GPR37 is processed in the N-terminal ectodomain by ADAM10 and furin.

GPR37 is an orphan G protein-coupled receptor (GPCR) implicated in several neurological diseases and important physiological pathways in the brain. We previously reported that its long N-terminal ectodomain undergoes constitutive metalloprotease-mediated cleavage and shedding, which have been rarely described for class A GPCRs. Here, we demonstrate that the protease that cleaves GPR37 at Glu167↓Gln168 is a disintegrin and metalloprotease 10 (ADAM10). This was achieved by employing selective inhibition, RNAi-mediated downregulation, and genetic depletion of ADAM10 in cultured cells as well as in vitro cleavage of the purified receptor with recombinant ADAM10. In addition, the cleavage was restored in ADAM10 knockout cells by overexpression of the wild type but not the inactive mutant ADAM10. Finally, postnatal conditional depletion of ADAM10 in mouse neuronal cells was found to reduce cleavage of the endogenous receptor in the brain cortex and hippocampus, confirming the physiological relevance of ADAM10 as a GPR37 sheddase. Additionally, we discovered that the receptor is subject to another cleavage step in cultured cells. Using site-directed mutagenesis, the site (Arg54↓Asp55) was localized to a highly conserved region at the distal end of the ectodomain that contains a recognition site for the proprotein convertase furin. The cleavage by furin was confirmed by using furin-deficient human colon carcinoma LoVo cells and proprotein convertase inhibitors. GPR37 is thus the first multispanning membrane protein that has been validated as an ADAM10 substrate and the first GPCR that is processed by both furin and ADAM10. The unconventional N-terminal processing may represent an important regulatory element for GPR37.

Novel congenital disorder of O-linked glycosylation caused by GALNT2 loss of function.

Congenital disorders of glycosylation are a growing group of rare genetic disorders caused by deficient protein and lipid glycosylation. Here, we report the clinical, biochemical, and molecular features of seven patients from four families with GALNT2-congenital disorder of glycosylation (GALNT2-CDG), an O-linked glycosylation disorder. GALNT2 encodes the Golgi-localized polypeptide N-acetyl-d-galactosamine-transferase 2 isoenzyme. GALNT2 is widely expressed in most cell types and directs initiation of mucin-type protein O-glycosylation. All patients showed loss of O-glycosylation of apolipoprotein C-III, a non-redundant substrate for GALNT2. Patients with GALNT2-CDG generally exhibit a syndrome characterized by global developmental delay, intellectual disability with language deficit, autistic features, behavioural abnormalities, epilepsy, chronic insomnia, white matter changes on brain MRI, dysmorphic features, decreased stature, and decreased high density lipoprotein cholesterol levels. Rodent (mouse and rat) models of GALNT2-CDG recapitulated much of the human phenotype, including poor growth and neurodevelopmental abnormalities. In behavioural studies, GALNT2-CDG mice demonstrated cerebellar motor deficits, decreased sociability, and impaired sensory integration and processing. The multisystem nature of phenotypes in patients and rodent models of GALNT2-CDG suggest that there are multiple non-redundant protein substrates of GALNT2 in various tissues, including brain, which are critical to normal growth and development.

Site-specific O-glycosylation of N-terminal serine residues by polypeptide GalNAc-transferase 2 modulates human δ-opioid receptor turnover at the plasma membrane.

G protein-coupled receptors (GPCRs) are an important protein family of signalling receptors that govern a wide variety of physiological functions. The capacity to transmit extracellular signals and the extent of cellular response are largely determined by the amount of functional receptors at the cell surface that is subject to complex and fine-tuned regulation. Here, we demonstrate that the cell surface expression level of an inhibitory GPCR, the human δ-opioid receptor (hδOR) involved in pain and mood regulation, is modulated by site-specific N-acetylgalactosamine (GalNAc) -type O-glycosylation. Importantly, we identified one out of the 20 polypeptide GalNAc-transferase isoforms, GalNAc-T2, as the specific regulator of O-glycosylation of Ser6, Ser25 and Ser29 in the N-terminal ectodomain of the receptor. This was demonstrated by in vitro glycosylation assays using peptides corresponding to the hδOR N-terminus, Vicia villosa lectin affinity purification of receptors expressed in HEK293 SimpleCells capable of synthesizing only truncated O-glycans, GalNAc-T edited cell line model systems, and site-directed mutagenesis of the putative O-glycosylation sites. Interestingly, a single-nucleotide polymorphism, at residue 27 (F27C), was found to alter O-glycosylation of the receptor in efficiency as well as in glycosite usage. Furthermore, flow cytometry and cell surface biotinylation assays using O-glycan deficient CHO-ldlD cells revealed that the absence of O-glycans results in decreased receptor levels at the plasma membrane due to enhanced turnover. In addition, mutation of the identified O-glycosylation sites led to a decrease in the number of ligand-binding competent receptors and impaired agonist-mediated inhibition of cyclic AMP accumulation in HEK293 cells. Thus, site-specific O-glycosylation by a selected GalNAc-T isoform can increase the stability of a GPCR, in a process that modulates the constitutive turnover and steady-state levels of functional receptors at the cell surface.

Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates ß1-Adrenergic Receptor N-terminal Cleavage.

The ß1-adrenergic receptor (ß1AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for ß-adrenergic antagonists, such as ß-blockers, relatively little is yet known about its regulation. We have shown previously that ß1AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O-glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O-glycosylates ß1AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O-glycosylation and proteolytic cleavage assays, a cell line deficient in O-glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of ß1AR. Furthermore, we demonstrate that impaired O-glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the ßAR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O-glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of ß1AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases.

ß-Adrenergic agonists mediate enhancement of ß1-adrenergic receptor N-terminal cleavage and stabilization in vivo and in vitro.

The ß(1)-adrenergic receptor (ß(1)AR) is the predominant ßAR in the heart and is the main target for ß-adrenergic antagonists, widely used in the treatment of cardiovascular diseases. Previously, we have shown that the human (h) ß(1)AR is cleaved in its N terminus by a metalloproteinase, both constitutively and in a receptor activation-dependent manner. In this study, we investigated the specific events involved in ß(1)AR regulation, focusing on the effects of long-term treatment with ß-adrenergic ligands on receptor processing in stably transfected human embryonic kidney 293(i) cells. The key findings were verified using the transiently transfected hß(1)AR and the endogenously expressed receptor in neonatal rat cardiomyocytes. By using flow cytometry and Western blotting, we demonstrated that isoproterenol, S-propranolol, CGP-12177 [4-[3-[(1,1-dimethylethyl)amino]2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one], pindolol, and timolol, which displayed agonistic properties toward the ß(1)AR in either the adenylyl cyclase or the mitogen-activated protein kinase signaling pathways, induced cleavage of the mature cell-surface receptor. In contrast, metoprolol, bisoprolol, and CGP-20712 [1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol], which showed no agonistic activity, had only a marginal or no effect. Importantly, the agonists also stabilized intracellular receptor precursors, possibly via their pharmacological chaperone action, and they stabilized the receptor in vitro. The opposing effects on the two receptor forms thus led to an increase in the amount of cleaved receptor fragments at the plasma membrane. The results underscore the pluridimensionality of ß-adrenergic ligands and extend this property from receptor activation and signaling to the regulation of ß(1)AR levels. This phenomenon may contribute to the exceptional resistance of ß(1)ARs to downregulation and tendency toward upregulation following long-term ligand treatments.

Cys-27 variant of human δ-opioid receptor modulates maturation and cell surface delivery of Phe-27 variant via heteromerization.

The important role of G protein-coupled receptor homo/heteromerization in receptor folding, maturation, trafficking, and cell surface expression has become increasingly evident. Here we investigated whether the human δ-opioid receptor (hδOR) Cys-27 variant that shows inherent compromised maturation has an effect on the behavior of the more common Phe-27 variant in the early secretory pathway. We demonstrate that hδOR-Cys-27 acts in a dominant negative manner and impairs cell surface delivery of the co-expressed hδOR-Phe-27 and impairs conversion of precursors to the mature form. This was demonstrated by metabolic labeling, Western blotting, flow cytometry, and confocal microscopy in HEK293 and human SH-SY5Y neuroblastoma cells using differentially epitope-tagged variants. The hδOR-Phe-27 precursors that were redirected to the endoplasmic reticulum-associated degradation were, however, rescued by a pharmacological chaperone, the opioid antagonist naltrexone. Co-immunoprecipitation of metabolically labeled variants revealed that both endoplasmic reticulum-localized precursors and mature receptors exist as homo/heteromers. The existence of homo/heteromers was confirmed in living cells by bioluminescence resonance energy transfer measurements, showing that the variants have a similar propensity to form homo/heteromers. By forming both homomers and heteromers, the hδOR-Cys-27 variant may thus regulate the levels of receptors at the cell surface, possibly leading to altered responsiveness to opioid ligands in individuals carrying the Cys-27 variant.

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

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

Phe27Cys polymorphism of the human delta opioid receptor predisposes cells to compromised calcium signaling.

