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.

Phosphorylation is considered a key event in the signalling and regulation of the μ opioid receptor (MOPr). Here we used mass spectroscopy to determine the phosphorylation status of the C-terminal tail of the rat MOPr expressed in HEK-293 cells. Under basal conditions, MOPr is phosphorylated on Ser363 and Thr370, while in the presence of morphine or [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO), the COOH-terminus is phosphorylated at three additional residues, Ser356, Thr357, and Ser375. Using N-terminal Glutathione S Transferase (GST) fusion proteins of the cytoplasmic, C-terminal tail of MOPr and point mutations of the same, we show that, in vitro, purified G protein-coupled receptor kinase 2 (GRK2) phosphorylates Ser375, PKC phosphorylates Ser363 whilst CaMKII phosphorylates Thr370. Phosphorylation of the GST fusion protein of the C-terminal tail of MOPr enhanced its ability to bind arrestin-2 and -3. Hence, our study identifies both the basal and agonist-stimulated phospho-acceptor sites in the C-terminal tail of MOPr, and suggests that the receptor is subject to phosphorylation and hence regulation by multiple protein kinases.

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).