Functional interaction between Vangl2 and N-cadherin regulates planar cell polarization of the developing neural tube and cochlear sensory epithelium.

Although the core constituents of the Wnt/planar cell polarity (PCP) signaling have been extensively studied, their downstream molecules and protein-protein interactions have not yet been fully elucidated. Here, we show genetic and molecular evidence that the PCP factor, Vangl2, functionally interacts with the cell-cell adhesion molecule, N-cadherin (also known as Cdh2), for typical PCP-dependent neural development. Vangl2 and N-cadherin physically interact in the neural plates undergoing convergent extension. Unlike monogenic heterozygotes, digenic heterozygous mice with Vangl2 and Cdh2 mutants exhibited defects in neural tube closure and cochlear hair cell orientation. Despite this genetic interaction, neuroepithelial cells derived from the digenic heterozygotes did not show additive changes from the monogenic heterozygotes of Vangl2 in the RhoA-ROCK-Mypt1 and c-Jun N-terminal kinase (JNK)-Jun pathways of Wnt/PCP signaling. Thus, cooperation between Vangl2 and N-cadherin is at least partly via direct molecular interaction; it is essential for the planar polarized development of neural tissues but not significantly associated with RhoA or JNK pathways.

Frequent Germline and Somatic Single Nucleotide Variants in the Promoter Region of the Ribosomal RNA Gene in Japanese Lung Adenocarcinoma Patients.

Ribosomal RNA (rRNA), the most abundant non-coding RNA species, is a major component of the ribosome. Impaired ribosome biogenesis causes the dysfunction of protein synthesis and diseases called "ribosomopathies," including genetic disorders with cancer risk. However, the potential role of rRNA gene (rDNA) alterations in cancer is unknown. We investigated germline and somatic single-nucleotide variants (SNVs) in the rDNA promoter region (positions -248 to +100, relative to the transcription start site) in 82 lung adenocarcinomas (LUAC). Twenty-nine tumors (35.4%) carried germline SNVs, and eight tumors (9.8%) harbored somatic SNVs. Interestingly, the presence of germline SNVs between positions +1 and +100 (n = 12; 14.6%) was associated with significantly shorter recurrence-free survival (RFS) and overall survival (OS) by univariate analysis (p < 0.05, respectively), and was an independent prognostic factor for RFS and OS by multivariate analysis. LUAC cell line PC9, carrying rDNA promoter SNV at position +49, showed significantly higher ribosome biogenesis than H1650 cells without SNV. Upon nucleolar stress induced by actinomycin D, PC9 retained significantly higher ribosome biogenesis than H1650. These results highlight the possible functional role of SNVs at specific sites of the rDNA promoter region in ribosome biogenesis, the progression of LUAC, and their potential prognostic value.

Vangl2 interaction plays a role in the proteasomal degradation of Prickle2.

The PET and LIM domain-containing protein, Prickle, plays a key role in planar cell polarity (PCP) in Drosophila. It has been reported that mutations in the PRICKLE2 gene, which encodes one of the human orthologues of Prickle, are associated with human diseases such as epilepsy and autism spectrum disorder. To develop preventive and therapeutic strategies for these intractable diseases, we studied the regulation of Prickle2 protein levels in transfected HEK293T cells. Prickle2 levels were negatively regulated by a physical interaction with another PCP protein, Van Gogh-like 2 (Vangl2). The Vangl2-mediated reduction in Prickle2 levels was, at least in part, relieved by proteasome inhibitors or by functional inhibition of the Cullin-1 E3 ubiquitin ligase. Furthermore, the expression of Vangl2 enhanced the polyubiquitination of Prickle2. This ubiquitination was partially blocked by co-expression of a ubiquitin mutant, which cannot be polymerised through their Lys48 residue to induce target proteins toward proteasomal degradation. Together, these results suggest that Prickle2 is polyubiquitinated by the Vangl2 interaction in a Cullin-1-dependent manner to limit its expression levels. This regulation may play a role in the local and temporal fine-tuning of Prickle protein levels during PCP signal-dependent cellular behaviours.

