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Protein kinase D and G?? 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.


Dane D. Jensen

Peishen Zhao

Nestor N. Jimenez-Vargas

Tina Marie Lieu

Marina Gerges

Holly R. Yeatman

Stephen J. Vanner

Daniel P. Poole

Nigel W. Bunnett


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 G?? 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 G??, a PKD activator, to the Golgi apparatus, determined by bioluminescence resonance energy transfer between G?-Venus and giantin-Rluc8. Inhibitors of PKD (CRT0066101) and G?? (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 G?? in agonist-evoked mobilization of intracellular PAR2 stores that is required for sustained signaling by extracellular proteases.


Jensen, D. D., Zhao, P., Jimenez-Vargas, N. N., Lieu, T. M., Gerges, M., Yeatman, H. R., …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.

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
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;
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