Rapid changes in shear stress induce dissociation of a G alpha(q/11)-platelet endothelial cell adhesion molecule-1 complex.

It has been recently shown that endothelial platelet endothelial cell adhesion molecule-1 (PECAM-1) expression is pro-atherogenic. PECAM-1 is involved in sensing rapid changes in fluid shear stress but the mechanisms for activating signalling complexes at the endothelial cell junction have yet to be elucidated. Additional studies suggest the activation of membrane-bound G proteins G alpha(q/11) also mediate flow-induced responses. Here, we investigated whether PECAM-1 and G alpha(q/11) could act in unison to rapidly respond to fluid shear stress. With immunohistochemistry, we observed a co-localization of G alpha(q/11) and PECAM-1 at the cell-cell junction in the atheroprotected section of mouse aortae. In contrast, G alpha(q/11) was absent from junctions in atheroprone areas as well as in all arterial sections of PECAM-1 knockout mice. In primary human endothelial cells, temporal gradients in shear stress led to a rapid dissociation of the G alpha(q/11)-PECAM-1 complex within 30 s and a partial relocalization of the G alpha(q/11) staining to perinuclear areas within 150 min, whereas transitioning fluid flow devoid of temporal gradients did not disrupt the complex. Inhibition of G protein activation eliminated temporal gradient flow-induced G alpha(q/11)-PECAM-1 dissociation. These results allow us to conclude that G alpha(q/11)-PECAM-1 forms a mechanosensitive complex and its localization suggests the G alpha(q/11)-PECAM-1 complex is a critical mediator of vascular diseases.

Regulation of G protein-coupled receptor activities by the platelet-endothelial cell adhesion molecule, PECAM-1.

It is becoming increasingly evident that the cell-cell junction is a major signaling center. Here we show that the Galphaq/11 subunit of heterotrimeric G proteins forms a complex with platelet-endothelial cell adhesion molecule 1 (PECAM-1), a junctional protein that has been shown to be involved in mechanosignaling in endothelial cells. To understand the role of PECAM-1 in this complex, we determined the critical regions of PECAM-1 involved in this interaction. By expressing truncated forms of PECAM-1 in human embryonic kidney (HEK293) cells, we found that the cytoplasmic domain of PECAM-1 is not required for its association with Galphaq/11. Domain swapping of PECAM-1 with intracellular cell adhesion molecule 1 (ICAM-1), a protein that does not form a complex with Galphaq/11, provides evidence that the extracellular domain of PECAM-1 is critical for this interaction. This result also suggests that PECAM-1 does not directly interact with Galphaq/11. Coexpression of bradykinin receptor B2 (BKRB2), a Galphaq/11-coupled receptor, with PECAM-1 enhances formation of the PECAM-1-Galphaq/11 complex, suggesting an interaction between PECAM-1 and BKRB2. Co-immunoprecipitation experiments indicate that these two molecules indeed form a complex when expressed in HEK293 cells. Activation of ERK1/2 by bradykinin in HUVEC is enhanced when PECAM-1 expression is inhibited by transfection of small interference RNA against PECAM-1. Taken together, our results provide evidence of interaction of PECAM-1 with BKRB2 and of its possible role in regulating G protein-coupled receptor (GPCR) and G protein functions.