82
PECAM-1/SHP-2 complexes formed as a result of PECAM-1
cross-linking36,39,40 should, in theory, be able to inhibit
signaling by nearby GPVI/FcR␥ chain complexes. Indeed,
several in vitro studies have demonstrated that activation of
PECAM-1 via mAb-induced PECAM-1 cross-linking inhibits
Ca2⫹ mobilization,40 granule secretion,40 aggregation,36,40 and
thrombus formation36 in collagen-activated platelets. The
extent to which PECAM-1 inhibits responses of platelets to
agonists that bind to non-ITAM– bearing receptors is, however, as yet unclear. One study found that mAb-induced
PECAM-1 cross-linking inhibited platelet activation induced
by binding of thrombin to the G-protein– coupled receptor,
PAR1, which is not thought to be regulated by ITIMs;
however, this study did not rule out nonspecific heterologous
desensitization as the mechanism of inhibition.40 In another
study, the anti–PECAM-1 mAb, AAP2, inhibited platelet
aggregation induced by all agonists examined, including
collagen, ADP, epinephrine, and thrombin; however, this
study did not discriminate between inhibition of PECAM-1–
mediated signaling versus adhesion.83
959
The notion that PECAM-1 inhibits specifically the action
of ITAM-bearing agonist receptors has recently gained support from studies that have had the opportunity to examine
blood cell function in PECAM-1– deficient mice. Compared
with wild-type cells, PECAM-1– deficient B cells84 and mast
cells85 fail to regulate signaling through the ITAM-bearing B
cell receptor and Fc⑀RI, respectively. Similar hyperactive
responses have been observed in PECAM-1– deficient platelets, which exhibit enhanced aggregation and granule secretion responses to GPVI/FcR␥ chain–specific agonists,36,86
whereas PECAM-1 deficiency has no measurable effect on in
vitro platelet responses to agonists that bind to G protein–
coupled receptors for ADP36,87 or thrombin.36,86 These data
suggest that, in vivo, platelet PECAM-1 plays a major role in
regulating signaling pathways of ITAM-containing, but not G
protein– coupled, receptors. Relative to its regulation of
ITAM-mediated cellular activation, however, it is important
to note that PECAM-1 inhibitory signaling in platelets can be
easily overcome by strong stimulation of the GPVI/FcR␥
chain collagen receptor.86 Thus, in situations where endothelial damage is severe enough to expose a high concentration
of underlying extracellular matrix, PECAM-1 will likely not
be able to inhibit platelet thrombus formation. This is a good
thing. This concept may explain the discrepancy between the
findings of Vollmar et al,88 who found that PECAM-1–
deficient and wild-type mice did not differ in either the rate or
extent of thrombus formation in blood vessels damaged by
exposure to laser light–induced injury to the endothelium,
versus those of Rosenblum and colleagues,89,90 who reported
that anti–PECAM-1 mAbs could inhibit platelet adhesion and
aggregation over mildly injured endothelial cell beds. Studies
using mild forms of vascular injury in PECAM-1– deficient
mice might be expected to yield similar results.
As these and other studies in PECAM-1– deficient mice
begin to emerge, opportunities to resolve several important
issues are likely to be realized. First, it is important to
determine the extent to which formation of PECAM-1/SHP-2
complexes is required to modulate platelet, B cell, and mast
cell activation in vivo. Expression of an ITIM-less form of
PECAM-1 in vivo would go a long way toward answering
this question. If SHP-2 binding to the PECAM-1 ITIMs is
important for the inhibitory activity of PECAM-1 in vivo,
specific roles for SHP-2 will have to be resolved. One
possibility is that PECAM-1 normally recruits SHP-2 and
triggers its phosphatase activity, resulting in dephosphorylation of nearby components of ITAM-dependent signal transduction pathways and diminished cellular responsiveness. In
cells expressing an ITIM-less form of PECAM-1, dephosphorylation of signal transduction pathway components
would be delayed, resulting in augmented cellular responsiveness. An alternative possibility is that SHP-2 normally
functions as a positive adapter or activating protein in growth
factor receptor signaling pathways91 from which PECAM-1
sequesters SHP-2 away. ITIM-less PECAM-1 would fail to
sequester SHP-2, again resulting in augmented cellular responses. Once the mechanism by which PECAM-1 exerts its
inhibitory function is more precisely understood, it may be
possible to exploit it to block pathologic cellular activation
(eg, to inhibit thrombosis, autoimmunity, or hypersensitivity)
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960
Arterioscler Thromb Vasc Biol.
June 2003
Figure 2. Possible molecular mechanisms by which PECAM-1 modulates
integrin function.
or constrain it to enhance desirable cell activation (eg, to
control bleeding or overcome immunosuppression).
