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.

The N-terminal domain of unknown function (DUF959) in collagen XVIII is intrinsically disordered and highly O-glycosylated.

Collagen XVIII (ColXVIII) is a non-fibrillar collagen and proteoglycan that exists in three isoforms: short, medium and long. The medium and long isoforms contain a unique N-terminal domain of unknown function, DUF959, and our sequence-based secondary structure predictions indicated that DUF959 could be an intrinsically disordered domain. Recombinant DUF959 produced in mammalian cells consisted of ∼50% glycans and had a molecular mass of 63 kDa. Circular dichroism spectroscopy confirmed the disordered character of DUF959, and static light scattering indicated a monomeric state for glycosylated DUF959 in solution. Small-angle X-ray scattering showed DUF959 to be a highly extended, flexible molecule with a maximum dimension of ∼23 nm. Glycosidase treatment demonstrated considerable amounts of O-glycosylation, and expression of DUF959 in HEK293 SimpleCells capable of synthesizing only truncated O-glycans confirmed the presence of N-acetylgalactosamine-type O-glycans. The DUF959 sequence is characterized by numerous Ser and Thr residues, and this accounts for the finding that half of the recombinant protein consists of glycans. Thus, the medium and long ColXVIII isoforms contain at their extreme N-terminus a disordered, elongated and highly O-glycosylated mucin-like domain that is not found in other collagens, and we suggest naming it the Mucin-like domain in ColXVIII (MUCL-C18). As intrinsically disordered regions and their post-translational modifications are often involved in protein interactions, our findings may point towards a role of the flexible mucin-like domain of ColXVIII as an interaction hub affecting cell signaling. Moreover, the MUCL-C18 may also serve as a lubricant at cell-extracellular matrix interfaces.

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.

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

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.

N-Glycan-dependent and -independent quality control of human δ opioid receptor N-terminal variants.

Quality control (QC) in the endoplasmic reticulum (ER) scrutinizes newly synthesized proteins and directs them either to ER export or ER-associated degradation (ERAD). Here, we demonstrate that the human δ-opioid receptor (hδOR) is subjected to ERQC in both N-glycan-dependent and -independent manners. This was shown by investigating the biosynthesis and trafficking of wild-type and non-N-glycosylated F27C variants in metabolic pulse-chase assays coupled with flow cytometry and cell surface biotinylation. Both QC mechanisms distinguished the minute one-amino acid difference between the variants, targeting a large fraction of hδOR-Cys(27) to ERAD. However, the N-glycan-independent QC was unable to compensate the N-glycan-dependent pathway, and some incompletely folded non-N-glycosylated hδOR-Cys(27) reached the cell surface in conformation incompatible with ligand binding. The turnover of receptors associating with the molecular chaperone calnexin (CNX) was significantly slower for the hδOR-Cys(27), pointing to an important role of CNX in the hδOR N-glycan-dependent QC. This was further supported by the fact that inhibiting the co-translational interaction of hδOR-Cys(27) precursors with CNX led to their ERAD. Opioid receptor pharmacological chaperones released the CNX-bound receptors to ER export and, furthermore, were able to rescue the Cys(27) variant from polyubiquitination and retrotranslocation to the cytosol whether carrying N-glycans or not. Taken together, the hδOR appears to rely primarily on the CNX-mediated N-glycan-dependent QC that has the capacity to assist in folding, whereas the N-glycan-independent mechanism constitutes an alternative, although less accurate, system for directing misfolded/incompletely folded receptors to ERAD, possibly in altered cellular conditions.

Targeting opioid receptors with pharmacological chaperones.

