Platelet/endothelial cell adhesion molecule-1 (PE-CAM-1) is a homophilic adhesion receptor that mediates leukocyte/endothelial cell interactions that take place during transendothelial migration. Recent reports have shown that the binding of certain anti-PECAM-1 antibodies results in up-regulation of integrin function on the surface of leukocytes and platelets, suggesting that PE-CAM-1 may be capable of transmitting information into the cell following its engagement. PECAM-1 isolated from resting or activated but nonaggregated platelets was phosphorylated predominantly on serine residues; however, PECAM-1 derived from activated, aggregated platelets was strongly phosphorylated on tyrosine. Synthetic tyrosine-phosphorylated peptides derived from five different regions within the cytoplasmic domain of PE-CAM-1 were screened for their ability to associate with cytoplasmic signaling molecules. The protein-tyrosine phosphatase SHP-2 was found to interact specifically with two different PECAM-1 phosphopeptides containing highly conserved phosphatase-binding motifs on PE-CAM-1 with the sequences VQpY 663 TEV and TVpY 686 SEV. More important, SHP-2 bound not only PECAM-1 phosphopeptides, but also became associated with full-length cellular PECAM-1 during the platelet aggregation process, and this interaction was mediated by the amino-terminal Src homology 2 domains of the phosphatase. Since SHP-2 normally serves as a positive regulator of signal transduction, its association with activated PECAM-1 suggests a number of potential mechanisms by which PE-CAM-1 engagement might be coupled to integrin activation in vascular cells.

The tetraspanin membrane protein CD151 is a broadly expressed molecule noted for its strong molecular associations with integrins, especially ␣3␤1, ␣6␤1, ␣7␤1, and ␣6␤4. In vitro functional studies have pointed to a role for CD151 in cell-cell adhesion, cell migration, platelet aggregation, and angiogenesis. It has also been implicated in epithelial tumor progression and metastasis. Here we describe the generation and initial characterization of CD151-null mice. The mice are viable, healthy, and fertile and show normal Mendelian inheritance. They have essentially normal blood and bone marrow cell counts and grossly normal tissue morphology, including hemidesmosomes in skin, and expression of ␣3 and ␣6 integrins. However, the CD151-null mice do show phenotypes in several different tissue types. An absence of CD151 leads to a minor abnormality in hemostasis, with CD151-null mice showing longer average bleeding times, greater average blood loss, and an increased incidence of rebleeding occurrences. CD151-null keratinocytes migrate poorly in skin explant cultures. Finally, CD151-null T lymphocytes are hyperproliferative in response to in vitro mitogenic stimulation.

Recent studies have shown that the Src homology-2 (SH2) domain-containing protein-tyrosine phosphatase, SHP-2, associates with the cytoplasmic domain of PECAM-1 as it becomes tyrosine-phosphorylated during platelet aggregation: a process that can be mimicked in part by small synthetic phosphopeptides corresponding to the cytoplasmic domain of PECAM-1 encompassing tyrosine residues Tyr-663 or Tyr-686. To further examine the molecular requirements for PECAM-1/SHP-2 interactions, we generated human embryonic kidney (HEK)-293 cell lines that stably expressed mutant forms of PECAM-1 harboring tyrosine to phenylalanine (Tyr 3 Phe) mutations in the cytoplasmic domain. Y663F and Y686F forms of PECAM-1 were tyrosine-phosphorylated to a somewhat lesser extent than wild-type PECAM-1, and a doubly substituted Y663,686F form of PECAM-1 failed to become tyrosine-phosphorylated, suggesting that the PECAM-1 cytoplasmic domain tyrosine residues 596, 636 and 701 do not serve as substrates for cellular kinases. Interestingly, SHP-2 binding was lost when either Tyr-663 or Tyr-686 were changed to phenylalanine, indicating that both residues are required for SHP-2/ PECAM-1 association. Although PECAM-1 phosphopeptides NSDVQpY 663 TEVQV and DTETVpY 686 SEVRK stimulated the catalytic activity of the phosphatase to a similar extent, surface plasmon resonance studies revealed that the Tyr-663-containing peptide had approximately 10-fold higher affinity for SHP-2 than did the Tyr-686 peptide. Finally, peptido-precipitation analysis showed that the NH 2 -terminal SH2 domain of SHP-2 reacted preferentially with the Tyr-663 PECAM-1 phosphopeptide, while the Tyr-686 phosphopeptide associated only with the COOH-terminal SH2 domain of the phosphatase. Together, these data provide a molecular model for PECAM-1/SHP-2 interactions that may shed light on the downstream events that follow PECAM-1-mediated interactions of vascular cells.Phosphorylation of proteins on tyrosine residues provides a key cellular control mechanism for intracellular signaling processes that regulate cell growth, proliferation, adhesion, differentiation, and metabolism (1, 2). The level of tyrosine phosphorylation of cellular proteins is controlled by the coordinated actions of protein-tyrosine kinases and protein-tyrosine phosphatases. Signal transmission by tyrosine phosphorylation is mediated by the binding of sequence-specific Src homology-2 (SH2) 1 domains present on cytosolic signaling molecules to phosphotyrosine (Tyr(P)) sites on activated receptors (3-7). These highly conserved protein modules play an important role in mediating protein-protein interactions and can regulate many facets of the signaling process (8, 9). The association of SH2-containing proteins with Tyr(P) sites on activated receptors can elicit biochemical changes within the cell, including regulating catalytic activity (10), directing subcellular localization (11), and enhancing tyrosine phosphorylation (12) to potentiate downstream signaling events.Platelet endothelial cell adhesion molecule-1 (P...

