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.
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.
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.
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.
Calcium/calmodulin dependent protein kinase IIδ (CaMKIIδ) acts as a molecular switch regulating cardiovascular Ca handling and contractility in health and disease. Activation of CaMKIIδ is also known to regulate cardiovascular inflammation and is reported to be required for pro-inflammatory NF-κB signalling. In this study the aim was to characterise how CaMKIIδ interacts with and modulates NF-κB signalling and whether this interaction exists in non-contractile cells of the heart. Recombinant or purified CaMKIIδ and the individual inhibitory -κB kinase (IKK) proteins of the NF-κB signalling pathway were used in autoradiography and Surface Plasmon Resonance (SPR) to explore potential interactions between both components. Primary adult rat cardiac fibroblasts were then used to study the effects of selective CaMKII inhibition on pharmacologically-induced NF-κB activation as well as interaction between CaMKII and specific IKK isoforms in a cardiac cellular setting. Autoradiography analysis suggested that CaMKIIδ phosphorylated IKKβ but not IKKα. SPR analysis further supported a direct interaction between CaMKIIδ and IKKβ but not between CaMKIIδ and IKKα or IKKγ. CaMKIIδ regulation of IκΒα degradation was explored in adult cardiac fibroblasts exposed to pharmacological stimulation. Cells were stimulated with agonist in the presence or absence of a CaMKII inhibitor, autocamtide inhibitory peptide (AIP). Selective inhibition of CaMKII resulted in reduced NF-κB activation, as measured by agonist-stimulated IκBα degradation. Importantly, and in agreement with the recombinant protein work, an interaction between CaMKII and IKKβ was evident following Proximity Ligation Assays in adult cardiac fibroblasts. This study provides new evidence supporting direct interaction between CaMKIIδ and IKKβ in pro-inflammatory signalling in cardiac fibroblasts and could represent a feature that may be exploited for therapeutic benefit.
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