G protein-coupled receptors (GPCRs) are polytopic membrane proteins that have a pivotal role in cellular signaling. Like other membrane proteins, they fold in the endoplasmic reticulum (ER) before they are transported to the plasma membrane. The ER quality control monitors the folding process and misfolded proteins and slowly folding intermediates are targeted to degradation in the cytosol via the ubiquitin-proteasome pathway. The high efficiency of the quality control machinery may lead to the disposal of potentially functional receptors. This is the major underlying course for loss-of-function conformational diseases, such as retinitis pigmentosa, nephrogenic diabetes insipidus and early onset obesity, which involve mutant GPCRs. During the past decade, it has become increasingly evident that small-molecular lipophilic and pharmacologically selective receptor ligands, called pharmacological chaperones (PCs), can rescue these mutant receptors from degradation by stabilizing newly synthesized receptors in the ER and enhancing their transport to the cell surface. This has raised the interesting prospect that PCs might have therapeutic value for the treatment of conformational diseases. At the same time, accumulating evidence has indicated that wild-type receptors might also be targeted by PCs, widening their therapeutic potential. This review focuses on one GPCR subfamily, opioid receptors that have been useful models to unravel the mechanism of action of PCs. In contrast to most other GPCRs, compounds that act as PCs for opioid receptors, including widely used opioid drugs, target wild-type receptors and their common natural variants.

Altered O-glycosylation of ß1-adrenergic receptor N-terminal single-nucleotide variants modulates receptor processing and functional activity.

N-terminal nonsynonymous single-nucleotide polymorphisms (SNPs) of G protein-coupled receptors (GPCRs) are common and often affect receptor post-translational modifications. Their functional implications are, however, largely unknown. We have previously shown that the human ß1-adrenergic receptor (ß1AR) is O-glycosylated in the N-terminal extracellular domain by polypeptide GalNAc transferase-2 that co-regulates receptor proteolytic cleavage. Here, we demonstrate that the common S49G and the rare A29T and R31Q SNPs alter these modifications, leading to distinct effects on receptor processing. This was achieved by in vitro O-glycosylation assays, analysis of native receptor N-terminal O-glycopeptides, and expression of receptor variants in cell lines and neonatal rat ventricular cardiomyocytes deficient in O-glycosylation. The SNPs eliminated (S49G) or introduced (A29T) regulatory O-glycosites that enhanced or inhibited cleavage at the adjacent sites (P52↓L53 and R31↓L32), respectively, or abolished the major site at R31↓L32 (R31Q). The inhibition of proteolysis of the T29 and Q31 variants correlated with increased full-length receptor levels at the cell surface. Furthermore, the S49 variant showed increased isoproterenol-mediated signaling in an enhanced bystander bioluminescence energy transfer ß-arrestin2 recruitment assay in a coordinated manner with the common C-terminal R389G polymorphism. As Gly at position 49 is ancestral in placental mammals, the results suggest that its exchange to Ser has created a ß1AR gain-of-function phenotype in humans. This study provides evidence for regulatory mechanisms by which GPCR SNPs outside canonical domains that govern ligand binding and activation can alter receptor processing and function. Further studies on other GPCR SNPs with clinical importance as drug targets are thus warranted.

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.

Allele-specific N-glycosylation delays human surfactant protein B secretion in vitro and associates with decreased protein levels in vivo.

BACKGROUND: Surfactant protein B (SP-B) is essential for normal lung function, and decreased concentrations of SP-B have a deleterious effect on pulmonary outcome. SP-B levels may correlate with variations in the encoding gene (SFTPB). SFTPB single-nucleotide polymorphism Ile131Thr affects proSP-B N-glycosylation in humans and the glycosylated Thr variant associates with pulmonary diseases. METHODS: We analyzed SP-B levels in amniotic fluid samples for associations with SFTPB polymorphisms and generated cell lines expressing either proSP-B/131Ile or proSP-B/131Thr for examining the effect of glycosylation on proSP-B secretion kinetics. To determine any transcription preference between Ile131Thr allelic variants, we used heterozygous human lungs for allelic expression imbalance assays. RESULTS: Protein levels correlated with Ile131Thr genotype and the lowest SP-B levels were observed in Thr/Thr homozygotes. Our results suggest that Ile131Thr variation-dependent N-glycosylation associates with decreased levels of SP-B, which is secreted from fetal lung to amniotic fluid. Glycosylated proSP-B/131Thr was secreted from transfected cells at a lower rate than nonglycosylated proSP-B/131Ile. Expression levels of the mRNA variants were equal. Secretion of the glycosylated variant was thus delayed in vitro by a posttranscriptional mechanism. CONCLUSION: These data support the hypothesis that proSP-B glycosylation due to Ile131Thr variation may have a causal role in genetic susceptibility to acute respiratory distress.

