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,Tinamarie Lieu,Marina Gerges,Holly R Yeatman,Meritxell Canals,Stephen J Vanner,Daniel P Poole,Nigel W Bunnett

doi:10.1074/jbc.m115.710681

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.

Highlights

The ability of cells to respond to extracellular signals depends, to a large degree, on the levels of receptors at the plasma membrane
We report the identification of a mechanism by which activation of a G protein-coupled receptors (GPCRs) at the cell surface triggers the rapid mobilization of trans-Golgi network (TGN) stores of fresh receptors, leading to replenishment of the plasma membrane with signaling-competent receptors
Our results show that proteolytic activation of protease-activated receptor-2 (PAR2) induces translocation of G␤␥ subunits to the Golgi apparatus and G␤␥mediated activation of protein kinase D (PKD) within the TGN

Summary

Introduction

The ability of cells to respond to extracellular signals depends, to a large degree, on the levels of receptors at the plasma membrane. 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 with a photoconvertible Kaede tag was expressed in KNRK cells to examine receptor translocation from the Golgi apparatus to the plasma membrane.

Results

Conclusion