Heparan Sulfates Mediate the Interaction Between PECAM-1 and the G{alpha}q/11 Subunits of Heterotrimeric G Proteins [Signal Transduction]

February 4th, 2014 by dela Paz, N. G., Melchior, B., Shayo, F. Y., Frangos, J. A.

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 alpha q and 11 (Gαq/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-1-Gα/11interaction is still unclear though 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-1-Gαq/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 in situ proximity ligation assay, we show that endogenous PECAM-1-Gαq/11 interactions in endothelial cells (ECs) are disrupted by both competitive inhibition and HS degradation. Furthermore, we identified the heparan sulfate proteoglycan (HSPG) syndecan-1 in complexes with PECAM-1 which are rapidly decreased in response to flow. Finally, we demonstrate that flow-induced Akt activation is attenuated in ECs in which PECAM-1 was knocked down and reconstituted with a binding mutant. Taken together, our results indicate that the PECAM-1-Gαq/11 mechanosensitive complex contains an endogenous HSPG with HS chains that are critical for junctional complex assembly and regulating the flow response.