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.

Human beta1-adrenergic receptor is subject to constitutive and regulated N-terminal cleavage.

The beta(1)-adrenergic receptor (beta(1)AR) is the predominant betaAR 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 beta(1)AR and reveal novel features relevant to its regulation using an inducible heterologous expression system in HEK293(i) 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 Asn(15). 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 Arg(31) and Leu(32) and Pro(52) and Leu(53), 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 betaAR 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 Arg(31)-Leu(32) cleavage site stabilized the mature receptor. We hypothesize that the N-terminal cleavage represents a novel regulatory mechanism of cell surface beta(1)ARs.

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.

Ecto-GPR37: a potential biomarker for Parkinson's disease.

OBJECTIVE: α-Synuclein has been studied as a potential biomarker for Parkinson's disease (PD) with no concluding results. Accordingly, there is an urgent need to find out reliable specific biomarkers for PD. GPR37 is an orphan G protein-coupled receptor that toxically accumulates in autosomal recessive juvenile parkinsonism. Here, we investigated whether GPR37 is upregulated in sporadic PD, and thus a suitable potential biomarker for PD. METHODS: GPR37 protein density and mRNA expression in postmortem substantia nigra (SN) from PD patients were analysed by immunoblot and RT-qPCR, respectively. The presence of peptides from the N-terminus-cleaved domain of GPR37 (i.e. ecto-GPR37) in human cerebrospinal fluid (CSF) was determined by liquid chromatography-mass spectrometric analysis. An engineered in-house nanoluciferase-based immunoassay was used to quantify ecto-GPR37 in CSF samples from neurological control (NC) subjects, PD patients and Alzheimer's disease (AD) patients. RESULTS: GPR37 protein density and mRNA expression were significantly augmented in sporadic PD. Increased amounts of ecto-GPR37 peptides in the CSF samples from PD patients were identified by mass spectrometry and quantified by the in-house ELISA method. However, the CSF total α-synuclein level in PD patients did not differ from that in NC subjects. Similarly, the cortical GPR37 mRNA expression and CSF ecto-GPR37 levels in AD patients were also unaltered. CONCLUSION: GPR37 expression is increased in SN of sporadic PD patients. The ecto-GPR37 peptides are significantly increased in the CSF of PD patients, but not in AD patients. These results open perspectives and encourage further clinical studies to confirm the validity and utility of ecto-GPR37 as a potential PD biomarker.

Association of the beta-1 adrenergic receptor carboxyl terminal variants with left ventricular hypertrophy among diabetic and non-diabetic survivors of acute myocardial infarction.

