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 (PAR) on nociceptors through distinct mechanisms. Whereas trypsin induces PAR coupling to Gα, Gα, and β-arrestins, cathepsin-S (CS) and neutrophil elastase (NE) cleave PAR at distinct sites and activate it by biased mechanisms that induce coupling to Gα, but not to Gα or β-arrestins. Because proteases activate PAR by irreversible cleavage, and activated PAR is degraded in lysosomes, sustained extracellular protease-mediated signaling requires mobilization of intact PAR from the Golgi apparatus or synthesis of new receptors by incompletely understood mechanisms. We found here that trypsin, CS, and NE stimulate PAR-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 PAR mobilization from the Golgi. Proteases also induced translocation of a photoconverted PAR-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, PAR responsiveness initially declined, consistent with PAR cleavage and desensitization, and then gradually recovered. Inhibitors of PKD, Gβγ, and protein translation inhibited recovery of PAR responsiveness. PKD and Gβγ inhibitors also attenuated protease-evoked mechanical allodynia in mice. We conclude that proteases that activate PAR by canonical and biased mechanisms stimulate PKD in the Golgi; PAR mobilization and synthesis repopulate the cell surface with intact receptors and sustain nociceptive signaling by extracellular proteases.