Dendritic Assembly of Heteromeric ��-Aminobutyric Acid Type B Receptor Subunits in Hippocampal Neurons

Understanding the mechanisms that control synaptic efficacy through the availability of neurotransmitter receptors depends on uncovering their specific intracellular trafficking routes. γ-Aminobutyric acid type B (GABA_B) receptors (GABA_BRs) are obligatory heteromers present at dendritic excitatory and inhibitory postsynaptic sites. It is unknown whether synthesis and assembly of GABA_BRs occur in the somatic endoplasmic reticulum (ER) followed by vesicular transport to dendrites or whether somatic synthesis is followed by independent transport of the subunits for assembly and ER export throughout the somatodendritic compartment. To discriminate between these possibilities we studied the association of GABA_BR subunits in dendrites of hippocampal neurons combining live fluorescence microscopy, biochemistry, quantitative colocalization, and bimolecular fluorescent complementation. We demonstrate that GABA_BR subunits are segregated and differentially mobile in dendritic intracellular compartments and that a high proportion of non-associated intracellular subunits exist in the brain. Assembled heteromers are preferentially located at the plasma membrane, but blockade of ER exit results in their intracellular accumulation in the cell body and dendrites. We propose that GABA_BR subunits assemble in the ER and are exported from the ER throughout the neuron prior to insertion at the plasma membrane. Our results are consistent with a bulk flow of segregated subunits through the ER and rule out a post-Golgi vesicular transport of preassembled GABA_BRs.The efficacy of synaptic transmission depends on the intracellular trafficking of neurotransmitter receptors (1, 2). The trafficking of glutamatergic and GABA_A⁶ receptors has been extensively studied, and their implications for synaptic plasticity have been well documented (3, 4). For example, differential trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors modifies synaptic strength and influences experience-dependent plasticity in vivo (5). The molecular mechanisms that govern the trafficking of metabotropic GABA_BRs and their consequences for synaptic inhibition remain less clear. In particular, limited information is available regarding the relationship between the trafficking of GABA_BRs and the topological complexity of the secretory pathway in neurons.GABA_BRs mediate the slow component of synaptic inhibition by acting on pre- and postsynaptic targets (6–8). They are implicated in epilepsy, anxiety, stress, sleep disorders, nociception, depression, and cognition (9). They also represent attractive targets for the treatment of withdrawal symptoms from drugs of addiction such as cocaine (10). They are obligatory heteromers composed of GABA_BR1 and GABA_BR2 subunits. GABA_BR1 contains an RXR-type sequence in the intracellular C-terminal domain that functions as an ER retention motif (11, 12). The ER retention sequence is masked upon assembly with GABA_BR2 resulting in the appearance of functional receptors at the plasma membrane. Only GABA_BR1 binds GABA with high affinity, whereas G protein signaling is exclusively mediated by the second and third intracellular loops of GABA_BR2 (13–15). GABA_BRs are located in dendrites and axons, but their distribution does not coincide with the active zone or the postsynaptic density. Rather, they are adjacent to both compartments constituting perisynaptic receptors (16, 17).If GABA_BR subunits are synthesized in the soma, at least two possibilities exist for their anterograde transport, assembly, and insertion in dendrites. First, the subunits may be synthesized in the cell body, assembled in the somatic ER, and targeted preassembled in post-Golgi vesicles to their site of insertion in dendrites. Alternatively, they may be synthesized in the soma and transported through the ER membrane as non-heteromeric subunits. In the latter scenario, newly assembled receptors may exit the ER throughout the somatodendritic compartment prior to insertion at the plasma membrane and diffuse laterally for retention at functional sites. No evidence exists to discriminate between these possibilities. We reasoned that a prevalence of associated subunits in post-Golgi vesicles in dendrites would favor the first alternative, whereas the existence of non-associated subunits in intracellular compartments would support a somatodendritic assembly mechanism. Here we explore the presence of associated GABA_BR subunits using fluorescence recovery after photobleaching (FRAP), biochemistry, and quantitative colocalization. In addition, we report for the first time the use of BiFC (18) to study GABA_BR assembly in neurons. Our results demonstrate that GABA_BR subunits are differentially mobile in dendrites and that a high proportion of non-associated subunits prevail in an intracellular fraction of the adult brain. They also show that GABA_BR subunits are heteromeric at the plasma membrane but segregated in intracellular compartments of dendrites of hippocampal neurons. Importantly, treatment with brefeldin A (BFA) or interference of the coatomer protein complex II impair ER export and result in the accumulation of assembled subunits in intracellular compartments throughout the somatodendritic arbor. We conclude that GABA_BR subunits are synthesized in the soma and remain segregated in intracellular compartments prior to somatodendritic assembly. Our observations rule out a post-Golgi vesicular transport of preassembled GABA_BRs and suggest an alternative mechanism of receptor targeting.     