Isozyme-Specific Role of SAD-A in Neuronal Migration During Development of Cerebral Cortex.

SAD kinases regulate presynaptic vesicle clustering and neuronal polarization. A previous report demonstrated that Sada-/- and Sadb-/- double-mutant mice showed perinatal lethality with a severe defect in axon/dendrite differentiation, but their single mutants did not. These results indicated that they were functionally redundant. Surprisingly, we show that on a C57BL/6N background, SAD-A is essential for cortical development whereas SAD-B is dispensable. Sada-/- mice died within a few days after birth. Their cortical lamination pattern was disorganized and radial migration of cortical neurons was perturbed. Birth date analyses with BrdU and in utero electroporation using pCAG-EGFP vector showed a delayed migration of cortical neurons to the pial surface in Sada-/- mice. Time-lapse imaging of these mice confirmed slow migration velocity in the cortical plate. While the neurites of hippocampal neurons in Sada-/- mice could ultimately differentiate in culture to form axons and dendrites, the average length of their axons was shorter than that of the wild type. Thus, analysis on a different genetic background than that used initially revealed a nonredundant role for SAD-A in neuronal migration and differentiation.

Prognostic Impact of Serum Albumin Concentration for Neurologically Favorable Outcome in Patients Treated with Targeted Temperature Management After Out-of-Hospital Cardiac Arrest: A Multicenter Prospective Study.

To assess whether serum albumin concentration measured upon hospital arrival was useful as an early prognostic biomarker for neurologically favorable outcome in out-of-hospital cardiac arrest (OHCA) patients treated with target temperature management (TTM). This prospective, multicenter observational study (The CRITICAL Study) carried out between July 1, 2012 and December 31, 2014 in Osaka Prefecture, Japan involving 13 critical care medical centers (CCMCs) and one non-CCMC with an emergency department. This study included patients ≥18 years of age who underwent an OHCA, for whom resuscitation was attempted by Emergency Medical Services personnel and were then transported to participating institutions, and who were then treated with TTM. Based on the serum albumin concentration upon hospital arrival, involved patients were divided into four quartiles (Q1-Q4) defined as Q1 (<3.0 g/dL), Q2 (≥3.0, <3.4 g/dL), Q3 (≥3.4, <3.8 g/dL), and Q4 (≥3.8 g/dL). The primary outcome of this study was 1-month survival with neurologically favorable outcome defined by cerebral performance category 1 or 2. During the study period, a total of 327 were eligible for our analysis. The overall proportion of neurologically favorable outcome was 33.0% (108/327). The Q4 group had the highest proportion of neurologically favorable outcome (52.5% [48/91]), followed by Q3 (34.5% [30/87]), Q2 (27.3% [21/77]), and Q1 (12.5% [9/72]). The multivariable logistic regression analysis demonstrated that the proportion of neurologically favorable outcome was significantly higher in the Q4 group than that in the Q1 group (adjusted odds ratio 10.39; 95% confidence interval 3.36-32.17). The adjusted proportion of neurologically favorable outcome increased in a stepwise fashion across increasing quartiles (p < 0.001). In this study, higher serum albumin concentration upon hospital arrival had a positive association with neurologically favorable outcome after OHCA in a dose-dependent manner.

Effect of Serum Albumin Concentration on Neurological Outcome After Out-of-Hospital Cardiac Arrest (from the CRITICAL [Comprehensive Registry of Intensive Cares for OHCA Survival] Study in Osaka, Japan).

