Accumulating evidence has indicated that membrane-permeable G protein-coupled receptor ligands can enhance cell surface targeting of their cognate wild-type and mutant receptors. This pharmacological chaperoning was thought to result from ligand-mediated stabilization of immature receptors in the endoplasmic reticulum (ER). In the present study, we directly tested this hypothesis using wild-type and mutant forms of the human ␦-opioid receptor as models. ER-localized receptors were isolated by expressing the receptors in HEK293 cells under tightly controlled tetracycline induction and blocking their ER export with brefeldin A. The ER-retained ␦-opioid receptor precursors were able to bind [ 3 H]diprenorphine with high affinity, and treatment of cells with an opioid antagonist naltrexone led to a 2-fold increase in the number of binding sites. After removing the transport block, the antagonist-mediated increase in the number of receptors was detectable at the cell surface by flow cytometry and cell surface biotinylation assay. Importantly, opioid ligands, both antagonists and agonists, were found to stabilize the ER-retained receptor precursors in an in vitro heat inactivation assay and the treatment enhanced dissociation of receptor precursors from the molecular chaperone calnexin. Thus, we conclude that pharmacological chaperones facilitate plasma membrane targeting of ␦-opioid receptors by binding and stabilizing receptor precursors, thereby promoting their release from the stringent ER quality control. Endoplasmic reticulum (ER)2 quality control, involving molecular chaperones and folding factors, scrutinizes newly synthesized proteins and allows only correctly folded and assembled ones to proceed through the secretory pathway (1). Proteins that do not fulfill the criteria of the quality control are targeted for retrotranslocation and degradation in the cytosol by the ER-associated degradation pathway (2). Since ER quality control relies on conformational rather than functional criteria, even minor changes in the primary structure of a protein can lead to intracellular retention, thus preventing the affected protein from reaching its correct location in the cell. Thus, even salvageable proteins that might be functionally active can be incorrectly directed for degradation. Such an etiology is the underlying cause for a growing number of congenital and acquired conformational diseases, including those that affect G protein-coupled receptors (GPCRs), cell surface seven-transmembrane domain proteins that mediate extracellular messages into intracellular responses. Examples include nephrogenic diabetes insipidus, retinitis pigmentosa, and familial obesity that are caused by mutant forms of the V2 vasopressin receptor, rhodopsin, and melanocortin receptor 4, respectively (3).Since many of the disease-causing proteins are not inherently nonfunctional, attempts to correct their folding and trafficking have attracted considerable attention. Several different ways to alleviate their incorrect cellular localization have ...

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.

†, ‡These authors contributed equally to this work.The human d opioid receptor (hdOR) is a G-proteincoupled receptor that is mainly involved in the modulation of pain and mood. Only one nonsynonymous single nucleotide polymorphism (T80G) has been described, causing Phe27Cys substitution in the receptor N-terminus and showing association with substance dependence. In this study, we expressed the two hdOR variants in a heterologous expression system with an identical genetic background. They differed greatly during early steps of biosynthesis, displaying a significant difference in the maturation efficiency (50% and 85% for the Cys27 and Phe27 variants, respectively). The Cys27 variant also showed accumulation in pre-Golgi compartments of the secretory pathway and impaired targeting to endoplasmic reticulum (ER)-associated degradation following longterm expression. In addition, the cell surface receptors of the Cys27 variant internalized constitutively. Replacement of phenylalanine with other amino acids revealed that cysteine at position 27 decreased the mature receptor/ precursor ratio most extensively, suggesting a thiolmediated retention of precursors in the ER. However, cysteine did not cause a major folding defect because pharmacological characteristics and the maturation kinetics of the variants were identical, and an opioid antagonist was able to enhance the maturation of both variants. We conclude that, instead of causing loss of function, Phe27Cys polymorphism of the hdOR causes a gain-of-function phenotype, which may have implications for the regulation of receptor expression at the cell surface and possibly also for the susceptibility to pathophysiological states.