Integrin Activation
More than 10 years ago, Tanaka et al92 noticed that addition
of anti–PECAM-1 mAbs to human T lymphocytes enhanced
their ability to bind to immobilized 1 integrin substrates,
such as fibronectin and VCAM-1, and proposed that
PECAM-1 could act as an integrin-function modulator. Since
that time, there have been numerous studies demonstrating
that addition of anti–PECAM-1 mAbs to the cell surface
results in upregulation of 1,93 2,94 –96 and 339,97 integrin
function, leading to speculation that PECAM-1 engagement
might influence diverse integrin-mediated processes, such as
thrombosis, cell migration, or transendothelial leukocyte
extravasation. Although the precise mechanism by which
PECAM-1 modulates integrin function is not known, several
clues have begun to emerge. First, PECAM-1–mediated
integrin activation seems to require an intact PECAM-1
cytoplasmic domain and its 2 ITIMs98 and that PECAM-1 be
induced to form lateral oligomers within the plane of the
plasma membrane.99 What subsequently occurs to effect
integrin activation is not clear, but recent studies by
Reedquist et al98 suggest that small GTPases may be involved. They found that addition of anti–PECAM-1 mAbs to
Jurkat T cells selectively activated Rap1, which, together
with its exchange factor CalDAG-GEFI, has been increasingly implicated in integrin activation,100,101 perhaps by
inducing integrin clustering (avidity modulation) or by inducing conformational changes in the integrin itself (affinity
modulation). Support for both of these mechanisms of action,
in fact, exists, because Poggi et al75 found that addition of
anti–PECAM-1 mAbs to human NK cells resulted in actin
rearrangements and recruitment of talin, a known integrinbinding102 and -activating103 protein, to membrane ruffles,
whereas Varon et al39 found that certain anti-PECAM-1
mAbs, when bound to platelets, induce exposure of ligandinduced binding sites (so-called LIBS epitopes) on the integrin ␣IIb3. Future studies using recently developed Rap1deficient mice may help to clarify the relationships between
Rap1 activation and cytoskeletal rearrangements, integrin
clustering, and integrin conformational changes in PECAM1–mediated modulation of integrin function.
In addition to affecting integrin activation, PECAM-1 may
also regulate integrin trafficking. A recent report by Dangerfield et al104 provides compelling evidence that PECAM-1/
PECAM-1 homophilic interactions may be required in vivo
for redistributing ␣61 from neutrophil intracellular granules
to the plasma membrane during the process of transendothelial migration,105 thereby facilitating ␣61-mediated neutrophil
migration through the laminin- and collagen-rich perivascular
basement membrane. Interestingly, endothelial cell surface
PECAM-1 seems to serve as a passive ligand for neutrophil
PECAM-1, which after engagement leads to neutrophil signal
transduction and subsequent integrin activation.106 The signaling pathway that regulates PECAM-1–mediated integrin
redistribution remains to be elucidated; however, given the
reported ability of PECAM-1 to activate Rap1, it is tempting
to speculate that PECAM-1 might similarly be activating 1 or
more Rab proteins107—related members of the Ras superfamily—to effect integrin-containing granule trafficking and
membrane fusion during neutrophil extravasation. A summary of molecular mechanisms by which PECAM-1 might
amplify integrin-mediated cell adhesion is shown in Figure 2.
Cell Survival
In addition to its role in vascular cell adhesion and signaling,
there is growing evidence that PECAM-1 may be able to
transduce signals that suppress programmed cell death. In
1999, Noble et al108 reported that PECAM-1/PECAM-1
homophilic interactions between monocytes and endothelial
cells reduced apoptotic endothelial cell death after serum
deprivation and speculated that PECAM-1 homophilic interactions might lead to the transmission of prosurvival signals.
Similar findings have been reported by Bird et al42 and by
Evans et al,109 each of whom found that endothelial cells
could be protected from serum deprivation–induced cell
death if first bound to PECAM-1/IgG or treated with an
anti–PECAM-1 monoclonal antibody. Although the molecular mechanisms by which PECAM-1 exerts its cytoprotective
effects are not yet known, Gao et al110 recently reported
preliminary findings showing that PECAM-1 can inhibit
cytochrome c release from mitochondria after exposure of
cells to a wide range of cytotoxic stimuli that activate Bax, a
proapoptotic pore-forming member of the Bcl-2 family that
plays a central role in mitochondria-dependent apoptosis.111
Interestingly, both extracellular homophilic binding function
and intact cytoplasmic ITIMs seem to be required for
PECAM-1 to suppress programmed cell death, because neither homophilic binding-crippled K89A14 nor ITIM-less
Y663,686F54 mutant forms of PECAM-1 were able to protect
cells from Bax-overexpression–induced apoptosis. Taken together with previous reports, these data suggest that signals
emanating from the PECAM-1 extracellular domain initiate
association of 1 or more cytosolic signaling molecules with
the PECAM-1 cytoplasmic tail, resulting in the transmission
of prosurvival signals that suppress the mitochondriadependent, Bax-mediated intrinsic apoptotic pathway. Candidate proteins/pathways that might be deserving of additional
investigation for their potential to support PECAM-1–mediated cell survival are illustrated in Figure 3.