Epitope-tagged Receptor Knock-in Mice Reveal That Differential Desensitization of ��2-Adrenergic Responses Is because of Ligand-selective Internalization

Although ligand-selective regulation of G protein-coupled receptor-mediated signaling and trafficking are well documented, little is known about whether ligand-selective effects occur on endogenous receptors or whether such effects modify the signaling response in physiologically relevant cells. Using a gene targeting approach, we generated a knock-in mouse line, in which N-terminal hemagglutinin epitope-tagged α_2A-adrenergic receptor (AR) expression was driven by the endogenous mouse α_2AAR gene locus. Exploiting this mouse line, we evaluated α_2AAR trafficking and α_2AAR-mediated inhibition of Ca²⁺ currents in native sympathetic neurons in response to clonidine and guanfacine, two drugs used for treatment of hypertension, attention deficit and hyperactivity disorder, and enhancement of analgesia through actions on the α_2AAR subtype. We discovered a more rapid desensitization of Ca²⁺ current suppression by clonidine than guanfacine, which paralleled a more marked receptor phosphorylation and endocytosis of α_2AAR evoked by clonidine than by guanfacine. Clonidine-induced α_2AAR desensitization, but not receptor phosphorylation, was attenuated by blockade of endocytosis with concanavalin A, indicating a critical role for internalization of α_2AAR in desensitization to this ligand. Our data on endogenous receptor-mediated signaling and trafficking in native cells reveal not only differential regulation of G protein-coupled receptor endocytosis by different ligands, but also a differential contribution of receptor endocytosis to signaling desensitization. Taken together, our data suggest that these HA-α_2AAR knock-in mice will serve as an important model in developing ligands to favor endocytosis or nonendocytosis of receptors, depending on the target cell and pathophysiology being addressed.G protein-coupled receptors (GPCRs)⁴ represent the largest family of cell surface receptors mediating responses to hormones, cytokines, neurotransmitters, and therapeutic agents (1). In addition to regulating downstream signaling, ligand binding to a receptor can initiate phosphorylation of the active conformation of the receptor by G protein receptor kinases (GRKs) and subsequent binding of arrestins, thus restricting the magnitude and duration of the ligand-evoked signaling responses (2, 3). Binding of arrestins to GPCRs also leads to GPCR internalization (4, 5), a process that has been proposed as a means to desensitize receptor signaling at the cell surface, resensitize receptors, and/or initiate intracellular signaling (6, 7).Different ligands are able to induce distinct signaling and internalization profiles of the same receptor (8-14). However, the lack of available tools to study trafficking of endogenous GPCRs in native target cells has limited our understanding of ligand-selective endocytosis profiles and the relative contribution of receptor endocytosis to desensitization in native biological settings.To specifically test hypotheses regarding ligand-selective effects on GPCR internalization, and functional consequences of this trafficking on signaling, we utilized a homologous recombination gene targeting strategy to introduce a hemagglutinin (HA) epitope-tagged wild type α_2A-adrenergic receptor (AR) into the mouse ADRA2A gene locus (“knock-in”). The α_2AAR is a prototypical GPCR that couples to the G_i/o subfamily of G proteins (15). Studies on genetically engineered mice made null or mutant for the α_2AAR have revealed that this subtype mediates the therapeutic effects of α₂-adrenergic agents on blood pressure, pain perception, volatile anesthetic sparing, analgesia, and working memory enhancement (16-18). Two classic α₂-ligands, clonidine and guanfacine, have been widely used to treat hypertension (19), attention deficit and hyperactivity disorder (20), and to elicit analgesia (19, 21) mediated via the α_2AAR. Clinically guanfacine has a much longer duration of action than clonidine (22-24); this longer duration of action cannot be accounted for by the pharmacokinetic profile of these agents in human beings, as both drugs have a half-life of 12-14 h (25, 26). Because ligand-induced desensitization and trafficking of GPCRs have been implicated as critical mechanisms for modulating response duration in vivo (3), one hypothesis underlying the difference in duration between clonidine and guanfacine is that clonidine provokes accelerated desensitization of the α_2AAR via one or several mechanisms, whereas guanfacine does not. Signaling desensitization in response to these two agonists has not been compared under the same experimental settings. To specifically test this hypothesis, we have exploited our HA-α_2AAR knock-in mice so that we could examine these properties of guanfacine and clonidine in native target cells.We compared internalization of the α_2AAR and inhibition of Ca²⁺ currents induced by clonidine and guanfacine in primary superior cervical ganglia (SCG) neurons, where the α_2AAR is the major adrenergic receptor subtype controlling norepinephrine release and sympathetic tone (17, 27). Our data revealed a differential regulation of α_2AAR trafficking and signaling duration by clonidine versus guanfacine, i.e. clonidine induced a more dramatic desensitization of the α_2AAR than guanfacine, and this desensitization was largely because of α_2AAR internalization. These studies reveal the powerful tool that the HA-α_2AAR knock-in mice provide for identifying endocytosis-dependent and -independent physiological phenomena for this receptor subtype as a first step in defining novel loci for therapeutic intervention in the α_2AAR signaling/trafficking cascade.     

The Asialoglycoprotein Receptor Regulates Levels of Plasma Glycoproteins Terminating with Sialic Acid ��2,6-Galactose

