Protein kinase D and Gβγ subunits mediate agonist-evoked translocation of protease-activated receptor-2 from the golgi apparatus to the plasma membrane

Dane D. Jensen, Peishen Zhao, Nestor N. Jimenez-Vargas, Tina Marie Lieu, Marina Gerges, Holly R. Yeatman, Meritxell Canals, Stephen J. Vanner, Daniel P. Poole, Nigel W. Bunnett

Research output: Contribution to journalArticle

Abstract

Agonist-evoked endocytosis of G protein-coupled receptors has been extensively studied. The mechanisms by which agonists stimulate mobilization and plasma membrane translocation of G protein-coupled receptors from intracellular stores are unexplored. Protease-activated receptor-2 (PAR2 ) traffics to lysosomes, and sustained protease signaling requires mobilization and plasma membrane trafficking of PAR2 from Golgi stores. We evaluated the contribution of protein kinase D (PKD) and Gβγ to this process. In HEK293 and KNRK cells, the PAR2 agonists trypsin and 2-furoyl-LIGRLO-NH2 activated PKD in the Golgi apparatus, where PKD regulates protein trafficking. PAR2 activation induced translocation of Gβγ, a PKD activator, to the Golgi apparatus, determined by bioluminescence resonance energy transfer between Gγ-Venus and giantin-Rluc8. Inhibitors of PKD (CRT0066101) and Gβγ (gallein) prevented PAR2 -stimulated activation of PKD. CRT0066101, PKD1 siRNA, and gallein all inhibited recovery of PAR2-evoked Ca2+ signaling. PAR2 with a photoconvertible Kaede tag was expressed in KNRK cells to examine receptor translocation from the Golgi apparatus to the plasma membrane. Irradiation of the Golgi region (405 nm) induced green-red photo-conversion of PAR2-Kaede. Trypsin depleted PAR2-Kaede from the Golgi apparatus and repleted PAR2-Kaede at the plasma membrane. CRT0066101 inhibited PAR2-Kaede translocation to the plasma membrane. CRT0066101 also inhibited sustained protease signaling to colonocytes and nociceptive neurons that naturally express PAR2 and mediate protease-evoked inflammation and nociception. Our results reveal a major role for PKD and Gβγ in agonist-evoked mobilization of intracellular PAR2 stores that is required for sustained signaling by extracellular proteases.

Original languageEnglish (US)
Pages (from-to)11285-11299
Number of pages15
JournalJournal of Biological Chemistry
Volume291
Issue number21
DOIs
StatePublished - May 20 2016

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Jensen, D. D., Zhao, P., Jimenez-Vargas, N. N., Lieu, T. M., Gerges, M., Yeatman, H. R., Canals, M., Vanner, S. J., Poole, D. P., & Bunnett, N. W. (2016). Protein kinase D and Gβγ subunits mediate agonist-evoked translocation of protease-activated receptor-2 from the golgi apparatus to the plasma membrane. Journal of Biological Chemistry, 291(21), 11285-11299. https://doi.org/10.1074/jbc.M115.710681