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.