The asialoglycoprotein receptor (ASGP-R) is an abundant, carbohydrate-specific, endocytic receptor expressed by parenchymal cells of the liver. We recently demonstrated that the ASGP-R mediates the clearance of glycoproteins bearing Siaα2,6GalNAc as well as those bearing terminal Gal or GalNAc. We now report that glycoproteins such as haptoglobin, serum amyloid protein (SAP), and carboxylesterase that bear oligosaccharides with terminal Siaα2,6Gal are elevated in the plasma of ASGP-R-deficient mice. Because of their abundance in plasma, glycoproteins bearing terminal Siaα2,6Gal will saturate the ASGP-R and compete with each other on the basis of their relative affinities for the ASGP-R and their relative abundance. We propose that the ASGP-R mediates the clearance of glycoproteins that bear oligosaccharides terminating with Siaα2,6Gal and thereby helps maintain the relative concentrations of these glycoproteins in the blood.The asialoglycoprotein receptor (ASGP-R)³ was initially identified and characterized by Ashwell and co-workers (1, 2) on the basis of its ability to rapidly remove glycoproteins bearing oligosaccharides terminating with β1,4-linked Gal from the circulation. The ASGP-R has been extensively characterized since its initial discovery; however, its biologic function in vivo has remained unclear. This endocytic receptor is highly abundant with 500,000 receptors expressed at the plasma membrane of hepatocytes (3–5) and is rapidly internalized (3, 6). The abundance of the ASGP-R and its rapid rate of internalization in combination with the large number of hepatocytes that are present in the liver, 1.35 × 10⁸/g of liver (7, 8), results in an enormous potential capacity to remove glycoproteins from the circulation. Until recently, mice that have had either subunit of the ASGP-R ablated, subunit 1 ASGP-R1(-/-) or subunit 2 ASGP-R2(-/-), have not been reported to have altered levels of circulating glycoproteins in their blood or to have a physiologic phenotype (9, 10). However, Grewal et al. (11) have reported that the ASGP-R plays a role in von Willebrand factor homeostasis and promotes thrombocytopenia during Steptococcus pneumoniae sepsis by removing platelets that have had their surface sialic acid removed by the bacterial neuraminidase.We recently established that glycoproteins bearing Asn-linked oligosaccharides terminating with the sequence Siaα2,6GalNAcβ1,4GlcNAc are recognized by the ASGP-R and rapidly removed from the blood (12, 13). Glycoproteins bearing terminal Siaα2,6GalNAcβ1,4GlcNAc are the first examples of endogenous glycoproteins that can be recognized by the ASGP-R without further modification; i.e. removal of terminal Sia. Glycoproteins bearing these structures, for example the prolactin-like proteins (14), glycodelin (15), urokinase (16), and glycoprotein hormones (17), are not highly abundant, suggesting that the ASGP-R recognizes and clears additional more abundant glycoproteins. The most likely candidates are glycoproteins bearing Asn-linked oligosaccharides that terminate with the sequence Siaα2,6Galβ1, 4GlcNAc. We have reported that the ASGP-R recognizes these structures with an avidity that is in the micromolar range (13). The avidity of the ASGP-R for structures terminating with Siaα2,6Galβ1,4GlcNAc is predicted to be sufficient to mediate binding and clearance of glycoproteins bearing structures terminating with Siaα2,6Galβ1,4GlcNAc from the blood. This concept is supported by indications that neo-glycoproteins bearing structures terminating with Siaα2,6Galβ1,4GlcNAc are removed from the blood at a faster rate than those bearing Siaα2,3Galβ1,4GlcNAc (18). Slow clearance of glycoproteins bearing Siaα2,6Galβ1,4GlcNAc, however, hampers accurate measurement of their half-lives by injection of radiolabeled ligands.We now report that multiple glycoproteins bearing oligosaccharides that terminate with Siaα2,6Galβ1,4GlcNAc are elevated in the plasma of ASGP-R-deficient ASGP-R2(-/-) mice as compared with wild-type (Wt) mice. The elevation of multiple glycoproteins bearing terminal Siaα2,6Galβ1,4GlcNAc supports our proposal that the ASGP-R accounts for the clearance of these glycoproteins. This previously undiscerned role of the ASGP-R now allows us to develop a model of how this receptor may contribute to the regulation of the concentration of many different glycoproteins in the blood.     

In Vitro Mutational Analysis of the β2 Adrenergic Receptor,an In Vivo Surrogate Odorant Receptor

Many G-protein coupled receptors (GPCRs), such as odorant receptors (ORs), cannot be characterized in heterologous cells because of their difficulty in trafficking to the plasma membrane. In contrast, a surrogate OR, the GPCR mouse β2-adrenergic-receptor (mβ2AR), robustly traffics to the plasma membrane. We set out to characterize mβ2AR mutants in vitro for their eventual use in olfactory axon guidance studies. We performed an extensive mutational analysis of mβ2AR using a Green Fluorescent Protein-tagged mβ2AR (mβ2AR::GFP) to easily assess the extent of its plasma membrane localization. In order to characterize mutants for their ability to successfully transduce ligand-initiated signal cascades, we determined the half maximal effective concentrations (EC50) and maximal response to isoprenaline, a known mβ2AR agonist. Our analysis reveals that removal of amino terminal (Nt) N-glycosylation sites and the carboxy terminal (Ct) palmitoylation site of mβ2AR do not affect its plasma membrane localization. By contrast, when both the Nt and Ct of mβ2AR are replaced with those of M71 OR, plasma membrane trafficking is impaired. We further analyze three mβ2AR mutants (RDY, E268A, and C327R) used in olfactory axon guidance studies and are able to decorrelate their plasma membrane trafficking with their capacity to respond to isoprenaline. A deletion of the Ct prevents proper trafficking and abolishes activity, but plasma membrane trafficking can be selectively rescued by a Tyrosine to Alanine mutation in the highly conserved GPCR motif NPxxY. This new loss-of-function mutant argues for a model in which residues located at the end of transmembrane domain 7 can act as a retention signal when unmasked. Additionally, to our surprise, amongst our set of mutations only Ct mutations appear to lower mβ2AR EC50s revealing their critical role in G-protein coupling. We propose that an interaction between the Nt and Ct is necessary for proper folding and/or transport of GPCRs.     