Protein palmitoylation is a reversible lipid modification that plays important roles for many proteins involved in signal transduction, but relatively little is known about the regulation of this modification and the cellular location where it occurs. We demonstrate that the human ␦ opioid receptor is palmitoylated at two distinct cellular locations in human embryonic kidney 293 cells and undergoes dynamic regulation at one of these sites. Although palmitoylation could be readily observed for the mature receptor (M r 55,000), [ 3 H]palmitate incorporation into the receptor precursor (M r 45,000) could be detected only following transport blockade with brefeldin A, nocodazole, and monensin, indicating that the modification occurs initially during or shortly after export from the endoplasmic reticulum. Blocking of palmitoylation with 2-bromopalmitate inhibited receptor cell surface expression, indicating that it is needed for efficient intracellular transport. However, cell surface biotinylation experiments showed that receptors can also be palmitoylated once they have reached the plasma membrane. At this location, palmitoylation is regulated in a receptor activation-dependent manner, as was indicated by the opioid agonist-promoted increase in the turnover of receptor-bound palmitate. This agonistmediated effect did not require receptor-G protein coupling and occurred at the cell surface without the need for internalization or recycling. The activation-dependent modulation of receptor palmitoylation may thus contribute to the regulation of receptor function at the plasma membrane.Palmitoylation, a post-translational lipid modification, plays an important role in the structure and function of a variety of proteins involved in cell signaling (1, 2). Among them several G protein-coupled receptors (GPCRs), 3 mainly belonging to family A, have been shown to be palmitoylated on one or more cysteine residues located in the proximal region of their C-terminal tail (2). The attached palmitate has therefore been proposed to promote the formation of a fourth cytoplasmic loop through its anchoring in the membrane (3). Consequently, palmitoylation may have a profound effect on the local conformation of this domain and possibly controls interaction of GPCRs with specific regulatory proteins. It has been shown to display diverse roles in GPCR function, including G protein coupling, desensitization, trafficking, and targeting of the receptors (2). Unlike other lipid modifications such as myristoylation and prenylation, palmitoylation is a highly dynamic modification. The 16-carbon saturated palmitate that is attached to the specific cysteine residues via an acyl-thioester bond has been demonstrated to turn over rapidly (4 -8) and palmitoylation/depalmitoylation cycles may therefore regulate protein function. It has been shown that the palmitoylation/depalmitoylation cycle is activation-dependent for several G protein ␣-subunits (4, 9 -11), endothelial nitric-oxide synthase (12), and several GPCRs (the ␤ 2 -adrenergic (5, 13), D 1...

Lehtonen, Siri T., Piia M. H. Markkanen, Mirva Peltoniemi, Sang Won Kang, and Vuokko L. Kinnula. Variable overoxidation of peroxiredoxins in human lung cells in severe oxidative stress.

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The respirable particles in both outdoor and indoor air contain several different components that are considered to have adverse health effects; e.g., polycyclic aromatic hydrocarbons (PAHs), various metals and microbial species. In this study, size segregated particle samples were collected for chemical, microbial and toxicological analyses from the indoor and outdoor air during each season of the year. The indoor sampling was carried out in a new, detached house with a novel sampling approach. The inorganic species accounted for 8-43% of the total respirable particles. The highest fine particle metal concentrations, both outdoors and indoors, were observed during summer, when the air quality was affected by wildfire smoke plumes, while in coarse particles the total metal concentrations were the highest during the spring, due to the high contribution from mineral dust. The PAH concentrations were 1.3 to 4.8 times higher in outdoor than in indoor air, and they were clearly the highest during winter, most probably due to residential heating, which is a major PAH source. PAHs with four rings had the largest contribution to the total PAHs. Microbial DNA was observed in all size classes, but the highest concentrations were measured in the coarse (PM 2.5-10 ) fraction. The microbial concentrations were higher in the indoor air samples during winter, while in the outdoor ones during summer.

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