The aim of this study was to assess whether serum albumin concentration upon hospital arrival had prognostic indications on out-of-hospital cardiac arrest (OHCA). This prospective, multicenter observational study conducted in Osaka, Japan (the CRITICAL [Comprehensive Registry of Intensive Cares for OHCA Survival] study), enrolled all patients with consecutive OHCA transported to 14 participating institutions. We included adult patients aged ≥18 years with nontraumatic OHCA who achieved return of spontaneous circulation and whose serum albumin concentration was available from July 2012 to December 2014. Based on the serum albumin concentration upon hospital arrival, patients were divided into quartiles (Q1 to Q4), namely, Q1 (<2.7 g/dl), Q2 (2.7 to 3.1 g/dl), Q3 (3.1 to 3.6 g/dl), and Q4 (≥3.6 g/dl). The primary outcome was 1-month survival with favorable neurological outcome (cerebral performance category scale 1 or 2). During the study period, a total of 1,269 patients with OHCA were eligible for our analyses. The highest proportion of favorable neurological outcome was 33.5% (109 of 325) in the Q4 group, followed by 13.2% (48 of 365), 5.0% (13 of 261), and 3.5% (11 of 318) in the Q3, Q2, and Q1 groups, respectively. In the multivariable logistic regression analysis, the proportion of favorable neurological outcome in the Q4 group was significantly higher, compared with that in the Q1 group (adjusted odds ratio 8.61; 95% confidence interval 4.28 to 17.33). The adjusted proportion of favorable neurological outcome increased in a stepwise manner across increasing quartiles (p for trend <0.001). Higher serum albumin concentration was significantly and independently associated with favorable neurological outcome in a dose-dependent manner.

Screening for hereditary angioedema (HAE) at 13 emergency centers in Osaka, Japan: A prospective observational study.

Hereditary angioedema (HAE) with deficiency of C1 inhibitor (C1-INH) is an autosomal-dominant disease characterized by recurrent episodes of potentially life-threatening angioedema. The objective is to study the incidence of HAE among patients who visit the emergency department.This was a 3-year prospective observational screening study involving 13 urban tertiary emergency centers in Osaka prefecture, Japan. Patients were included if they met the following criteria: unexplained edema of the body, upper airway obstruction accompanied by edema, anaphylaxis, acute abdomen with intestinal edema (including ileus and acute pancreatitis), or asthma attack. C1-INH activity and C4 level were measured at the time of emergency department admission during the period between July 2011 and June 2014.This study comprised 66 patients with a median age of 54.0 (IQR: 37.5-68.3) years. Three patients were newly diagnosed as having HAE, and 1 patient had already been diagnosed as having HAE. C1-INH activity levels of the patients with HAE were below the detection limit (<25%), whereas those of non-HAE patients (n = 62) were 106% (IQR: 85.5%-127.0%) (normal range, 70%-130%). The median level of C4 was significantly lower in the patients with HAE compared with those without HAE (1.2 [IQR: 1-3] mg/dL vs 22 [IQR: 16.5-29.5] mg/dL, P < 0.01) (normal range, 17-45 mg/dL).Three patients with undiagnosed HAE were diagnosed as having HAE in the emergency department during the 3-year period. If patients have signs and symptoms suspicious of HAE, the levels of C1-INH activity and C4 should be measured.

Rationale, design, and profile of Comprehensive Registry of In-Hospital Intensive Care for OHCA Survival (CRITICAL) study in Osaka, Japan.