Cadherin Switching and Activation of ��-Catenin Signaling Underlie Proinvasive Actions of Calcitonin-Calcitonin Receptor Axis in Prostate Cancer

Calcitonin, a neuroendocrine peptide, and its receptor are localized in the basal epithelium of benign prostate but in the secretory epithelium of malignant prostates. The abundance of calcitonin and calcitonin receptor mRNA displays positive correlation with the Gleason grade of primary prostate cancers. Moreover, calcitonin increases tumorigenicity and invasiveness of multiple prostate cancer cell lines by cyclic AMP-dependent protein kinase-mediated actions. These actions include increased secretion of matrix metalloproteinases and urokinase-type plasminogen activator and an increase in prostate cancer cell invasion. Activation of calcitonin-calcitonin receptor autocrine loop in prostate cancer cell lines led to the loss of cell-cell adhesion, destabilization of tight and adherens junctions, and internalization of key integral membrane proteins. In addition, the activation of calcitonin-calcitonin receptor axis induced epithelial-mesenchymal transition of prostate cancer cells as characterized by cadherin switch and the expression of the mesenchymal marker, vimentin. The activated calcitonin receptor phosphorylated glycogen synthase kinase-3, a key regulator of cytosolic β-catenin degradation within the WNT signaling pathway. This resulted in the accumulation of intracellular β-catenin, its translocation in the nucleus, and transactivation of β-catenin-responsive genes. These results for the first time identify actions of calcitonin-calcitonin receptor axis on prostate cancer cells that lead to the destabilization of cell-cell junctions, epithelial-to-mesenchymal transition, and activation of WNT/β-catenin signaling. The results also suggest that cyclic AMP-dependent protein kinase plays a key role in calcitonin receptor-induced destabilization of cell-cell junctions and activation of WNT-β-catenin signaling.Prostate cancer (PC)² is the most commonly diagnosed cancer and the second leading cause of cancer deaths in men in the United States (1, 2). Although androgen ablation therapy is effective in men with advanced disease for some time, the disease subsequently progresses to the androgen-independent stage. The population of prostate cells expressing neuroendocrine factors such as calcitonin (CT) also increases during this progression (3–5). At this stage, the disease is metastatic and chemoresistant. Present evidence suggests that cancer metastasis is usually preceded by the disruption of normal cell-cell adhesion and the loss of integrity of the primary tumor site (6, 7). This process may include several genetic, molecular, and morphological changes characterized by epithelial-to-mesenchymal transition (EMT) (8–10). The EMT is characterized by the loss of cell polarity, altered cell-cell and cell-matrix adhesion, and acquisition of migratory, mesenchymal phenotype. Other reported changes include down-regulation of E-cadherin, induction of N-cadherin, release of β-catenin from junctional complexes, and its translocation to the nucleus (11–13). However, the precise molecular mechanisms associated with this process are obscure.Several growth factors, including hepatocyte growth factor, transforming growth factor-β, vascular endothelial growth factor, and epidermal growth factor, have been reported to induce EMT in tumor cell lines (14–16). We have shown that the expression of CT and its G protein-coupled receptor (CTR) is remarkably higher in advanced PCs, and the CT-CTR autocrine axis is a potent stimulator of PC cell tumorigenicity, invasion, and metastasis (4, 17–19). Although CT-stimulated increase in the motility and invasion of PC cells may be mediated by CT-stimulated secretion of matrix metalloproteinases and urokinase-type plasminogen activator, the precise molecular mechanisms preceding these CTR actions remain to be elucidated (18, 20). We tested the hypothesis that CT induces biochemical and morphological changes associated with EMT to increase the invasiveness of PC cells.Our results indicate that activation of the CT-CTR autocrine axis in prostate cancer cells induced several changes associated with EMT such as remodeling of tight and adherens junctions, cadherin switching, and activation of WNT/β-catenin signaling. In contrast, the silencing of the CT-CTR axis reversed this process. Moreover, cyclic AMP-dependent protein kinase (PKA) plays a key role in this CT-CTR-mediated process. This is the first study demonstrating the action of prostate CTR on junctional complexes and WNT/β-catenin signaling of PC cell lines.     

The Octopamine Receptor Octβ2R Regulates Ovulation in Drosophila melanogaster

Oviposition is induced upon mating in most insects. Ovulation is a primary step in oviposition, representing an important target to control insect pests and vectors, but limited information is available on the underlying mechanism. Here we report that the beta adrenergic-like octopamine receptor Octβ2R serves as a key signaling molecule for ovulation and recruits protein kinase A and Ca²⁺/calmodulin-sensitive kinase II as downstream effectors for this activity. We found that the octβ2r homozygous mutant females are sterile. They displayed normal courtship, copulation, sperm storage and post-mating rejection behavior but were unable to lay eggs. We have previously shown that octopamine neurons in the abdominal ganglion innervate the oviduct epithelium. Consistently, restored expression of Octβ2R in oviduct epithelial cells was sufficient to reinstate ovulation and full fecundity in the octβ2r mutant females, demonstrating that the oviduct epithelium is a major site of Octβ2R’s function in oviposition. We also found that overexpression of the protein kinase A catalytic subunit or Ca²⁺/calmodulin-sensitive protein kinase II led to partial rescue of octβ2r’s sterility. This suggests that Octβ2R activates cAMP as well as additional effectors including Ca²⁺/calmodulin-sensitive protein kinase II for oviposition. All three known beta adrenergic-like octopamine receptors stimulate cAMP production in vitro. Octβ1R, when ectopically expressed in the octβ2r’s oviduct epithelium, fully reinstated ovulation and fecundity. Ectopically expressed Octβ3R, on the other hand, partly restored ovulation and fecundity while OAMB-K3 and OAMB-AS that increase Ca²⁺ levels yielded partial rescue of ovulation but not fecundity deficit. These observations suggest that Octβ2R have distinct signaling capacities in vivo and activate multiple signaling pathways to induce egg laying. The findings reported here narrow the knowledge gap and offer insight into novel strategies for insect control.     

