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Contributions of protein kinases and ?-arrestin to termination of protease-activated receptor 2 signaling.

ABSTRACT: Activated Gq protein-coupled receptors (GqPCRs) can be desensitized by phosphorylation and ?-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates Gq and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3). We used fluorescent protein-tagged optical probes to monitor several consequences of PAR2 signaling, including PIP2 depletion and ?-arrestin translocation in real time. During continuous activation of PAR2, PIP2 was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of ?-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP2 restoration significantly and even adding a delayed secondary phase of further PIP2 depletion. These effects of ?-arrestin knockdown on PIP2 recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient ?-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein-coupled receptor kinase potentiated the PIP2 depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited ?-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by ?-arrestin to explain why knockdown of ?-arrestin led to secondary depletion of PIP2. Indeed, artificial recruitment of PIP5K removed the secondary loss of PIP2 completely. Altogether, our experimental and theoretical approaches demonstrate roles and dynamics of the protein kinases, ?-arrestin, and PIP5K in the desensitization of PAR2.

SUBMITTER: Jung SR

PROVIDER: S-EPMC4772372 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling.

Jung Seung-Ryoung SR Seo Jong Bae JB Deng Yi Y Asbury Charles L CL Hille Bertil B Koh Duk-Su DS

The Journal of general physiology 20160301 3

Activated Gq protein-coupled receptors (GqPCRs) can be desensitized by phosphorylation and β-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates Gq and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3). We used fluorescent protein-tagg ...[more]

PMID: 26927499

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Project description:Given the limited options and often harmful side effects of current analgesics and the suffering caused by the opioid crisis, new classes of pain therapeutics are needed. Protease-activated receptors (PARs), particularly PAR2, are implicated in a variety of pathologies, including pain. Since the discovery of the role of PAR2 in pain, development of potent and specific antagonists has been slow. In this study, we describe the in vivo characterization of a novel small molecule/peptidomimetic hybrid compound, C781, as a β-arrestin-biased PAR2 antagonist. In vivo behavioral studies were done in mice using von Frey filaments and the Mouse Grimace Scale. Pharmacokinetic studies were done to assess pharmacokinetic/pharmacodynamic relationship in vivo. We used both prevention and reversal paradigms with protease treatment to determine whether C781 could attenuate protease-evoked pain. C781 effectively prevented and reversed mechanical and spontaneous nociceptive behaviors in response to small molecule PAR2 agonists, mast cell activators, and neutrophil elastase. The ED50 of C781 (intraperitoneal dosing) for inhibition of PAR2 agonist (20.9 ng 2-AT)-evoked nociception was 6.3 mg/kg. C781 was not efficacious in the carrageenan inflammation model. Pharmacokinetic studies indicated limited long-term systemic bioavailability for C781 suggesting that optimizing pharmacokinetic properties could improve in vivo efficacy. Our work demonstrates in vivo efficacy of a biased PAR2 antagonist that selectively inhibits β-arrestin/MAPK signaling downstream of PAR2. Given the importance of this signaling pathway in PAR2-evoked nociception, C781 exemplifies a key pharmacophore for PAR2 that can be optimized for clinical development. PERSPECTIVE: Our work provides evidence that PAR2 antagonists that only block certain aspects of signaling by the receptor can be effective for blocking protease-evoked pain in mice. This is important because it creates a rationale for developing safer PAR2-targeting approaches for pain treatment.

Dataset Information

Contributions of protein kinases and ?-arrestin to termination of protease-activated receptor 2 signaling.

Publications

Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling.

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