Heparan Sulfates Mediate the Interaction between Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) and the Gαq/11 Subunits of Heterotrimeric G Proteins

Background: The mechanisms by which the PECAM-1G(q/11) mechanosensitive complex mediates endothelial flow responses remain unclear. Results: The PECAM-1G(q/11) complex contains heparan sulfate proteoglycans (HSPGs) and is disrupted by inhibition of HS. Conclusion: The interaction between PECAM-1 and G(q/11) may be mediated by the HS of the HSPG syndecan-1. Significance: Targeting specific HSPGs may be an effective strategy for the therapeutic treatment of vascular diseases. The endothelial cell-cell junction has emerged as a major cell signaling structure that responds to shear stress by eliciting the activation of signaling pathways. Platelet endothelial cell adhesion molecule-1 (PECAM-1) and heterotrimeric G protein subunits G(q) and 11 (G(q/11)) are junctional proteins that have been independently proposed as mechanosensors. Our previous findings suggest that they form a mechanosensitive junctional complex that discriminates between different flow profiles. The nature of the PECAM-1G(q/11) interaction is still unclear although it is likely an indirect association. Here, we investigated the role of heparan sulfates (HS) in mediating this interaction and in regulating downstream signaling in response to flow. Co-immunoprecipitation studies show that PECAM-1G(q/11) binding is dramatically decreased by competitive inhibition with heparin, pharmacological inhibition with the HS antagonist surfen, and enzymatic removal of HS chains with heparinase III treatment as well as by site-directed mutagenesis of basic residues within the extracellular domain of PECAM-1. Using an in situ proximity ligation assay, we show that endogenous PECAM-1G(q/11) interactions in endothelial cells are disrupted by both competitive inhibition and HS degradation. Furthermore, we identified the heparan sulfate proteoglycan syndecan-1 in complexes with PECAM-1 that are rapidly decreased in response to flow. Finally, we demonstrate that flow-induced Akt activation is attenuated in endothelial cells in which PECAM-1 was knocked down and reconstituted with a binding mutant. Taken together, our results indicate that the PECAM-1G(q/11) mechanosensitive complex contains an endogenous heparan sulfate proteoglycan with HS chains that is critical for junctional complex assembly and regulating the flow response.