A Human Platelet Receptor Protein Microarray Identifies the High Affinity Immunoglobulin E Receptor Subunit α (FcεR1α) as an Activating Platelet Endothelium Aggregation Receptor 1 (PEAR1) Ligand

Genome-wide association studies to identify loci responsible for platelet function and cardiovascular disease susceptibility have repeatedly identified polymorphisms linked to a gene encoding platelet endothelium aggregation receptor 1 (PEAR1), an “orphan” cell surface receptor that is activated to stabilize platelet aggregates. To investigate how PEAR1 signaling is initiated, we sought to identify its extracellular ligand by creating a protein microarray representing the secretome and receptor repertoire of the human platelet. Using an avid soluble recombinant PEAR1 protein and a systematic screening assay designed to detect extracellular interactions, we identified the high affinity immunoglobulin E (IgE) receptor subunit α (FcεR1α) as a PEAR1 ligand. FcεR1α and PEAR1 directly interacted through their membrane-proximal Ig-like and 13th epidermal growth factor domains with a relatively strong affinity (K_D ∼ 30 nm). Precomplexing FcεR1α with IgE potently inhibited the FcεR1α-PEAR1 interaction, and this was relieved by the anti-IgE therapeutic omalizumab. Oligomerized FcεR1α potentiated platelet aggregation and led to PEAR1 phosphorylation, an effect that was also inhibited by IgE. These findings demonstrate how a protein microarray resource can be used to gain important insight into the function of platelet receptors and provide a mechanistic basis for the initiation of PEAR1 signaling in platelet aggregation.Platelets play a vital role in preserving blood circulation in response to vessel injury by detecting lesions, aggregating to form a hemostatic plug, and nucleating the formation of a fibrin-rich, injury-occluding clot. Although necessary to prevent blood loss at sites of tissue trauma, clot formation must also be attenuated to prevent blockage of the vasculature serving vital organs that would cause life-threatening ischemia and infarction. Inappropriate platelet aggregation and vessel occlusion, often triggered by atherosclerotic plaque rupture, is a major pathological process that is a major contributor to cardiovascular disease, which is the leading cause of mortality worldwide (1). With the eventual aim of guiding the development of new treatments and diagnostic assays, genome-wide association studies using large patient cohorts have identified several genetic loci that are associated with cardiovascular disease susceptibility and platelet function (2, 3). Among the candidate genes identified, polymorphisms linked to PEAR1 have been repeatedly linked to natural variation in response to platelet agonists in several independent studies (3–7). PEAR1 encodes platelet endothelium activation receptor 1 (PEAR1;¹ also known as multiple epidermal growth factor-like domain protein 12 (MEGF12) or JEDI-1), a platelet cell surface receptor that was originally identified as a protein phosphorylated in response to platelet aggregation (8, 9). PEAR1 is expressed at low levels on the surface of circulating platelets but is significantly up-regulated during platelet activation when released from cytoplasmic α-granules (8). Consistent with polymorphisms linked to PEAR1 being associated with cardiovascular disease and platelet function, PEAR1-mediated signaling was shown to reinforce and stabilize the interactions between platelets within a forming aggregate (8). PEAR1 is an orphan receptor, and an important unanswered question in understanding the mechanism of PEAR1 function during platelet aggregation, therefore, is the identification of its activating ligand.Identifying interactions between membrane-embedded receptor proteins is technically challenging, and many commonly used approaches such as biochemical purifications are generally not suitable to detect them. This is largely due to the amphipathic nature of membrane-embedded proteins that makes them difficult to solubilize in detergents that retain their native conformation and the fact that their extracellular interactions are often highly transient, having half-lives of just fractions of a second (10). To address these issues, we and others have developed assays based on detecting direct protein interactions between the entire ectodomains of cell surface receptors expressed as soluble recombinant proteins in eukaryotic cells (11–14). Using this approach, binding avidity can be increased by the purposeful inclusion of oligomerizing tags to overcome the fleeting nature of these interactions. In our assay, avidity-based extracellular interaction screen (AVEXIS), arrays of monomeric biotinylated “bait” proteins are screened against multimerized, enzyme-tagged, highly avid “preys” (11, 15); a schematic of the assay is shown in supplemental Fig. S1. The likelihood that the extracellular binding functions of receptors are preserved is increased by expressing whole ectodomains in mammalian cells so that structurally critical posttranslational modifications such as disulfide bonds are faithfully added. Consequently, this method has identified interactions that have subsequently been demonstrated to be essential for cellular recognition processes in vivo (16–18).In this study, we have compiled a protein resource representing the cell surface receptor repertoire and secretome of the human platelet that will be useful to identify intercellular interactions important for platelet biology. As an example, we identify the activating ligand for PEAR1 as the high affinity immunoglobulin E (IgE) receptor subunit α (FcεR1α) and show that multimerized FcεR1α potentiated platelet aggregation and led to PEAR1 phosphorylation, an effect that was specifically inhibited by IgE.     

