The Parkinson’s-disease-associated receptor GPR37 undergoes metalloproteinase-mediated N-terminal cleavage and ectodomain shedding

Abstract The G-protein-coupled receptor 37 ( GPR37) has been implicated in the juvenile form of Parkinson’s disease, in dopamine signalling and in the survival of dopaminergic cells in animal models. The structure and function of the receptor, however, have remained enigmatic. Here, we demonstrate that although GPR37 matures and is exported from the endoplasmic reticulum in a normal manner upon heterologous expression in HEK293 and SH-SY5Y cells, its long extracellular N-terminus is subject to metalloproteinase-mediated limited proteolysis between E167 and Q168. The proteolytic processing is a rapid and efficient process that occurs constitutively. Moreover, the GPR37 ectodomain is released from cells by shedding, a phenomenon rarely described for GPCRs. Immunofluorescence microscopy further established that although full-length receptors are present in the secretory pathway until the trans-Golgi network, GPR37 is expressed at the cell surface predominantly in the N-terminally truncated form. This notion was verified by flow cytometry and cell surface biotinylation assays. These new findings on the GPR37 N-terminal limited proteolysis may help us to understand the role of this GPCR in the pathophysiology of Parkinson’s disease and in neuronal function in general

Distinct subcellular localization for constitutive and agonist-modulated palmitoylation of the human delta opioid receptor

Abstract 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 humanδ 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 (Mr 55,000), [3H]palmitate incorporation into the receptor precursor (Mr 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 agonist-mediated 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

Opioid receptor pharmacological chaperones act by binding and stabilizing newly synthesized receptors in the endoplasmic reticulum

Abstract 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 δ-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 δ-opioid receptor precursors were able to bind [³H]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 δ-opioid receptors by binding and stabilizing receptor precursors, thereby promoting their release from the stringent ER quality control

Inefficient maturation of the rat luteinizing hormone receptor:a putative way to regulate receptor numbers at the cell surface

Abstract Increasing evidence suggests that the folding and maturation of monomeric proteins and assembly of multimeric protein complexes in the endoplasmic reticulum (ER) may be inefficient not only for mutants that carry changes in the primary structure but also for wild type proteins. In the present study, we demonstrate that the rat luteinizing hormone receptor, a G protein-coupled receptor, is one of these proteins that matures inefficiently and appears to be very prone to premature degradation. A substantial portion of the receptors in stably transfected human embryonic kidney 293 cells existed in immature form of Mr 73,000, containing high mannose-type N-linked glycans. In metabolic pulse-chase studies, only ∼20% of these receptor precursors were found to gain hormone binding ability and matured to a form of Mr 90,000, containing bi- and multiantennary sialylated N-linked glycans. The rest had a propensity to form disulfide-bonded complexes with a Mr 120,000 protein in the ER membrane and were eventually targeted for degradation in proteasomes. The number of membrane-bound receptor precursors increased when proteasomal degradation was inhibited, and no cytosolic receptor forms were detected, suggesting that retrotranslocation of the misfolded/incompletely folded receptors is tightly coupled to proteasomal function. Furthermore, a proteasomal blockade was found to increase the number of receptors that were capable of hormone binding. Thus, these results raise the interesting possibility that luteinizing hormone receptor expression at the cell surface may be controlled at the ER level by regulating the number of newly synthesized proteins that will mature and escape the ER quality control and premature degradation

Cysteine 27 variant of the delta-opioid receptor affects amyloid precursor protein processing through altered endocytic trafficking

Agonist-induced activation of the delta-opioid receptor (deltaOR) was recently shown to augment beta- and gamma-secretase activities, which increased the production of beta-amyloid peptide (Abeta), known to accumulate in the brain tissues of Alzheimer's disease (AD) patients. Previously, the deltaOR variant with a phenylalanine at position 27 (deltaOR-Phe27) exhibited more efficient receptor maturation and higher stability at the cell surface than did the less common cysteine (deltaOR-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 deltaOR-Cys27, but not deltaOR-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 beta-amyloid 40 levels were decreased. These changes upon deltaOR-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 deltaOR-specific ligands or a c-Src tyrosine kinase inhibitor suppressed the deltaOR-Cys27-induced APP phenotype. These data suggest that an increased constitutive internalization and/or concurrent signaling of the deltaOR-Cys27 variant affects APP processing through altered endocytic trafficking of APP

