Agonist-induced activation of the ␦-opioid receptor (␦OR) was recently shown to augment ␤-and ␥-secretase activities, which increased the production of ␤-amyloid peptide (A␤), known to accumulate in the brain tissues of Alzheimer's disease (AD) patients. Previously, the ␦OR variant with a phenylalanine at position 27 (␦OR-Phe27) exhibited more efficient receptor maturation and higher stability at the cell surface than did the less common cysteine (␦OR-Cys27) variant. For this study, we expressed these variants in human SH-SY5Y and HEK293 cells expressing exogenous or endogenous amyloid precursor protein (APP) and assessed the effects on APP processing. Expression of ␦OR-Cys27, but not ␦OR-Phe27, resulted in a robust accumulation of the APP C83 C-terminal fragment and the APP intracellular domain, while the total soluble APP and, particularly, the ␤-amyloid 40 levels were decreased. These changes upon ␦OR-Cys27 expression coincided with decreased localization of APP C-terminal fragments in late endosomes and lysosomes. Importantly, a long-term treatment with a subset of ␦OR-specific ligands or a c-Src tyrosine kinase inhibitor suppressed the ␦OR-Cys27-induced APP phenotype. These data suggest that an increased constitutive internalization and/or concurrent signaling of the ␦OR-Cys27 variant affects APP processing through altered endocytic trafficking of APP.Alzheimer's disease (AD) is the most common neurodegenerative disorder in the aging population. It is neuropathologically characterized by well-known hallmarks, such as extracellular amyloid plaques and intraneuronal neurofibrillary tangles, composed of ␤-amyloid peptide (A␤) and hyperphosphorylated tau, respectively. A␤ is generated from the amyloid precursor protein (APP) after sequential cleavages by ␤ (BACE1)-and ␥-secretases. It is a well-established fact that the molecular mechanisms underlying AD pathogenesis involve alterations in APP processing which lead to increased A␤ production or, alternatively, decreased enzymatic degradation and clearance of A␤ (39). To facilitate the design of novel intervention approaches for AD, it is important to identify and functionally characterize genetic alterations which play a role in AD pathogenesis. A plausible candidate in this context is the OPRD1 gene, encoding the ␦-opioid receptor (␦OR), which was recently shown to form a complex with ␤-and ␥-secretases (28, 40). Following agonist-induced activation, ␦OR mediates coendocytic sorting of this complex to late endosomes and lysosomes (LEL) (28,40), in which compartments A␤ production primarily takes place. Conversely, ␤-and ␥-secretase activities as well as A␤ levels were found to be significantly reduced in transgenic APP/PS1⌬E9 mice (overexpressing human APP with the Swedish mutation together with human presenilin-1 harboring the exon 9 deletion) treated with a selective nonpeptide antagonist for ␦OR (40). These results suggest that the amyloidogenic processing of APP is enhanced upon ␦OR activation and that the selective antagonist-mediated modulation of ␦OR ma...

Edited by F. Anne StephensonThe ␤ 1 -adrenergic receptor (␤ 1 AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for ␤-adrenergic antagonists, such as ␤-blockers, relatively little is yet known about its regulation. We have shown previously that ␤ 1 AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O-glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O-glycosylates ␤ 1 AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O-glycosylation and proteolytic cleavage assays, a cell line deficient in O-glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of ␤ 1 AR. Furthermore, we demonstrate that impaired O-glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the ␤AR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O-glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of ␤ 1 AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases. ␤-Adrenergic receptors (␤ARs)4 are G protein-coupled receptors (GPCRs) that activate intracellular signaling pathways mainly via the stimulatory G s protein after binding of agonists such as adrenaline and noradrenaline (1, 2). The ␤ARs exist as three subtypes, ␤ 1 , ␤ 2 , and ␤ 3 , and the former two are important in the regulation of the excitation-contraction coupling of the myocardium. The ␤ 1 AR is the predominant ␤AR subtype expressed in the heart and the main mediator of the endogenous catecholamine-stimulated positive chronotropy and inotropy (1, 2). Thus, it is the most important target receptor for ␤-adrenergic antagonists, also called ␤-blockers, which are widely used in the treatment of cardiac diseases, such as chronic heart failure, coronary artery disease, arrhythmias, and hypertension. However, these therapeutic agents have limited effectiveness in some patients and also exert adverse effects. Consequently, there is a growing need to better understand the underlying mechanisms in cardiac function and disease to develop alternative and more individualized treatment options that can improve clinical outcomes.During chronic heart failure, a persistent compensatory increase of catecholamines causes ␤ 1 AR desensitization and down-regulation. The density of ␤ 1 ARs at the plasma membrane is reduced by 50%, whereas that of ␤...

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