The Endoplasmic Reticulum Ca2+-pump SERCA2b Interacts with G Protein-coupled Receptors and Enhances their Expression at the Cell Surface

Tuusa, Jussi; Markkanen, Piia; Apaja, Pirjo M.; Hakalahti, Anna E.; Petäjä-Repo, Ulla E.

doi:10.1016/j.jmb.2007.02.108

Cited by 20 publications

(33 citation statements)

References 57 publications

(70 reference statements)

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“…1B). As we have shown previously (18,23), one major species of M r 61,000 and two minor ones of M r 47,000 and M r 51,000 were immunoprecipitated from induced cells that were transfected with the wild-type receptor construct (Fig. 1B, lane 2).…”

Section: Results H␦or Carries Two N-linked Glycans Both Of Which Aresupporting

confidence: 78%

“…The total pool of receptors was purified by two-step immunoprecipitation using anti-FLAG M2 antibody, and the calnexin-interacting receptor pool was recovered from the denatured anti-calnexin immunoprecipitate by second immunoprecipitation with anti-FLAG M2 antibody. As we have previously reported (18,23), the wild-type receptor precursor was readily recovered from the calnexin immunoprecipitate, whereas only a trace amount of the non-N-glycosylated receptor was recovered (Fig. 3B, lanes 1 and 4, respectively).…”

Section: Receptorsupporting

confidence: 78%

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N-Glycan-mediated Quality Control in the Endoplasmic Reticulum Is Required for the Expression of Correctly Folded δ-Opioid Receptors at the Cell Surface

Markkanen

Petäjä-Repo

2008

Journal of Biological Chemistry

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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 ␦-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 ␦-opioid receptor is needed to maintain the expression of fully functional and stable receptor molecules at the cell surface.

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Section: Results H␦or Carries Two N-linked Glycans Both Of Which Aresupporting

confidence: 78%

Section: Receptorsupporting

confidence: 78%

N-Glycan-mediated Quality Control in the Endoplasmic Reticulum Is Required for the Expression of Correctly Folded δ-Opioid Receptors at the Cell Surface

Markkanen

Petäjä-Repo

2008

Journal of Biological Chemistry

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“…The eluted samples were diluted 10-fold with DDM buffer supplemented with 0.1% BSA and used for a second immunoprecipitation with the anti-APP-CTF antibody (A8717) coupled to protein A-Sepharose (GE Healthcare), followed by washes as described above and elution with the SDS-PAGE sample buffer. Aliquots of immunoprecipitates and of whole-cell extracts containing equal amounts of protein were separated by 10% SDS-PAGE and analyzed by Western blotting or fluorography as described previously (41).…”

Section: Methodsmentioning

confidence: 99%

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

Sarajärvi

Tuusa

Haapasalo

et al. 2011

Molecular and Cellular Biology

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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...

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“…This ER phenotype has a condensed shape instead of the typical, expanded reticular network ( e.g. , Chapple and Cheetham 2003 ;Robert et al 2005 ;Tuusa et al 2007 ;Duvernay et al 2009 ;Málaga-Diéguez et al 2010 ) . The microscopic representation is reminiscent of organized structures of the smooth endoplasmic reticulum (OSERs, Fig.…”

Section: A Consideration Regarding the Experimental Assessment Of Er mentioning

confidence: 97%

“…under conditions where UPR might occur. By contrast, the interaction was much weaker with an endogenous receptor (luteotropin receptor in ovarian follicular cells); although expressed to high levels the endogenous receptor was subject to ef fi cient maturation (thus synthesized under conditions of less ER-stress; Tuusa et al 2007 ) .…”

Section: A Consideration Regarding the Experimental Assessment Of Er mentioning

confidence: 99%

ER-Bound Steps in the Biosynthesis of G Protein-Coupled Receptors

Nanoff

Freissmuth

2012

Subcellular Biochemistry

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The polypeptide of a G protein-coupled receptor is inserted into the membrane of the endoplasmic reticulum while being translated and this process by itself may be sufficient to establish the proper receptor fold. X-ray structures reveal a common polypeptide topology with little variation in the alignment and orientation of the seven transmembrane segments, the proximal carboxyl terminus (C-tail) and parts of the extracellular loops. These define a structural core the stability of which probably represents a major criterion for the receptor to pass endoplasmic reticulum (ER) quality control; point mutations affecting the structure of the core have an extraordinary chance of causing receptor retention. In contrast, cytoplasmic loops 2 and 3 and the distal C-tail are poorly ordered at least in the absence of an interaction partner. Similarly, the amino terminal tail of rhodopsin-related receptors (but not of receptor subtypes where ligand binding requires a stable fold of the N-tail) is unlikely to establish a stable fold. These segments can cause ER retention when mutated to inappropriately expose hydrophobic peptide patches; to prevent protein aggregation chaperone molecules attach to them thus initiating selection for ER-associated degradation. It is less clear however if there are additional mechanisms to specifically survey the transmembrane core at the level of the lipid bilayer or if insufficient packing is detected due to misalignment of the cytoplasmic or extracellular face of the receptor.

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The Endoplasmic Reticulum Ca2+-pump SERCA2b Interacts with G Protein-coupled Receptors and Enhances their Expression at the Cell Surface

Cited by 20 publications

References 57 publications

N-Glycan-mediated Quality Control in the Endoplasmic Reticulum Is Required for the Expression of Correctly Folded δ-Opioid Receptors at the Cell Surface

N-Glycan-mediated Quality Control in the Endoplasmic Reticulum Is Required for the Expression of Correctly Folded δ-Opioid Receptors at the Cell Surface

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

ER-Bound Steps in the Biosynthesis of G Protein-Coupled Receptors

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