Protease-activated Receptor 2 (PAR2) Protein and Transient Receptor Potential Vanilloid 4 (TRPV4) Protein Coupling Is Required for Sustained Inflammatory Signaling*
G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR 2 ), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR 2 regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR 2 agonist stimulated a transient increase in [Ca 2+ ] i . TRPV4 expression led to a markedly sustained increase in [Ca 2+ ] i . Removal of extracellular Ca 2+ and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A 2 and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR 2 generates arachidonic acid-derived lipid mediators, such as 5′,6′-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR 2 -induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR 2 was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR 2 signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR 2 -stimulated neurogenic inflammation. Thus, PAR 2 activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR 2 . Background: Receptors activate channels of sensory nerves to cause inflammation and pain by unknown mechanisms. Results: Protease-activated receptor 2 (PAR 2 ) stimulated transient receptor potential vanilloid 4 (TRPV4) by generation of channel agonists. This required a key TRPV4 tyrosine and induced inflammation. Conclusion: PAR 2 opens TRPV4 by functional coupling. Significance: Antagonism of PAR 2 -TRPV4 coupling could alleviate inflammation and pain.