Proper Restoration of Excitation-Contraction Coupling in the Dihydropyridine Receptor ��1-null Zebrafish Relaxed Is an Exclusive Function of the ��1a Subunit

The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) β_1a subunit. Lack of β_1a results in (i) reduced membrane expression of the pore forming DHPR α_1S subunit, (ii) elimination of α_1S charge movement, and (iii) impediment of arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine receptor (RyR1), a structural prerequisite for skeletal muscle-type excitation-contraction (EC) coupling. In this study we used relaxed larvae and isolated myotubes as expression systems to discriminate specific functions of β_1a from rather general functions of β isoforms. Zebrafish and mammalian β_1a subunits quantitatively restored α_1S triad targeting and charge movement as well as intracellular Ca²⁺ release, allowed arrangement of DHPRs in tetrads, and most strikingly recovered a fully motile phenotype in relaxed larvae. Interestingly, the cardiac/neuronal β_2a as the phylogenetically closest, and the ancestral housefly β_M as the most distant isoform to β_1a also completely recovered α_1S triad expression and charge movement. However, both revealed drastically impaired intracellular Ca²⁺ transients and very limited tetrad formation compared with β_1a. Consequently, larval motility was either only partially restored (β_2a-injected larvae) or not restored at all (β_M). Thus, our results indicate that triad expression and facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common features of all tested β subunits, whereas the efficient arrangement of DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the β_1a isoform. Consequently, we postulate a model that presents β_1a as an allosteric modifier of α_1S conformation enabling skeletal muscle-type EC coupling.Excitation-contraction (EC)³ coupling in skeletal muscle is critically dependent on the close interaction of two distinct Ca²⁺ channels. Membrane depolarizations of the myotube are sensed by the voltage-dependent 1,4-dihydropyridine receptor (DHPR) in the sarcolemma, leading to a rearrangement of charged amino acids (charge movement) in the transmembrane segments S4 of the pore-forming DHPR α_1S subunit (1, 2). This conformational change induces via protein-protein interaction (3, 4) the opening of the sarcoplasmic type-1 ryanodine receptor (RyR1) without need of Ca²⁺ influx through the DHPR (5). The release of Ca²⁺ from the sarcoplasmic reticulum via RyR1 consequently induces muscle contraction. The protein-protein interaction mechanism between DHPR and RyR1 requires correct ultrastructural targeting of both channels. In Ca²⁺ release units (triads and peripheral couplings) of the skeletal muscle, groups of four DHPRs (tetrads) are coupled to every other RyR1 and hence are geometrically arranged following the RyR-specific orthogonal arrays (6).The skeletal muscle DHPR is a heteromultimeric protein complex, composed of the voltage-sensing and pore-forming α_1S subunit and auxiliary subunits β_1a, α₂δ-1, and γ₁ (7). While gene knock-out of the DHPR γ₁ subunit (8, 9) and small interfering RNA knockdown of the DHPR α₂δ-1 subunit (10-12) have indicated that neither subunit is essential for coupling of the DHPR with RyR1, the lack of the α_1S or of the intracellular β_1a subunit is incompatible with EC coupling and accordingly null model mice die perinatally due to asphyxia (13, 14). β subunits of voltage-gated Ca²⁺ channels were repeatedly shown to be responsible for the facilitation of α₁ membrane insertion and to be potent modulators of α₁ current kinetics and voltage dependence (15, 16). Whether the loss of EC coupling in β₁-null mice was caused by decreased DHPR membrane expression or by the lack of a putative specific contribution of the β subunit to the skeletal muscle EC coupling apparatus (17, 18) was not clearly resolved. Recently, other β-functions were identified in skeletal muscle using the β₁-null mutant zebrafish relaxed (19, 20). Like the β₁-knock-out mouse (14) zebrafish relaxed is characterized by complete paralysis of skeletal muscle (21, 22). While β₁-knock-out mouse pups die immediately after birth due to respiratory paralysis (14), larvae of relaxed are able to survive for several days because of oxygen and metabolite diffusion via the skin (23). Using highly differentiated myotubes that are easy to isolate from these larvae, the lack of EC coupling could be described by quantitative immunocytochemistry as a moderate ∼50% reduction of α_1S membrane expression although α_1S charge movement was nearly absent, and, most strikingly, as the complete lack of the arrangement of DHPRs in tetrads (19). Thus, in skeletal muscle the β subunit enables EC coupling by (i) enhancing α_1S membrane targeting, (ii) facilitating α_1S charge movement, and (iii) enabling the ultrastructural arrangement of DHPRs in tetrads.The question arises, which of these functions are specific for the skeletal muscle β_1a and which ones are rather general properties of Ca²⁺ channel β subunits. Previous reconstitution studies made in the β₁-null mouse system (24, 25) using different β subunit constructs (26) did not allow differentiation between β-induced enhancement of non-functional α_1S membrane expression and the facilitation of α_1S charge movement, due to the lack of information on α_1S triad expression levels. Furthermore, the β-induced arrangement of DHPRs in tetrads was not detected as no ultrastructural information was obtained.In the present study, we established zebrafish mutant relaxed as an expression system to test different β subunits for their ability to restore skeletal muscle EC coupling. Using isolated myotubes for in vitro experiments (19, 27) and complete larvae for in vivo expression studies (28-31) and freeze-fracture electron microscopy, a clear differentiation between the major functional roles of β subunits was feasible in the zebrafish system. The cloned zebrafish β_1a and a mammalian (rabbit) β_1a were shown to completely restore all parameters of EC coupling when expressed in relaxed myotubes and larvae. However, the phylogenetically closest β subunit to β_1a, the cardiac/neuronal isoform β_2a from rat, as well as the ancestral β_M isoform from the housefly (Musca domestica), could recover functional α_1S membrane insertion, but led to very restricted tetrad formation when compared with β_1a, and thus to impaired DHPR-RyR1 coupling. This impairment caused drastic changes in skeletal muscle function.The present study shows that the enhancement of functional α_1S membrane expression is a common function of all the tested β subunits, from β_1a to even the most distant β_M, whereas the effective formation of tetrads and thus proper skeletal muscle EC coupling is an exclusive function of the skeletal muscle β_1a subunit. In context with previous studies, our results suggest a model according to which β_1a acts as an allosteric modifier of α_1S conformation. Only in the presence of β_1a, the α_1S subunit is properly folded to allow RyR1 anchoring and thus skeletal muscle-type EC coupling.     