BACKGROUND: The beta-1 adrenergic receptor (beta1AR) plays a fundamental role in the regulation of cardiovascular functions. It carries a nonsynonymous single nucleotide polymorphism in its carboxyl terminal tail (Arg389Gly), which has been shown to associate with various echocardiographic parameters linked to left ventricular hypertrophy (LVH). Diabetes mellitus (DM), on the other hand, represents a risk factor for LVH. We investigated the possible association between the Arg389Gly polymorphism and LVH among non-diabetic and diabetic acute myocardial infarction (AMI) survivors. METHODS: The study population consisted of 452 AMI survivors, 20.6% of whom had diagnosed DM. Left ventricular parameters were measured with two-dimensional guided M-mode echocardiography 2-7 days after AMI, and the Arg389Gly polymorphism was determined using a polymerase chain reaction-restriction fragment length polymorphism assay. RESULTS: The Arg389 homozygotes in the whole study population had a significantly increased left ventricular mass index (LVMI) when compared to the Gly389 carriers (either Gly389 homozygotes or Arg389/Gly389 heterozygotes) [62.7 vs. 58.4, respectively (p = 0.023)]. In particular, the Arg389 homozygotes displayed thicker diastolic interventricular septal (IVSd) measures when compared to the Gly389 carriers [13.2 vs. 12.3 mm, respectively (p = 0.004)]. When the euglycemic and diabetic patients were analyzed separately, the latter had significantly increased LVMI and diastolic left ventricular posterior wall (LVPWd) values compared to the euglycemic patients [LVMI = 69.1 vs. 58.8 (p = 0.001) and LVPWd = 14.2 vs. 12.3 mm (p < 0.001), respectively]. Furthermore, among the euglycemic patients, the Arg389 homozygotes displayed increased LVMI and IVSd values compared to the Gly389 carriers [LVMI = 60.6 vs. 56.3, respectively (p = 0.028) and IVSd = 13.1 vs. 12.0 mm, respectively (p = 0.001)]. There was no difference in the LVMI and IVSd values between the diabetic Arg389 homozygotes and Gly389 carriers. CONCLUSIONS: The data suggest an association between the beta1AR Arg389Gly polymorphism and LVH, particularly the septal hypertrophy. The Arg389 variant appears to confer a higher risk of developing LVH than the corresponding Gly389 variant among patients who have suffered AMI. This association cannot be considered to be universal, however, since it does not appear to exist among diabetic AMI survivors.

G Protein-Coupled Receptors in the Sweet Spot: Glycosylation and other Post-translational Modifications.

Post-translational modifications (PTMs) are a fundamental phenomenon across all classes of life and several hundred different types have been identified. PTMs contribute widely to the biological functions of proteins and greatly increase their diversity. One important class of proteins regulated by PTMs, is the cell surface expressed G protein-coupled receptors (GPCRs). While most PTMs have been shown to exert distinct biological functions, we are only beginning to approach the complexity that the potential interplay between different PTMs may have on biological functions and their regulation. Importantly, PTMs and their potential interplay represent an appealing mechanism for cell and tissue specific regulation of GPCR function and may partially contribute to functional selectivity of some GPCRs. In this review we highlight examples of PTMs located in GPCR extracellular domains, with special focus on glycosylation and the potential interplay with other close-by PTMs such as tyrosine sulfation, proteolytic cleavage, and phosphorylation.

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.

The endoplasmic reticulum Ca2+-pump SERCA2b interacts with G protein-coupled receptors and enhances their expression at the cell surface.

Calcium (Ca(2+)) plays a pivotal role in both cellular signaling and protein synthesis. However, it is not well understood how calcium metabolism and synthesis of secreted and membrane-bound proteins are related. Here we demonstrate that the sarco(endo)plasmic reticulum Ca(2+) ATPase 2b (SERCA2b), which maintains high Ca(2+) concentration in the lumen of the endoplasmic reticulum, interacts specifically with the human delta opioid receptor during early steps of receptor biogenesis in human embryonic kidney 293 cells. The interaction involves newly synthesized incompletely folded receptor precursors, because the association between the delta opioid receptor and SERCA2b (i) was short-lived and took place soon after receptor translation, (ii) was not affected by misfolding of the receptor, and (iii) decreased if receptor folding was enhanced by opioid receptor pharmacological chaperone. The physical association with SERCA2b was found to be a universal feature among G protein-coupled receptors within family A and was shown to occur also between the endogenously expressed luteinizing hormone receptor and SERCA2b in rat ovaries. Importantly, active SERCA2b rather than undisturbed Ca(2+) homeostasis was found to be essential for delta opioid receptor biogenesis, as inhibition of its Ca(2+) pumping activity by thapsigargin reduced the interaction and impaired the efficiency of receptor maturation, two phenomena that were not affected by a Ca(2+) ionophore A23187. Nevertheless, inhibition of SERCA2b did not compromise the functionality of receptors that were able to mature. Thus, we propose that the association with SERCA2b is required for efficient folding and/or membrane integration of G protein-coupled receptors.

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.