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

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

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.

Citation

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

• Journal Article Type: Article
• Acceptance Date: Mar 17, 2016
• Online Publication Date: Mar 30, 2016
• Publication Date: Mar 30, 2016
• Deposit Date: Jan 17, 2020
• Journal: Journal of Biological Chemistry
• Print ISSN: 0021-9258
• Electronic ISSN: 1083-351X
• Publisher: American Society for Biochemistry and Molecular Biology
• Peer Reviewed: Peer Reviewed
• Volume: 291
• Issue: 21
• Pages: 11285-11299
• DOI: https://doi.org/10.1074/jbc.M115.710681
• Keywords: Chemical activation; Energy transfer; Enzyme activity; Enzymes; Membranes; Molecular biology; Proteins; Signaling; Bioluminescence resonance energy transfer; Extracellular protease; G protein coupled receptors; Intracellular stores; Membrane trafficking
• Public URL: https://nottingham-repository.worktribe.com/output/1311915
• Publisher URL: https://www.scopus.com/inward/record.uri?eid=2-s2.0-84971673244&doi=10.1074%2fjbc.M115.710681&partnerID=40&md5=38bb18a82a25f1cfbcfb04fe8054570a