Platelet endothelial cell adhesion molecule-1 (PE-CAM-1/CD31) is a member of the immunoglobulin (Ig) superfamily that has distinctive features of an immunoreceptor based upon its genomic structure and the presence of intrinsic immunoreceptor tyrosine inhibitory motifs (ITIMs) in its ligand binding polypeptide. This has lead to its subclassi¢cation into the Ig-ITIM superfamily. Its amino-terminal Ig-like domain of PE-CAM-1 is necessary for its homophilic binding, which plays an important role in cell^cell interactions. Its intracellular ITIMs serve as sca¡olds for recruitment of signalling molecules including protein-tyrosine phosphatases to mediate its inhibitory co-receptor activity. Increasing evidence has implicated PE-CAM-1 in a plethora of biological phenomena, including modulation of integrin-mediated cell adhesion, transendothelial migration, angiogenesis, apoptosis, cell migration, negative regulation of immune cell signalling, autoimmunity, macrophage phagocytosis, IgE-mediated anaphylaxis and thrombosis. In this review, we discuss some of the new developments attributed to this molecule and its unique roles in biology. ß

The functional importance of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) in platelets is unclear. Because PECAM-1 represents a newly assigned immunoglobulin-ITIM superfamily member expressed on the surface of platelets, it was hypothesized that it may play an important regulatory role in modulating ITAM-bearing receptors such as collagen (GP)VI receptor and Fc␥RIIA. To examine the functional role of PECAM-1 in regulating platelet-collagen interactions, 2 different approaches were applied using recombinant human PECAM-1-immunoglobulin chimeras and platelets derived from PECAM-1-deficient mice. Stimulation of platelets by collagen-, (GP)VI-selective agonist, collagen-related peptide (CRP)-, and PECAM-1-immunoglobulin chimera induced tyrosine phosphorylation of PECAM-1 in a time-and dose-dependent manner. Activation of PECAM-1 directly through the addition of soluble wild-type PECAM-1-immunoglobulin chimera, but not mutant K89A PECAM-1-immunoglobulin chimera that prevents homophilic binding, was found to inhibit collagen-and CRP-induced platelet aggregation. PECAM-1-deficient platelets displayed enhanced platelet aggregation and secretion responses on stimulation with collagen and CRP, though the response to thrombin was unaffected. Under conditions of flow, human platelet thrombus formation on a collagen matrix was reduced in a dose-dependent manner by human PECAM-1-immunoglobulin chimera. Platelets derived from PECAM-1-deficient mice form larger thrombi when perfused over a collagen matrix under flow at a shear rate of 1800 seconds ؊1 compared to wild-type mice. Collectively, these results indicate that PECAM-1 serves as a physiological negative regulator of platelet-collagen interactions that may function to negatively limit growth of platelet thrombi on collagen surfaces.