GEC1-κ Opioid Receptor Binding Involves Hydrophobic Interactions: GEC1 HAS CHAPERONE-LIKE EFFECT*S?

We demonstrated previously that the protein GEC1 (glandular epithelial cell 1) bound to the human κ opioid receptor (hKOPR) and promoted cell surface expression of the receptor by facilitating its trafficking along the secretory pathway. Here we showed that three hKOPR residues (Phe³⁴⁵, Pro³⁴⁶, and Met³⁵⁰) and seven GEC1 residues (Tyr⁴⁹, Val⁵¹, Leu⁵⁵, Thr⁵⁶, Val⁵⁷, Phe⁶⁰, and Ile⁶⁴) are indispensable for the interaction. Modeling studies revealed that the interaction was mediated via direct contacts between the kinked hydrophobic fragment in hKOPR C-tail and the curved hydrophobic surface in GEC1 around the S2 β-strand. Intramolecular Leu⁴⁴-Tyr¹⁰⁹ interaction in GEC1 was important, likely by maintaining its structural integrity. Microtubule binding mediated by the GEC1 N-terminal domain was essential for the GEC1 effect. Expression of GEC1 also increased cell surface levels of the GluR1 subunit and the prostaglandin EP3.f receptor, which have FPXXM and FPXM sequences, respectively. With its widespread distribution in the nervous system and its predominantly hydrophobic interactions, GEC1 may have chaperone-like effects for many cell surface proteins along the biosynthesis pathway.κ opioid receptor (KOPR)² is one of the three major types of opioid receptors mediating effects of opioid drugs and endogenous opioid peptides. Stimulation of KOPR generates many effects in vivo, for example antinociception (especially for visceral chemical pain, antipruritis, and water diuresis (1). The KOPR agonist nalfurafine (TRK-820) is used clinically in Sweden for the treatment of uremic pruritus in kidney dialysis patients (2). Because KOPR agonists produce profound sedative effects, it has been proposed that KOPR agonists may be useful in treating mania, antagonists as anti-depressants, and partial agonists for the management of mania depression (3). KOPR antagonists may also be useful for curbing cocaine craving and as anti-anxiety drugs (4, 5).KOPR, a member of the rhodopsin subfamily of the seven-transmembrane receptor superfamily, is coupled preferentially to pertussis toxin-sensitive G proteins, namely G_i/o proteins (6). KOPR has been found to interact with several non-G protein-binding partners, such as Na⁺,H⁺-exchanger regulatory factor-1/ezrin-radixin-moesin-binding phosphoprotein-50 and the δ opioid receptor. These interactions have influence on signal transduction and trafficking of the receptor (7–9). By yeast two-hybrid (Y2H) assay using the hKOPR C-tail to screen a human brain cDNA library, we identified GEC1, also named GABA_A receptor-associated protein like 1 (GABARAPL1), to be a binding partner of hKOPR (10).GEC1 cDNA was first cloned as an early estrogen-regulated mRNA from guinea pig endometrial glandular epithelial cells by Pellerin et al. (11). Subsequently, it was cloned from other species, including human and house mouse (12). Interestingly, the amino acid sequences of GEC1 are completely conserved among all these species except orangutan, in which Arg⁹⁹ substitutes for His⁹⁹. Northern blot and immunoblotting analyses revealed that it has widespread tissue distribution (12–14). In particular, GEC1 was found to be abundant in the central nervous system and expressed throughout the rat brain (14, 15). This wide tissue distribution and the high sequence identity across species strongly suggest that GEC1 has important biological functions in mammalian cells.Based on sequence similarity, GEC1 is classified as a member of microtubule-associated proteins (MAPs), which also include GABA_A receptor-associated protein (GABARAP), Golgi-associated ATPase enhancer of 16 kDa (GATE16), GABARAP-like 3 (GABARAPL3), light chain 3 (LC3) of MAP 1A/1B, and the yeast autophagy protein 8 (Atg8) (12, 13). Among these homologues, GEC1 share the highest identity with GABARAPL3 (93%), followed by GABARAP (86%), GATE16 (61%), Atg8 (55%), and LC3 (∼30%).A growing body of evidence shows that this protein family is closely related to two distinct biological functions. Studies mainly on GABARAP, GATE16, and GEC1 indicate that they promote intracellular protein trafficking by enhancing vesicle fusion (10, 16–21). In addition, they facilitate degradation of proteins and intracellular organelles via autophagy-related pathways, which is bolstered largely by research on Atg8 and LC3 (22, 23).We previously reported that GEC1 interacted with the hKOPR C-tail and enhanced cell surface levels of hKOPR stably expressed in CHO cells. GEC1 expression enhances hKOPR expression through facilitating its anterograde trafficking along the protein biosynthesis pathway without affecting degradation of the receptor (10). This represented the first biological function reported for GEC1. Mansuy et al. (24) demonstrated that GEC1 interacted with tubulin and promoted microtubule bundling in vitro, and that green fluorescence protein-tagged GEC1 was localized in the perinuclear vesicles with a scattered pattern. Our electron microscopic studies in the rat brain showed that GEC1 was associated with ER, Golgi apparatus, endosome-like vesicles, and plasma membranes and scattered in cytoplasm in neurons (14). In addition, N-ethylmaleimide-sensitive factor, a protein critical for intracellular membrane-trafficking events, binds directly to GEC1 (10).In this study, we employed Y2H techniques to determine the amino acid residues in both GEC1 and hKOPR C-tail involved in the interaction. Further studies were then carried out in mammalian cells to examine if elimination of the interaction affected the effect of GEC1 on hKOPR expression. In addition, we generated a molecular model of GEC1 based on the x-ray crystal structure of GABARAP and found that the residues involved in hKOPR binding formed hydrophobic patches on the exterior surface of GEC1. Moreover, we found that the cytosolic tail of AMPA receptor subunit GluR1 has the same FPXXM motif as that found in the hKOPR C-tail to be involved in GEC1 binding and that GEC1 expression up-regulated GluR1.     