BACKGROUND: We established a multi-center, prospective cohort that could provide appropriate therapeutic strategies such as criteria for the introduction and the effectiveness of in-hospital advanced treatments, including percutaneous coronary intervention (PCI), target temperature management, and extracorporeal cardiopulmonary resuscitation (ECPR) for out-of-hospital cardiac arrest (OHCA) patients. METHODS: In Osaka Prefecture, Japan, we registered all consecutive patients who were suffering from an OHCA for whom resuscitation was attempted and who were then transported to institutions participating in this registry since July 1, 2012. A total of 11 critical care medical centers and one hospital with an emergency care department participated in this registry. The primary outcome was neurological status after OHCA, defined as cerebral performance category (CPC) scale. RESULTS: A total of 688 OHCA patients were documented between July 2012 and December 2012. Of them, 657 were eligible for our analysis. Patients' average age was 66.2 years old, and male patients accounted for 66.2 %. The proportion of OHCAs having a cardiac origin was 50.4 %. The proportion as first documented rhythm of ventricular fibrillation/pulseless ventricular tachycardia was 11.6 %, pulseless electrical activity 23.4 %, and asystole 54.5 %. After hospital arrival, 10.5 % received defibrillation, 90.8 % tracheal intubation, 3.0 % ECPR, 3.5 % PCI, and 83.1 % adrenaline administration. The proportions of 90-day survival and CPC 1/2 at 90 days after OHCAs were 5.9 and 3.0 %, respectively. CONCLUSIONS: The Comprehensive Registry of In-hospital Intensive Care for OHCA Survival (CRITICAL) study will enroll over 2000 OHCA patients every year. It is still ongoing without a set termination date in order to provide valuable information regarding appropriate therapeutic strategies for OHCA patients (UMIN000007528).

The planar cell polarity protein Vangl2 is involved in postsynaptic compartmentalization.

The excitatory postsynaptic region of the vertebrate hippocampus is usually compartmentalized into the postsynaptic density (PSD) and N-cadherin-rich domain, which is important for synaptic adhesion. However, the molecular mechanisms underlying the compartment formation are unknown. In the present report, we show that the planar cell polarity (PCP) protein Van Gogh-like 2 (Vangl2) plays a role in this regionalization. In cultured rat hippocampal neurons that were subjected to Vangl2 expression silencing, the formed clusters of PSD-95, one of the major scaffolding proteins in PSD, tended to overlap with those of N-cadherin. Further, in the dendrites of these neurons, the immunofluorescence of PSD-95 was to some extent diffused, without a significant change in the total signal. Because Vangl2 physically interacts with both PSD-95 and N-cadherin in vivo, these results suggest that a PCP-related direct molecular mechanism underlies the horizontal polarization of the postsynaptic regions.

PDZ interaction of Vangl2 links PSD-95 and Prickle2 but plays only a limited role in the synaptic localisation of Vangl2.

Postsynaptic density-95/Discs large/Zonula occludens-1 (PDZ) domain-mediated protein interactions play pivotal roles in various molecular biological events, including protein localisation, assembly, and signal transduction. Although the vertebrate regulator of planar cell polarity Van Gogh-like 2 (Vangl2) was recently described as a postsynaptic molecule with a PDZ-binding motif, the role of its PDZ interaction at the synapse is unknown. In this report, we demonstrate that the PDZ interaction was dispensable for the normal cluster formation of Vangl2 and not absolutely required for the synapse-associated localisation of Vangl2 in cultured hippocampal neurons. We further showed that the synaptic localisation of Vangl2 was categorised into two types: overlapping co-localisation with postsynaptic density (PSD)-95 or highly correlated but complementary pattern of association with PSD-95. Only the former was significantly sensitive to deletion of the PDZ-binding motif. In addition, the PDZ interaction enhanced the protein interactions between PSD-95 and Prickle2, which is another planar cell polarity factor that is localised at the postsynaptic density. Taken together with our recent report that the density of PSD-95 clusters was reduced in Vangl2-silenced neurons, these results suggest that Vangl2 determines the complex formation and clustering of postsynaptic molecules for synaptogenesis in mammalian brains.

Vangl2 regulates E-cadherin in epithelial cells.

