Protease‐Activated Receptor Dimerization and Signaling

Thrombin activates endothelial cells by proteolytic cleavage of a family of specialized G‐protein coupled receptors, protease‐activated receptors (PARs), causing an increase in lung vascular endothelial permeability and may contribute to the formation of pulmonary edema associated complications in lung injury. Recombinant expression systems, transgenic animal models, and mouse studies have demonstrated an interdependency between receptor isoforms and their associated signaling networks which cannot be recapitulated by their individual signaling pathways. To address this, we postulate that these receptors form distinct homo‐ and heterodimers resulting in a functional receptor complex with unique signaling properties. We show here for the first time direct evidence that these receptors form distinct homo‐ and heterodimers via adaptation of a new approach involving bioluminescent resonance energy transfer, BRET. We have also begun to address the consequence of receptor dimerization on G‐protein coupling, or functional selectivity, by a similar, BRET‐based approach involving labeled G‐proteins and receptors. These studies provide evidence for a functional role of thrombin receptor dimerization in endothelial cell signaling and shed light on the possible mechanisms involved in receptor activation and G‐protein selectivity.