title: Protein Kinase D and Gβγ Subunits Mediate Agonist-evoked Translocation of Protease-activated Receptor-2 from the Golgi Apparatus to the Plasma Membrane.
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 Ca(2+) 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.
authors.affiliation: From the Monash Institute of Pharmaceutical Sciences and Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville, Victoria 3052, Australia.
authors.forename: Dane D
authors.initials: DD
authors.lastname: Jensen
authors.affiliation: From the Monash Institute of Pharmaceutical Sciences and Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville, Victoria 3052, Australia.
authors.forename: Peishen
authors.initials: P
authors.lastname: Zhao
authors.affiliation: the Gastrointestinal Diseases Research Unit, Division of Gastroenterology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Nestor N
authors.initials: NN
authors.lastname: Jimenez-Vargas
authors.affiliation: From the Monash Institute of Pharmaceutical Sciences and Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville, Victoria 3052, Australia.
authors.forename: TinaMarie
authors.initials: T
authors.lastname: Lieu
authors.affiliation: From the Monash Institute of Pharmaceutical Sciences and.
authors.forename: Marina
authors.initials: M
authors.lastname: Gerges
authors.affiliation: From the Monash Institute of Pharmaceutical Sciences and.
authors.forename: Holly R
authors.initials: HR
authors.lastname: Yeatman
authors.affiliation: From the Monash Institute of Pharmaceutical Sciences and Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville, Victoria 3052, Australia.
authors.forename: Meritxell
authors.initials: M
authors.lastname: Canals
authors.affiliation: the Gastrointestinal Diseases Research Unit, Division of Gastroenterology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Vanner
authors.affiliation: From the Monash Institute of Pharmaceutical Sciences and Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville, Victoria 3052, Australia, the Departments of Anatomy and Neuroscience and daniel.poole@monash.edu.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Poole
authors.affiliation: From the Monash Institute of Pharmaceutical Sciences and Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville, Victoria 3052, Australia, Pharmacology and Therapeutics, University of Melbourne, Parkville, Victoria 3010, Australia, and nigel.bunnett@monash.edu.
authors.forename: Nigel W
authors.initials: NW
authors.lastname: Bunnett
mesh_terms: D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D020013:Calcium Signaling; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000037:antagonists & inhibitors / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D044464:Receptor, PAR-2 / Q000819:agonists* / Q000378:metabolism*; D014966:Xanthenes / Q000494:pharmacology
chemical_list: C551536:CRT 0066101; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D044464:Receptor, PAR-2; D014966:Xanthenes; C007820:gallein; C089212:protein kinase D; D011493:Protein Kinase C
keywords: 7-helix receptor; Golgi; protein kinase D (PKD); protein trafficking (Golgi); receptor desensitization