Cell Surface Glycosaminoglycans Do Not Serve as Ligands for PECAM-1

Abstract

Previous studies have suggested that PECAM-1 mediates cellular interactions via both homophilic and heterophilic adhesive mechanisms. Cell surface glycoaminoglycans have been implicated as one of the heterophilic ligands for PECAM-1. To determine whether PECAM-1 is capable of interacting directly with glycosaminoglycans, we examined the adhesive properties of multiple monovalent and multivalent forms of this adhesion molecule. We found that the binding of a bivalent PECAM-1/IgG chimeric protein or multivalent PECAM-1-containing proteoliposomes to multiple different cell lines was 1) strictly dependent upon cell surface expression of PECAM-1 and 2) unaffected by the presence of excess heparin or heparan sulfate. The extracellular domain of PECAM-1 failed to interact specifically with heparinSepharose, 3 H-labeled heparin, or a heparin-bovine serum albumin conjugate. In addition, an amino acid sequence motif inadvertently created by the juxtaposition of PECAM-1 and IgG sequences within the hinge region of certain PECAM-1/IgG chimeric constructs was found to confer glycosaminoglycan binding properties not normally present within the extracellular domain of the native molecule. Together, these data suggest that the mechanism by which heparin is able to affect PECAM-1-dependent cell-cell adhesion is indirect and occurs via inhibition of events that occur downstream from PECAM-1 engagement.PECAM-1 (CD31) is a 130-kDa member of the Ig gene superfamily that is constitutively expressed on the surface of circulating platelets, monocytes, neutrophils, and selected T cell subsets. It is also present at relatively high concentration at the cell junctions of all continuous endothelium, both in vivo and in cell culture (for a recent review on the biology of PECAM-1, see Ref. 1). Because of its presence on these vascular cells, PECAM-1 has been implicated in mediating a number of cellular interactions, most notably those that take place between leukocytes and the vessel wall during the process of transendothelial migration (2-9) and between adjacent endothelial cells during the process of angiogenesis (10 -12).A number of different cell surface components have been implicated as counterreceptors or cellular targets for PECAM-1. Albelda et al. (13) found that PECAM-1 became concentrated at cell-cell borders only if both cells expressed PECAM-1, and they were the first to propose, based on this observation, that PECAM-1-mediated cellular interactions might operate homophilically, i.e. via PECAM-1/PECAM-1 intermolecular contacts. In support of this hypothesis, PECAM-1-containing proteoliposomes were recently shown to be able to self-associate in a concentration-dependent, divalent cationindependent, manner (14), providing direct experimental support that PECAM-1 is capable of interacting with itself. Homophilic binding activity requires amino-terminal Ig homology domains 1 and 2 (14, 15), and specific residues within Ig domain 1 that participate in PECAM-1/PECAM-1 interactions have recently been identified by Newton et al. (16). ...

Evidence That the Cellular Ligand for the Human NK Cell Activation Receptor NKp30 Is Not a Heparan Sulfate Glycosaminoglycan

“…These experiments illustrate that specificity must be controlled in binding studies using recombinant protein/human IgG1 Fc chimeras. In fact, nonspecific binding of PECAM-1/Fc chimeras was noted in an early study by Sun et al (26), and the potential for nonspecific binding of Fc chimeras to FcRs was noted in a recent study by Stark et al (14).…”

Angiogenesis in Platelet Endothelial Cell Adhesion Molecule-1-Null Mice

“…This suggests that PECAM-1-dependent heterophilic ligand interactions 2,6 with constituents of the extracellular matrix may be involved in the stimulation of filopodia formation by PECAM-1, although the existence of these interactions has been questioned. 4 Small GTPases of the Rho superfamily (RhoA, Rac1, and Cdc42) have been linked to actin cytoskeletal remodeling and morphological changes involved in cell motility, with Cdc42 implicated in the formation of filopodia. [31][32][33][34][35] Our data indicating that the protein levels of Cdc42 are increased in cells expressing PECAM-1 ( Figure 9) are therefore consistent with a role for PECAM-1 in the formation of filopodia.…”

“…[2][3][4][5][6] To determine whether PECAM-1-dependent ligand interactions might play a role in the ability of PECAM-1 to promote endothelial filopodia formation, we studied the effects of two functionally distinct antibodies: mAb 37, which blocks heterophilic binding; and mAb 62, which blocks both homophilic and heterophilic bindings. 49 We found that the initial formation of filopodia by HUVEC was suppressed by both antibodies but not by control IgG (Table 1).…”

“…1 Although it was originally recognized as a protein capable of binding interactions with itself and possibly other non-PECAM-1 molecules, [2][3][4][5][6] there is now a significant body of evidence that PECAM-1 also participates in signaling cascades. 7,8 Of note, PECAM-1 has two tyrosine residues (Y663 and Y686) that each fall within a conserved signaling sequence known as the immunoreceptor tyrosine-based inhibitory motif.…”

The Neutrophil-specific Antigen CD177 Is a Counter-receptor for Platelet Endothelial Cell Adhesion Molecule-1 (CD31)

“…mAb Gi18 directed against the first two domains of PECAM-1 was produced and characterized in our laboratory (29). mAbs PECAM 1.1 and PECAM 1.2 recognizing domains 5 and 6 of PECAM-1, respectively, have been previously described (24,30). mAbs 68-5H11 and AK-4 against E-and P-selectin, respectively, were purchased from BD Pharmingen (Heidelberg, Germany).…”