Allosteric Regulation of G‐protein Coupled Receptor Signaling by Receptor Hetero‐Dimerization

Divergent signaling pathways can be induced through individual G‐protein coupled receptors (GPCRs). To address molecular mechanisms governing this, we studied the interaction between the GPCR protease‐activated receptor 1 (PAR‐1) and the transmembrane endothelial protein C receptor (EPCR). Proteases such as thrombin and activated protein C (aPC), the ligand for EPCR, activate PAR‐1 by cleaving its N‐terminus to produce the same tethered‐ligand yet result in opposing signaling networks. We used bioluminescent resonance energy transfer to show that in the absence of protease ligands, unactivated PAR‐1 dimerizes with EPCR. However, proteolytically activated PAR‐1*/EPCR interaction was maintained with aPC but not thrombin. Using transendothelial electrical resistance we showed that both aPC and thrombin induce G 13 signaling. However, aPC failed to induce G q ‐mediated intracellular calcium mobilization and the pro‐inflammatory G q /NF‐κB pathway compared to thrombin. aPC‐induced PAR‐1/G q signaling appears to be impaired by aPC‐bound EPCR and is relieved when EPCR is depleted using siRNA. Since thrombin alone cannot maintain PAR‐1*/EPCR interaction, both G q and G 13 signaling are enabled which leads to barrier dysfunction and inflammation. The PAR‐1/EPCR interface may represent an allosteric regulatory site capable of functionally modulating PAR‐1 signaling.