Abstract: The β1-adrenergic receptor (β1AR) is the predominant βAR in the heart and is the main target for β-adrenergic antagonists, widely used in the treatment of cardiovascular diseases. Previously, we have shown that the human (h)β1AR is cleaved in its N-terminus by a metalloproteinase, both constitutively and in a receptor activation dependent manner. Here, we investigated the specific events involved in β1AR regulation, focusing on the effects of long-term treatment with β-adrenergic ligands on receptor processing in stably transfected HEK293i cells. The key findings were verified using the transiently transfected hβ1AR and the endogenously expressed receptor in neonatal rat cardiomyocytes. By using flow cytometry and Western blotting we demonstrate that isoproterenol, S-propranolol, CGP-12177, pindolol and timolol, which displayed agonistic properties towards β1AR in either the adenylyl cyclase or the mitogen-activated protein kinase signaling pathways, induced cleavage of the mature cell surface receptor. In contrast, metoprolol, bisoprolol and CGP-20712 that showed no agonistic activity had only marginal or no effect. Importantly, the agonists also stabilized intracellular receptor precursors, possibly via their pharmacological chaperone action, and they also stabilized the receptor in vitro. The opposing effects on the two receptor forms thus led to an increase in the amount of cleaved receptor fragments at the plasma membrane. The results underscore the pluridimensionality of β-adrenergic ligands and extend this property from receptor activation and signaling to the regulation of β1AR levels. This phenomenon may contribute to the exceptional resistance of β1ARs to down-regulation and tendency toward up-regulation following long-term ligand treatments.
Abstract: The important role of G protein-coupled receptor homo/heteromerization in receptor folding, maturation, trafficking, and cell surface expression has become increasingly evident. Here we investigated whether the human δ-opioid receptor (hδOR) Cys-27 variant that shows inherent compromised maturation has an effect on the behavior of the more common Phe-27 variant in the early secretory pathway. We demonstrate that hδOR-Cys-27 acts in a dominant negative manner and impairs cell surface delivery of the co-expressed hδOR-Phe-27 and impairs conversion of precursors to the mature form. This was demonstrated by metabolic labeling, Western blotting, flow cytometry, and confocal microscopy in HEK293 and human SH-SY5Y neuroblastoma cells using differentially epitope-tagged variants. The hδOR-Phe-27 precursors that were redirected to the endoplasmic reticulum-associated degradation were, however, rescued by a pharmacological chaperone, the opioid antagonist naltrexone. Co-immunoprecipitation of metabolically labeled variants revealed that both endoplasmic reticulum-localized precursors and mature receptors exist as homo/heteromers. The existence of homo/heteromers was confirmed in living cells by bioluminescence resonance energy transfer measurements, showing that the variants have a similar propensity to form homo/heteromers. By forming both homomers and heteromers, the hδOR-Cys-27 variant may thus regulate the levels of receptors at the cell surface, possibly leading to altered responsiveness to opioid ligands in individuals carrying the Cys-27 variant.
Abstract: Agonist-induced activation of the δ-opioid receptor (δOR) was recently shown to augment β- and γ-secretase activities, which increased the production of β-amyloid peptide (Aβ), known to accumulate in the brain tissues of Alzheimer's disease (AD) patients. Previously, the δOR variant with a phenylalanine at position 27 (δOR-Phe27) exhibited more efficient receptor maturation and higher stability at the cell surface than did the less common cysteine (δOR-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 δOR-Cys27, but not δOR-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 β-amyloid 40 levels were decreased. These changes upon δOR-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 δOR-specific ligands or a c-Src tyrosine kinase inhibitor suppressed the δOR-Cys27-induced APP phenotype. These data suggest that an increased constitutive internalization and/or concurrent signaling of the δOR-Cys27 variant affects APP processing through altered endocytic trafficking of APP.
