Protein kinase D and Gβγ mediate sustained nociceptive signaling by biased agonists of protease-activated receptor-2.

Proteases sustain hyperexcitability and pain by cleaving protease-activated receptor-2 (PAR₂) on nociceptors through distinct mechanisms. Whereas trypsin induces PAR₂ coupling to Gα_q, Gα_s, and β-arrestins, cathepsin-S (CS) and neutrophil elastase (NE) cleave PAR₂ at distinct sites and activate it by biased mechanisms that induce coupling to Gα_s, but not to Gα_q or β-arrestins. Because proteases activate PAR₂ by irreversible cleavage, and activated PAR₂ is degraded in lysosomes, sustained extracellular protease-mediated signaling requires mobilization of intact PAR₂ from the Golgi apparatus or de novo synthesis of new receptors by incompletely understood mechanisms. We found here that trypsin, CS, and NE stimulate PAR₂-dependent activation of protein kinase D (PKD) in the Golgi of HEK293 cells, in which PKD regulates protein trafficking. The proteases stimulated translocation of the PKD activator Gβγ to the Golgi, coinciding with PAR₂ mobilization from the Golgi. Proteases also induced translocation of a photoconverted PAR₂-Kaede fusion protein from the Golgi to the plasma membrane of KNRK cells. After incubation of HEK293 cells and dorsal root ganglia neurons with CS, NE, or trypsin, PAR₂ responsiveness initially declined, consistent with PAR₂ cleavage and desensitization, and then gradually recovered. Inhibitors of PKD, Gβγ, and protein translation inhibited recovery of PAR₂ responsiveness. PKD and Gβγ inhibitors also attenuated protease-evoked mechanical allodynia in mice. We conclude that proteases that activate PAR₂ by canonical and biased mechanisms stimulate PKD in the Golgi; PAR₂ mobilization and de novo synthesis repopulate the cell surface with intact receptors and sustain nociceptive signaling by extracellular proteases.

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