Identification of the Lipopolysaccharide Core of Yersinia pestis and Yersinia pseudotuberculosis as the Receptor for Bacteriophage ��A1122

φA1122 is a T7-related bacteriophage infecting most isolates of Yersinia pestis, the etiologic agent of plague, and used by the CDC in the identification of Y. pestis. φA1122 infects Y. pestis grown both at 20°C and at 37°C. Wild-type Yersinia pseudotuberculosis strains are also infected but only when grown at 37°C. Since Y. pestis expresses rough lipopolysaccharide (LPS) missing the O-polysaccharide (O-PS) and expression of Y. pseudotuberculosis O-PS is largely suppressed at temperatures above 30°C, it has been assumed that the phage receptor is rough LPS. We present here several lines of evidence to support this. First, a rough derivative of Y. pseudotuberculosis was also φA1122 sensitive when grown at 22°C. Second, periodate treatment of bacteria, but not proteinase K treatment, inhibited the phage binding. Third, spontaneous φA1122 receptor mutants of Y. pestis and rough Y. pseudotuberculosis could not be isolated, indicating that the receptor was essential for bacterial growth under the applied experimental conditions. Fourth, heterologous expression of the Yersinia enterocolitica O:3 LPS outer core hexasaccharide in both Y. pestis and rough Y. pseudotuberculosis effectively blocked the phage adsorption. Fifth, a gradual truncation of the core oligosaccharide into the Hep/Glc (l-glycero-d-manno-heptose/d-glucopyranose)-Kdo/Ko (3-deoxy-d-manno-oct-2-ulopyranosonic acid/d-glycero-d-talo-oct-2-ulopyranosonic acid) region in a series of LPS mutants was accompanied by a decrease in phage adsorption, and finally, a waaA mutant expressing only lipid A, i.e., also missing the Kdo/Ko region, was fully φA1122 resistant. Our data thus conclusively demonstrated that the φA1122 receptor is the Hep/Glc-Kdo/Ko region of the LPS core, a common structure in Y. pestis and Y. pseudotuberculosis.     

O-GlcNAcylation-Inducing Treatments Inhibit Estrogen Receptor α Expression and Confer Resistance to 4-OH-Tamoxifen in Human Breast Cancer-Derived MCF-7 Cells

O-GlcNAcylation (addition of N-acetyl-glucosamine on serine or threonine residues) is a post-translational modification that regulates stability, activity or localization of cytosolic and nuclear proteins. O-linked N-acetylgluocosmaine transferase (OGT) uses UDP-GlcNAc, produced in the hexosamine biosynthetic pathway to O-GlcNacylate proteins. Removal of O-GlcNAc from proteins is catalyzed by the β-N-Acetylglucosaminidase (OGA). Recent evidences suggest that O-GlcNAcylation may affect the growth of cancer cells. However, the consequences of O-GlcNAcylation on anti-cancer therapy have not been evaluated. In this work, we studied the effects of O-GlcNAcylation on tamoxifen-induced cell death in the breast cancer-derived MCF-7 cells. Treatments that increase O-GlcNAcylation (PUGNAc and/or glucosoamine) protected MCF-7 cells from death induced by tamoxifen. In contrast, inhibition of OGT expression by siRNA potentiated the effect of tamoxifen on cell death. Since the PI-3 kinase/Akt pathway is a major regulator of cell survival, we used BRET to evaluate the effect of PUGNAc+glucosamine on PIP₃ production. We observed that these treatments stimulated PIP₃ production in MCF-7 cells. This effect was associated with an increase in Akt phosphorylation. However, the PI-3 kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, which abolished the effect of PUGNAc+glucosamine on Akt phosphorylation, did not impair the protective effects of PUGNAc+glucosamine against tamoxifen-induced cell death. These results suggest that the protective effects of O-GlcNAcylation are independent of the PI-3 kinase/Akt pathway. As tamoxifen sensitivity depends on the estrogen receptor (ERα) expression level, we evaluated the effect of PUGNAc+glucosamine on the expression of this receptor. We observed that O-GlcNAcylation-inducing treatment significantly reduced the expression of ERα mRNA and protein, suggesting a potential mechanism for the decreased tamoxifen sensitivity induced by these treatments. Therefore, our results suggest that inhibition of O-GlcNAcylation may constitute an interesting approach to improve the sensitivity of breast cancer to anti-estrogen therapy.