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N-glycan-mediated quality control in the endoplasmic reticulum is required for the expression of correctly folded delta-opioid receptors at the cell surface.

ABSTRACT: A great majority of G protein-coupled receptors are modified by N-glycosylation, but the functional significance of this modification for receptor folding and intracellular transport has remained elusive. Here we studied these phenomena by mutating the two N-terminal N-glycosylation sites (Asn(18) and Asn(33)) of the human delta-opioid receptor, and expressing the mutants from the same chromosomal integration site in stably transfected inducible HEK293 cells. Both N-glycosylation sites were used, and their abolishment decreased the steady-state level of receptors at the cell surface. However, pulse-chase labeling, cell surface biotinylation, and immunofluorescence microscopy revealed that this was not because of intracellular accumulation. Instead, the non-N-glycosylated receptors were exported from the endoplasmic reticulum with enhanced kinetics. The results also revealed differences in the significance of the individual N-glycans, as the one attached to Asn(33) was found to be more important for endoplasmic reticulum retention of the receptor. The non-N-glycosylated receptors did not show gross functional impairment, but flow cytometry revealed that a fraction of them was incapable of ligand binding at the cell surface. In addition, the receptors that were devoid of N-glycans showed accelerated turnover and internalization and were targeted for lysosomal degradation. The results accentuate the importance of protein conformation-based screening before export from the endoplasmic reticulum, and demonstrate how the system is compromised when N-glycosylation is disrupted. We conclude that N-glycosylation of the delta-opioid receptor is needed to maintain the expression of fully functional and stable receptor molecules at the cell surface.

SUBMITTER: Markkanen PM

PROVIDER: S-EPMC2662010 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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N-glycan-mediated quality control in the endoplasmic reticulum is required for the expression of correctly folded delta-opioid receptors at the cell surface.

Markkanen Piia M H PM Petäjä-Repo Ulla E UE

The Journal of biological chemistry 20080814 43

A great majority of G protein-coupled receptors are modified by N-glycosylation, but the functional significance of this modification for receptor folding and intracellular transport has remained elusive. Here we studied these phenomena by mutating the two N-terminal N-glycosylation sites (Asn(18) and Asn(33)) of the human delta-opioid receptor, and expressing the mutants from the same chromosomal integration site in stably transfected inducible HEK293 cells. Both N-glycosylation sites were used ...[more]

PMID: 18703511

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Project description:The δ opioid receptor (δR) is a promising alternate target for pain management because δR agonists show decreased abuse potential compared with current opioid analgesics that target the μ opioid receptor. A critical limitation in developing δR as an analgesic target, however, is that δR agonists show relatively low efficacy in vivo, requiring the use of high doses that often cause adverse effects, such as convulsions. Here we tested whether intracellular retention of δR in sensory neurons contributes to this low δR agonist efficacy in vivo by limiting surface δR expression. Using direct visualization of δR trafficking and localization, we define a phosphatase and tensin homolog (PTEN)-regulated checkpoint that retains δR in the Golgi and decreases surface delivery in rat and mice sensory neurons. PTEN inhibition releases δR from this checkpoint and stimulates delivery of exogenous and endogenous δR to the neuronal surface both in vitro and in vivo PTEN inhibition in vivo increases the percentage of TG neurons expressing δR on the surface and allows efficient δR-mediated antihyperalgesia in mice. Together, we define a critical role for PTEN in regulating the surface delivery and bioavailability of the δR, explain the low efficacy of δR agonists in vivo, and provide evidence that active δR relocation is a viable strategy to increase δR antinociception.SIGNIFICANCE STATEMENT Opioid analgesics, such as morphine, which target the μ opioid receptor (μR), have been the mainstay of pain management, but their use is highly limited by adverse effects and their variable efficacy in chronic pain. Identifying alternate analgesic targets is therefore of great significance. Although the δ opioid receptor (δR) is an attractive option, a critical limiting factor in developing δR as a target has been the low efficacy of δR agonists. Why δR agonists show low efficacy is still under debate. This study provides mechanistic and functional data that intracellular localization of δR in neurons is a key factor that contributes to low agonist efficacy, and presents a proof of mechanism that relocating δR improves efficacy.

Dataset Information

N-glycan-mediated quality control in the endoplasmic reticulum is required for the expression of correctly folded delta-opioid receptors at the cell surface.

Publications

N-glycan-mediated quality control in the endoplasmic reticulum is required for the expression of correctly folded delta-opioid receptors at the cell surface.

Similar Datasets

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