Cysteine 27 Variant of the δ-Opioid Receptor Affects Amyloid Precursor Protein Processing through Altered Endocytic Trafficking

Abstract

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...

Molecular Pharmacology ofδ-Opioid Receptors

“…Thus, the least frequent variant Cys27 (Gelernter and Kranzler, 2000) displays greater precursor retention in the ER and enhanced turnover of mature surface receptors compared with the more common Phe27 allele (Leskelä et al, 2009). These differences have been interpreted as manifestation of a gain-of-function phenotype with possible pathophysiological consequences (Leskelä et al, 2009), a hypothesis that was verified by showing that human neuronal cell lines that expressed the Cys27 variant displayed altered endocytic trafficking and abnormal processing of amyloid precursor protein (Sarajärvi et al, 2011).…”

“…2) (Gelernter and Kranzler, 2000). Both alleles of the nonsynonymous mutations display similar pharmacological and signaling properties (Leskelä et al, 2009), but their maturation and ligandindependent trafficking differ (Leskelä et al, 2009(Leskelä et al, , 2012Sarajärvi et al, 2011). Thus, the least frequent variant Cys27 (Gelernter and Kranzler, 2000) displays greater precursor retention in the ER and enhanced turnover of mature surface receptors compared with the more common Phe27 allele (Leskelä et al, 2009).…”

Cys-27 Variant of Human δ-Opioid Receptor Modulates Maturation and Cell Surface Delivery of Phe-27 Variant via Heteromerization

“…In our recent study we demonstrated that h␦OR F27C polymorphism might represent a risk factor for Alzheimer disease as heterozygotes were overrepresented among the Alzheimer disease patients in two independent study populations. We also observed that overexpression of the Cys-27 variant, but not that of the Phe-27 one, caused major changes in processing of the exogenously or endogenously expressed amyloid precursor protein in SH-SY5Y and HEK293 cells (3). This occurred most likely via a mechanism that relates to the enhanced constitutive internalization of h␦OR (4).…”

“…The human ␦-opioid receptor (h␦OR) has a common single-nucleotide polymorphism (T80G) that results in the replacement of phenylalanine (Phe) with cysteine (Cys) at the amino acid position 27 in the N-terminal domain of the receptor. The allelic frequency of the less common Cys-27 variant varies depending on the ethnic background and is around 10% in Caucasians (2,3). Recently, we demonstrated that the Cys-27 variant shows an altered trafficking profile when expressed in HEK293 and CHO cells (4).…”

N-Glycan-dependent and -independent Quality Control of Human δ Opioid Receptor N-terminal Variants

“…The human ␦OR (h␦OR) carries two N-glycans in its extracellular N-terminal domain at Asn 18 and Asn 33 (23), flanking a polymorphic site at position 27 (24). Unlike the Phe 27 variant, the less common Cys 27 variant (allelic frequency in Caucasians is ϳ10% (24,25)) matures inefficiently and is partially targeted for premature ERAD (26 -28). Removal of the two N-glycosylation sites of the h␦OR-Cys 27 variant by site-directed mutagenesis leads to a further decrease in receptor cell surface expression (23).…”