Human β1-adrenergic receptor is subject to constitutive and regulated N-terminal cleavage

Abstract The β₁-adrenergic receptor (β₁AR) is the predominant βAR in the heart, mediating the catecholamine-stimulated increase in cardiac rate and force of contraction. Regulation of this important G protein-coupled receptor is nevertheless poorly understood. We describe here the biosynthetic profile of the human β₁AR and reveal novel features relevant to its regulation using an inducible heterologous expression system in HEK293i cells. Metabolic pulse-chase labeling and cell surface biotinylation assays showed that the synthesized receptors are efficiently and rapidly transported to the cell surface. The N terminus of the mature receptor is extensively modified by sialylated mucin-type O-glycosylation in addition to one N-glycan attached to Asn15. Furthermore, the N terminus was found to be subject to limited proteolysis, resulting in two membrane-bound C-terminal fragments. N-terminal sequencing of the fragments identified two cleavage sites between Arg³¹ and Leu³² and Pro⁵² and Leu⁵³, which were confirmed by cleavage site and truncation mutants. Metalloproteinase inhibitors were able to inhibit the cleavage, suggesting that it is mediated by a matrix metalloproteinase or a disintegrin and metalloproteinase (ADAM) family member. Most importantly, the N-terminal cleavage was found to occur not only in vitro but also in vivo. Receptor activation mediated by the βAR agonist isoproterenol enhanced the cleavage in a concentration- and time-dependent manner, and it was also enhanced by direct stimulation of protein kinase C and adenylyl cyclase. Mutation of the Arg³¹–Leu³² cleavage site stabilized the mature receptor. We hypothesize that the N-terminal cleavage represents a novel regulatory mechanism of cell surface β₁ARs

Improving anatomical stature estimation method:the relationship between living stature and intervertebral disc thickness

Abstract Anatomical stature estimation methods reconstruct stature for skeletal specimens by adding up the heights of skeletal elements contributing to stature. In addition, these estimations factor in a certain amount of soft tissue known as “soft tissue correction”. Our study focuses on the relationship between living stature and one of the major soft tissue contributors to stature: the intervertebral disc thickness/height. The purpose of this study was to clarify whether intervertebral disc thickness is greater in tall individuals and whether there is a linear correlation between stature and intervertebral disc height. To conduct this study, we utilized a subsample of the Northern Finland Birth Cohort of 1966 (n = 12,058) with known stature. We measured vertebral heights and intervertebral disc heights from low back MRI examination performed at the age of 46 years (n = 200). All subjects were considered healthy with no spinal injuries or pathologies. Our results clearly indicate that stature and intervertebral disc height have positive, statistically significant association. According to our results it is advisable to take into account the individual’s skeletal height when soft tissue corrections for anatomical stature estimations are performed. Further studies utilizing full body MRI are needed to produce more accurate soft tissue corrections

Autoantibodies against the immunodominant bullous pemphigoid epitopes are rare in patients with dermatitis herpetiformis and coeliac disease

Abstract Dermatitis herpetiformis (DH) is an extraintestinal manifestation of coeliac disease (CD). Patients with DH have an elevated risk of development of another autoimmune blistering skin disease, bullous pemphigoid (BP). In this study we investigated whether patients with DH and CD (mean age for both 49 years) have circulating autoantibodies against BP180, the major BP autoantigen. ELISA tests showed that only a few DH (3/46) and CD (2/43) patients had BP180-NC16A IgG autoantibodies. Immunoblotting found that more than half of the DH samples contained IgG autoantibodies against full-length BP180. Epitope mapping with 13 fusion proteins covering the BP180 polypeptide revealed that in DH and CD patients, IgG autoantibodies did not target the NC16A or other epitopes typical of BP but recognized other intracellular and mid-extracellular regions of BP180. None of the analyzed DH and CD patients with either ELISA or immunoblotting positivity had IgG or IgA reactivity against the cutaneous basement membrane in indirect immunofluorescence analysis or skin symptoms characteristic of BP. Although only a minority of middle-aged DH patients had IgG autoantibodies against the immunodominant epitopes of BP180, our results do not exclude the possibility that intermolecular epitope spreading could explain the switch from DH to BP in elderly patients