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

Peishen Zhao, Luke A. Pattison, Dane D. Jensen, Nestor N. Jimenez-Vargas, Rocco Latorre, Tinamarie Lieu, Josue O. Jaramillo, Cintya Lopez-Lopez, Daniel P. Poole, Stephen J. Vanner, Brian L. Schmidt, Nigel W. Bunnett

Research output: Contribution to journalArticlepeer-review

Abstract

Proteases sustain hyperexcitability and pain by cleaving protease-activated receptor-2 (PAR2) on nociceptors through distinct mechanisms. Whereas trypsin inducesPAR2 coupling to Gαq,Gαs, 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 Gαs, but not to Gαq or β-arrestins. Because proteases activate PAR2 by irreversible cleavage, and activated PAR2 is degraded in lysosomes, sustained extracellular proteasemediated 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 PAR 2-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 activatePAR2 by canonical and biased mechanisms stimulate PKD in the Golgi; PAR2 mobilization and de novo synthesis repopulate the cell surface with intact receptorsandsustain nociceptive signaling by extracellular proteases.

Original languageEnglish (US)
Pages (from-to)10649-10662
Number of pages14
JournalJournal of Biological Chemistry
Volume294
Issue number27
DOIs
StatePublished - Jul 5 2019

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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