Other Functions Mediated by the PECAM-1
Cytoplasmic Domain
As noted above, the cytoplasmic ITIMs of PECAM-1 become
tyrosine phosphorylated in response to numerous forms of
cellular activation, including thrombin- or collagen-induced
platelet aggregation, exposure to oxidative injury, and as a
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Signal Transduction Pathways Mediated by PECAM-1
961
Figure 3. Signaling pathways emanating from PECAM-1 that might mediate resistance to apoptosis.
consequence of T or B cell receptor and mast cell Fc⑀
receptor cross-linking. In each case, tyrosine phosphorylation
of the PECAM-1 cytoplasmic domain results in recruitment
and activation of the protein-tyrosine phosphatase, SHP-2.
The downstream effectors of the PECAM-1/SHP-2 signaling
complex, however, have not yet been described in any of
these experimental systems. Progress in this area has recently
been made by Osawa et al,41 who discovered that formation
of the PECAM-1/SHP-2 signaling complex is required for
extracellular signal-regulated kinase (Erk) to become activated when endothelial cells are subjected to fluid shear
stress. Erk is an early shear stress response element that
regulates transcription of multiple genes, some of which are
thought to be involved in atherogenesis, and an earlier study
had shown the potential for certain PECAM-1 isoforms to
activate Erk.112 Interestingly, the signaling pathway from
PECAM-1/SHP-2 to Erk activation seems to involve several
steps, including (1) shear-induced perturbation of the plasma
membrane, causing (2) activation of one or more tyrosine
kinases, which (3) phosphorylate PECAM-1 cytoplasmic
ITIM tyrosine residues, resulting in (4) recruitment of SHP-2
and Gab1, a multisite PH-domain– containing adaptor protein
involved in growth factor–induced ERK activation,113 initiating (5) SHP-2/Gab-mediated activation of Erk, probably via
the ability of Gab to associate with Shc,114 which in turn
recruits the Grb2-Sos Ras activation complex. Taken together, these data implicate PECAM-1 as a mechanoresponsive cell-surface receptor that, together with SHP-2 and
Gab1, regulates Erk-mediated endothelial responses to fluid
shear stress.
One of the more intriguing and novel functions of
PECAM-1–mediated signal transduction relates to the ability
of PECAM-1 to send “leave me alone” signals from healthy
cells to potentially hostile cellular adversaries. Thus, Brown
et al115 recently reported that homophilic interactions between
PECAM-1–positive macrophages and PECAM-1-positive
leukocytes results in the transmission of signals that facilitate
either active detachment if the leukocyte is viable or phagocytic ingestion if the leukocyte is apoptotic. The nature of the
PECAM-1–mediated release versus tethering signal has not
been fully explored, but it is known to require PECAM-1
cytoplasmic ITIMs, as neither a Y 663,686 F- nor a
glycophosphatidylinositol-linked form of PECAM-1 was able
to mediate attachment of apoptotic cells or detachment of
viable ones. SHP-2 binding to PECAM-1 was also associated
with release signaling, although a causative relationship was
not established. It will be interesting in the future to determine the relationships, if any, between PECAM-1–mediated
detachment signals, PECAM-1–mediated integrin activation,
and PECAM-1–mediated inhibitory signaling.
Concluding Remarks
Contributions from many laboratories over the past 15 years
have revealed that PECAM-1 serves several distinct roles in
the biology of blood and vascular cells, some no doubt related
to its adhesive properties, and others as a result of it ability to
transduce signals into cells. The biochemical pathways
through which PECAM-1 inhibits cellular activation mediated by ITAM-bearing agonist receptors, modulates integrin
function, regulates vascular integrity, and controls cell survival, however, are just now beginning to be defined. Studies
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962
Arterioscler Thromb Vasc Biol.
June 2003
in mice and other well-defined experimental systems using
selected PECAM-1 variants lacking either adhesive or signaling capacity, or both, will be required to elucidate the
manner by which PECAM-1 functions in thrombosis, inflammation, cell survival, and the immune response.
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Signal Transduction Pathways Mediated by PECAM-1: New Roles for an Old Molecule in
Platelet and Vascular Cell Biology
Peter J. Newman and Debra K. Newman
Arterioscler Thromb Vasc Biol. 2003;23:953-964; originally published online April 10, 2003;
doi: 10.1161/01.ATV.0000071347.69358.D9
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