Proteinase-activated receptors 4 (PAR₄) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR₄ remain unknown. Here, we report novel features of the intracellular trafficking of PAR₄ to the plasma membrane. PAR₄ was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR₄ protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R¹⁸³AR → A¹⁸³AA), mutation of which allowed efficient membrane delivery of PAR₄. Interestingly, co-expression with PAR₂ facilitated plasma membrane delivery of PAR₄, an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR₂ and PAR₄. PAR₂ also enhanced glycosylation of PAR₄ and activation of PAR₄ signaling. Our results identify a novel regulatory role for PAR₂ in the anterograde traffic of PAR₄. PAR₂ was shown to both facilitate and abrogate protein interactions with PAR₄, impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR₄ in normal physiology and disease.

Background and purpose: Proteinase‐activated receptor 2 (PAR₂) is a G‐protein coupled receptor associated with many pathophysiological functions. To date, the development of PAR₂antagonists has been limited. Here, we identify a number of novel peptide‐mimetic PAR₂antagonists 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 PAR₂agonist peptides were screened for inhibition of PAR₂‐induced calcium mobilisation in human keratinocytes. Representative compounds were further evaluated by radioligand binding and inhibition of NFκB transcriptional activity and IL‐8 production. The vascular effects of the antagonists were assessed usingin vitroandin vivomodels.Key results: Two compounds, K‐12940 and K‐14585, significantly reduced SLIGKV‐induced Ca²⁺mobilisation in primary human keratinocytes. Both K‐12940 and K‐14585 exhibited competitive inhibition for the binding of a high‐affinity radiolabelled PAR₂‐ligand, [³H]‐2‐furoyl‐LIGRL‐NH₂, to human PAR₂with K_ivalues of 1.94 and 0.627 µM respectively. NFκB reporter activity and IL‐8 production were also significantly reduced. Furthermore, relaxation of rat‐isolated aorta induced by SLIGRL‐NH₂was inhibited competitively by K‐14585. K‐14585 also significantly lowered plasma extravasation in the dorsal skin of guinea pigs and reduced salivation in mice.Conclusions and implications: K‐12940 and K‐14585 antagonized PAR₂competitively, resulting in inhibition of PAR₂‐mediated signalling and physiological responses bothin vitroandin vivo. These peptide‐mimetic PAR₂antagonists could be useful in evaluating PAR₂‐mediated biological events and might lead to a new generation of therapeutically useful antagonists.

We have recently shown in a patient with mild bleeding that the PDZ-binding motif of the platelet G protein-coupled P2Y₁₂ receptor (P2Y₁₂R) is required for effective receptor traffic in human platelets. In this study we show for the first time that the PDZ motif-binding protein NHERF1 exerts a major role in potentiating G protein-coupled receptor (GPCR) internalization. NHERF1 interacts with the C-tail of the P2Y₁₂R and unlike many other GPCRs, NHERF1 interaction is required for effective P2Y₁₂R internalization. In vitro and prior to agonist stimulation P2Y₁₂R/NHERF1 interaction requires the intact PDZ binding motif of this receptor. Interestingly on receptor stimulation NHERF1 no longer interacts directly with the receptor but instead binds to the receptor via the endocytic scaffolding protein arrestin. These findings suggest a novel model by which arrestin can serve as an adaptor to promote NHERF1 interaction with a GPCR to facilitate effective NHERF1-dependent receptor internalization.

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), P2Y1 and P2Y12. 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 P2Y1 and P2Y12 receptor regulation, with particular emphasis on the structural motifs within the P2Y12 receptor, which are required to maintain regulatory protein interaction. The implications of these findings for platelet responsiveness are also discussed.