A quarter of the human population with European background carries at least one allele of the OPRD1 gene that encodes the delta opioid receptor with cysteine at the amino acid position 27 (hδOR(Cys27)) instead of the evolutionary conserved phenylalanine (hδOR(Phe27)). The two variants have indistinguishable pharmacological properties but, importantly, hδOR(Cys27) differs from hδOR(Phe27) in having low maturation efficiency, lower stability at the cell surface and pronounced intracellular location. Both variants were previously shown to interact with the Sarco(endo)plasmic reticulum Ca²+ ATPase (SERCA) 2b in the early phase of their biosynthesis. We analyzed by pulse-chase assays, whether cellular signaling can affect hδOR(Cys27) maturation. Neither activation of the receptor by a δOR-specific agonist Leu-enkephalin, induction of intracellular calcium (Ca²+) release by ATP nor the direct stimulation of SERCA 2b by protein kinase C activation affected receptor maturation in HEK-293 cells. No signaling-mediated regulation of receptor maturation could therefore be demonstrated. Instead, we found by using single cell Ca²+ measurements that over-expression of hδOR(Cys27), but not hδOR(Phe27), compromised ATP-induced intracellular Ca²+-signaling. Furthermore, hδOR(Cys27) precursors showed slower dissociation from SERCA2b and hδOR(Cys27) expression caused down-regulation of the homocysteine-inducible endoplasmic reticulum-resident ubiquitin domain-like member 1 protein (HERP). We suggest that aging individuals with at least one hδOR(Cys27) encoding allele might have lowered threshold for Ca²+ dysregulation in neurons expressing hδOR.

Human delta opioid receptor biogenesis is regulated via interactions with SERCA2b and calnexin.

Sarco(endo)plasmic reticulum calcium ATPase (SERCA)2b maintains the cellular Ca(2+) homeostasis by transferring Ca(2+) from the cytosol to the lumen of the endoplasmic reticulum (ER). Recently, SERCA2b has also been shown to be involved in the biosynthesis of secreted and membrane proteins via direct protein-protein interactions, involving components of the ER folding and quality-control machinery, as well as newly synthesized G protein-coupled receptors. Here we demonstrate that the human delta opioid receptor (hdeltaOR) exists in a ternary complex with SERCA2b and the ER molecular chaperone calnexin. The interaction between SERCA2b and hdeltaOR in vivo did not require calnexin as it was independent of the C-terminal calnexin-interacting domain of SERCA2b. However, the receptor was able to mediate co-immunoprecipitation of calnexin with the C-terminally truncated SERCA2b. The association of SERCA2b with hdeltaOR was regulated in vitro by Ca(2+) and ATP in a manner that was opposite to the calnexin-hdeltaOR interaction. Importantly, co-expression of the catalytically inactive SERCA2b(D351A) or calnexin binding-compromised SERCA2bDeltaC mutants with the receptor decreased the expression of mature receptors in a manner that did not directly relate to changes in the ER Ca(2+) concentration. We conclude that dynamic interactions among SERCA2b, calnexin and the hdeltaOR precursor orchestrate receptor biogenesis and are regulated by Ca(2+) and ATP. We further hypothesize that the primary role of SERCA2b in this process is to act as a Ca(2+) sensor in the vicinity of active translocons, integrating protein folding with local fluctuations of ER Ca(2+) levels.

Phe27Cys polymorphism alters the maturation and subcellular localization of the human delta opioid receptor.

The human delta opioid receptor (hdeltaOR) is a G-protein-coupled 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 hdeltaOR 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 long-term 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 thiol-mediated 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 hdeltaOR 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.

Luteinizing hormone receptor ectodomain splice variant misroutes the full-length receptor into a subcompartment of the endoplasmic reticulum.

The luteinizing hormone receptor (LHR) is a G protein-coupled receptor that is expressed in multiple RNA messenger forms. The common rat ectodomain splice variant is expressed concomitantly with the full-length LHR in tissues and is a truncated transcript corresponding to the partial ectodomain with a unique C-terminal end. Here we demonstrate that the variant alters the behavior of the full-length receptor by misrouting it away from the normal secretory pathway in human embryonic kidney 293 cells. The variant was expressed as two soluble forms of M(r) 52,000 and M(r) 54,000, but although the protein contains a cleavable signal sequence, no secretion to the medium was observed. Only a very small fraction of the protein was able to gain hormone-binding ability, suggesting that it is retained in the endoplasmic reticulum (ER) by its quality control due to misfolding. This was supported by the finding that the variant was found to interact with calnexin and calreticulin and accumulated together with these ER chaperones in a specialized juxtanuclear subcompartment of the ER. Only proteasomal blockade with lactacystin led to accumulation of the variant in the cytosol. Importantly, coexpression of the variant with the full-length LHR resulted in reduction in the number of receptors that were capable of hormone binding and were expressed at the cell surface and in targeting of immature receptors to the juxtanuclear ER subcompartment. Thus, the variant mediated misrouting of the newly synthesized full-length LHRs may provide a way to regulate the number of cell surface receptors.

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

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 M(r) 73,000, containing high mannose-type N-linked glycans. In metabolic pulse-chase studies, only approximately 20% of these receptor precursors were found to gain hormone binding ability and matured to a form of M(r) 90,000, containing bi- and multiantennary sialylated N-linked glycans. The rest had a propensity to form disulfide-bonded complexes with a M(r) 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.