TY - JOUR
T1 - Protein kinase D and Gβγ mediate sustained nociceptive signaling by biased agonists of protease-activated receptor-2
AU - Zhao, Peishen
AU - Pattison, Luke A.
AU - Jensen, Dane D.
AU - Jimenez-Vargas, Nestor N.
AU - Latorre, Rocco
AU - Lieu, Tinamarie
AU - Jaramillo, Josue O.
AU - Lopez-Lopez, Cintya
AU - Poole, Daniel P.
AU - Vanner, Stephen J.
AU - Schmidt, Brian L.
AU - Bunnett, Nigel W.
N1 - Publisher Copyright:
© 2019 Zhao et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2019/7/5
Y1 - 2019/7/5
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.RA118.006935
DO - 10.1074/jbc.RA118.006935
M3 - Article
C2 - 31142616
AN - SCOPUS:85069045524
SN - 0021-9258
VL - 294
SP - 10649
EP - 10662
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 27
ER -