Background:The PDZ-binding motif of the P2Y 12 receptor regulates correct receptor traffic in human platelets. Results: The PDZ-binding protein NHERF1 binds to the P2Y 12 receptor to promote agonist-dependent internalization. Conclusion: Arrestin scaffolds NHERF1 to the P2Y 12 receptor to facilitate effective NHERF1-dependent receptor internalization. Significance: A novel model of arrestin-dependent GPCR internalization.

Background and purpose: Proteinase-activated receptor 2 (PAR2) is a G-protein coupled receptor associated with many pathophysiological functions. To date, the development of PAR2 antagonists has been limited. Here, we identify a number of novel peptide-mimetic PAR2 antagonists and demonstrate inhibitory effects on PAR2-mediated intracellular signalling pathways and vascular responses. Experimental approach: The peptide-mimetic compound library based on the structures of PAR2 agonist peptides were screened for inhibition of PAR2-induced calcium mobilisation in human keratinocytes. Representative compounds were further evaluated by radioligand binding and inhibition of NFkB transcriptional activity and IL-8 production. The vascular effects of the antagonists were assessed using in vitro and in vivo models.

Together, these results indicate that CLCb phosphorylation acts as a discriminator for the endocytosis of specific GPCRs.

Background: Bioinformatic analysis revealed that PAR 4 possesses an ER retention motif. Results: PAR 2 both abrogates and facilitates chaperone protein interaction with PAR 4 to allow PAR 4 to evade ER retention and be delivered to the plasma membrane. Conclusion: PAR 2 regulates PAR 4 localization and cell signaling through heterodimerization. Significance: Impact upon understanding PAR 2 and PAR 4 in inflammation where clear roles are defined.

Intensity-and amplitude-weighted average lifetimes, denoted as τ I and τ A hereafter, are useful indicators for revealing Förster resonance energy transfer (FRET) or fluorescence quenching behaviors. In this work, we discussed the differences between τ I and τ A and presented several model-free lifetime determination algorithms (LDA), including the center-of-mass, phasor, and integral equation methods for fast τ I and τ A estimations. For model-based LDAs, we discussed the model-mismatch problems, and the results suggest that a bi-exponential model can well approximate a signal following a multi-exponential model. Depending on the application requirements, suggestions about the LDAs to be used are given. The instrument responses of the imaging systems were included in the analysis. We explained why only using the τ I model for FRET analysis can be misleading; both τ I and τ A models should be considered. We also proposed using τ A /τ I as a new indicator on two-photon fluorescence lifetime images, and the results show that τ A /τ I is an intuitive tool for visualizing multi-exponential decays.

Platelets are critical for haemostasis, however inappropriate activation can lead to the development of arterial thrombosis, which can result in heart attack and stroke. ADP is a key platelet agonist that exerts its actions via stimulation of two surface GPCRs (G-protein-coupled receptors), P2Y(1) and P2Y(12). Similar to most GPCRs, P2Y receptor activity is tightly regulated by a number of complex mechanisms including receptor desensitization, internalization and recycling. In the present article, we review the molecular mechanisms that underlie P2Y(1) and P2Y(12) receptor regulation, with particular emphasis on the structural motifs within the P2Y(12) receptor, which are required to maintain regulatory protein interaction. The implications of these findings for platelet responsiveness are also discussed.

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Consumption of ethanol is a considerable risk factor for death in heroin overdose. We sought to determine whether a mildly intoxicating concentration of ethanol could alter morphine tolerance at the cellular level. In rat locus coeruleus (LC) neurons, tolerance to morphine was reversed by acute exposure of the brain slice to ethanol (20 mM).