Jensen Dane D, Zhao Peishen, Jimenez-Vargas Nestor N, Lieu TinaMarie, Gerges Marina, Yeatman Holly R, Canals Meritxell, Vanner Stephen J, Poole Daniel P, Bunnett Nigel W
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 Gastrointestinal Diseases Research Unit, Division of Gastroenterology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
J Biol Chem. 2016 May 20;291(21):11285-99. doi: 10.1074/jbc.M115.710681. Epub 2016 Mar 30.
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.
G蛋白偶联受体激动剂诱发的内吞作用已得到广泛研究。激动剂刺激G蛋白偶联受体从细胞内储存库动员并转位至质膜的机制尚未得到探索。蛋白酶激活受体2（PAR2）会转运至溶酶体，而持续的蛋白酶信号传导需要PAR2从高尔基体储存库动员并转运至质膜。我们评估了蛋白激酶D（PKD）和Gβγ在此过程中的作用。在HEK293和KNRK细胞中，PAR2激动剂胰蛋白酶和2-呋喃甲酰-LIGRLO-NH2在高尔基体中激活PKD，而PKD在高尔基体中调节蛋白质转运。通过Gγ-金星蛋白和巨蛋白-Rluc8之间的生物发光共振能量转移确定，PAR2激活诱导PKD激活剂Gβγ转位至高尔基体。PKD抑制剂（CRT0066101）和Gβγ抑制剂（加列因）可阻止PAR2刺激的PKD激活。CRT0066101、PKD1小干扰RNA和加列因均抑制PAR2诱发的Ca(2+)信号恢复。在KNRK细胞中表达带有光转换型Kaede标签的PAR2，以检查受体从高尔基体转位至质膜的情况。对高尔基体区域（405 nm）进行照射可诱导PAR2-Kaede发生绿-红光电转换。胰蛋白酶使PAR2-Kaede从高尔基体中耗尽，并在质膜处补充PAR2-Kaede。CRT0066101抑制PAR2-Kaede转位至质膜。CRT0066101还抑制对结肠细胞和伤害性神经元的持续蛋白酶信号传导，这些细胞天然表达PAR2并介导蛋白酶诱发的炎症和伤害感受。我们的结果揭示了PKD和Gβγ在激动剂诱发的细胞内PAR2储存库动员中的主要作用，这是细胞外蛋白酶持续信号传导所必需的。
