Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization

Abstract

Thrombin activates endothelial cell signaling by cleaving the protease-activated receptor-1 (PAR1). However, the function of the apparently nonsignaling receptor PAR3 also expressed in endothelial cells is unknown. We demonstrate here the crucial role of PAR3 in potentiating the responsiveness of PAR1 to thrombin. We tested the hypothesis that PAR1/PAR3 heterodimerization and its effect in modifying G protein selectivity was responsible for PAR3 regulation of PAR1 sensitivity. Using bioluminescent resonance energy transfer-2, we showed that PAR1 had comparable dimerization affinity for PAR3 as for itself. We observed increased G␣13 coupling between the PAR1/3 heterodimer compared with the PAR1/1 homodimer. Moreover, knockdown of PAR3 moderated the PAR1-activated increase in endothelial permeability. These results demonstrate a role of PAR3 in allosterically regulating PAR1 signaling governing increased endothelial permeability. Because PAR3 is a critical determinant of PAR1 function, targeting of PAR3 may mitigate the effects of PAR1 in activating endothelial responses such as vascular inflammation.T hrombin generation during thrombosis contributes to the pathophysiology of multiple thrombosis-related diseases such as acute lung injury resulting increased endothelial permeability (1), vascular inflammation (1, 2), and edema formation (for recent reviews, see refs. 3 and 4). These complications are the result of thrombin activation of protease-activated receptor-1 (PAR1) (5). Cleavage of the extracellular N terminus of PAR1 initiates signaling (6) by the concomitant activation of heterotrimeric GTP-binding proteins G␣ q and G␣ 12/13 (7-9). The mechanisms by which PAR1 selectively activates G protein signaling remain unknown. Because the nonsignaling receptor PAR3 is also expressed in endothelial cells (10), we surmised that PAR1 and PAR3 are capable of interacting in such manner as to regulate PAR1 signaling. We found that PAR3 directly dimerizes with PAR1 to induce a specific PAR1/G␣ 13 -binding conformation that favors G␣ 13 activation. We propose a model of PAR1 activation involving the interaction with PAR3, which alters the selectivity of PAR1 for G␣ 13 coupling and promotes endothelial barrier dysfunction. PAR3 functions as an essential allosteric modulator of PAR1 signaling through PAR1/3 dimerization and favors a distinct G␣ 13 -activated downstream pathway. Results PAR3 Knockdown in Endothelial Cells Shifts the Potency of ThrombinActivation of PAR1. Endothelial cells express PAR1, -2, and -3, but not PAR4 (10), and of these only PAR1 and PAR3 are thrombinsensitive (11). To address whether PAR3 alters thrombin signaling in endothelial cells, siRNA knockdown was used to reduce thrombin receptor expression in human pulmonary artery endothelial cells (HPAECs). PAR1 and PAR3 suppression was determined by semiquantitative RT-PCR. siRNA against PAR3 did not affect PAR1 expression, whereas it reduced PAR3 expression 8-fold. Conversely, siRNA against PAR1 only modestly affected PAR3 expression, whereas it suppres...

Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics

“…The co-immunoprecipitation and FRET data demonstrate that PAR1 self-associates to form a homodimer or homooligomer, consistent with previous BRET experiments in HEK cells (55). Although the exact structure and relative orientation of the homodimer are difficult to determine with certainty, the direct binding data presented here indicate that a stable homodimer may form with each i3 loop interacting with an H8 helix across the dimer interface.…”

“…homodimer (55) in HEK cells. Similarly, PAR1 has been documented to heterodimerize with the closely related PAR2 and PAR4 receptors on endothelial cells and/or platelets (22,26).…”

Thrombin Promotes Release of ATP from Lung Epithelial Cells through Coordinated Activation of Rho- and Ca2+-dependent Signaling Pathways

“…PAR3 suppression resulted in ϳ50% reduction of transendothelial electrical resistance, while PAR1 suppression completely reduced transendothelial electrical resistance in response to thrombin. Based on bioluminescent resonance energy transfer-2 measurements, the authors concluded that PAR3 dimerizes with and regulates PAR1 signaling (65). Our data, illustrating that (i) PAR1AP and PAR4AP fail to promote cellular responses in A549 cells, (ii) PAR3 is the only PAR transcript present in these cells, and (iii) PAR3 siRNA decreased thrombin-evoked responses, indicate that PAR3 is the major thrombin receptor functionally present in these cells.…”

Dynamic interactions ofPlasmodiumspp. with vascular endothelium

“…Thrombin is known to induce permeability through PAR-1 which heterodimerizes with PAR-3 leading to G a13 -dependent calcium signaling, actin stress fiber formation and transient increases in endothelial permeability. 70 Moreover, a subset of parasites from patients with CM has been shown to bind to EPCR, and may compete with APC for the binding to this receptor. 55,71,72 As APC exerts a protective effect on thrombin induced barrier dysfunction, IRBC adhesion may exaggerate the barrier dysfunction elicited by thrombin.…”