E-cadherin belongs to the classic cadherin subfamily of calcium-dependent cell adhesion molecules and is crucial for the formation and function of epithelial adherens junctions. In this study, we demonstrate that Vangl2, a vertebrate regulator of planar cell polarity (PCP), controls E-cadherin in epithelial cells. E-cadherin co-immunoprecipitates with Vangl2 from embryonic kidney extracts, and this association is also observed in transfected fibroblasts. Vangl2 enhances the internalization of E-cadherin when overexpressed. Conversely, the quantitative ratio of E-cadherin exposed to the cell surface is increased in cultured renal epithelial cells derived from Vangl2(Lpt/+) mutant mice. Interestingly, Vangl2 is also internalized through protein traffic involving Rab5- and Dynamin-dependent endocytosis. Taken together with recent reports regarding the transport of Frizzled3, MMP14 and nephrin, these results suggest that one of the molecular functions of Vangl2 is to enhance the internalization of specific plasma membrane proteins with broad selectivity. This function may be involved in the control of intercellular PCP signalling or in the PCP-related rearrangement of cell adhesions.

The Wnt/planar cell polarity pathway component Vangl2 induces synapse formation through direct control of N-cadherin.

Although regulators of the Wnt/planar cell polarity (PCP) pathway are widely expressed in vertebrate nervous systems, their roles at synapses are unknown. Here, we show that Vangl2 is a postsynaptic factor crucial for synaptogenesis and that it coprecipitates with N-cadherin and PSD-95 from synapse-rich brain extracts. Vangl2 directly binds N-cadherin and enhances its internalization in a Rab5-dependent manner. This physical and functional interaction is suppressed by ß-catenin, which binds the same intracellular region of N-cadherin as Vangl2. In hippocampal neurons expressing reduced Vangl2 levels, dendritic spine formation as well as synaptic marker clustering is significantly impaired. Furthermore, Prickle2, another postsynaptic PCP component, inhibits the N-cadherin-Vangl2 interaction and is required for normal spine formation. These results demonstrate direct control of classic cadherin by PCP factors; this control may play a central role in the precise formation and maturation of cell-cell adhesions at the synapse.

A simple and highly efficient method to identify the integration site of a transgene in the animal genome.

Because genetic manipulation occasionally disrupts the expression of the neighboring genes, the chromosomal locus where the transgene has been integrated should be identified in the use of transgenic organisms. By using a new blend of thermostable DNA polymerase, we established a highly efficient method of inverse polymerase chain reaction for this purpose. By using this protocol, we successfully determined the vector integration sites of 2 mouse lines, NSE-tTA and tetO-Cre, the combination of which is a useful tool in neuroscience research. On the basis of this information, we quantified the relative expression amount of the chromosomal genes adjacent to these transgenes and found that the insertion of the tetO-Cre vector significantly altered the mRNA level of one of the examined genes. Considering the potential risk of the insertion effect, we recommend that the vector integration sites of any transgenic lines should be determined routinely by using this method, and that the expression levels of their neighboring genes should be determined.

SAD kinases control the maturation of nerve terminals in the mammalian peripheral and central nervous systems.

Axons develop in a series of steps, beginning with specification, outgrowth, and arborization, and terminating with formation and maturation of presynaptic specializations. We found previously that the SAD-A and SAD-B kinases are required for axon specification and arborization in subsets of mouse neurons. Here, we show that following these steps, SAD kinases become localized to synaptic sites and are required within presynaptic cells for structural and functional maturation of synapses in both peripheral and central nervous systems. Deleting SADs from sensory neurons can perturb either axonal arborization or nerve terminal maturation, depending on the stage of deletion. Thus, a single pair of kinases plays multiple, sequential roles in axonal differentiation.

Current termination of resuscitation (TOR) guidelines predict neurologically favorable outcome in Japan.

BACKGROUND: It is unclear whether the basic life support (BLS) and advanced life support (ALS) pre-hospital termination of resuscitation (TOR) rules developed in North America can be applied successfully to patients with out-of-hospital cardiac arrest (OHCA) in other countries. OBJECTIVES: To assess the performance of the BLS and ALS TOR in Japan. METHODS: Retrospective nationwide, population-based, observational cohort study of consecutive OHCA patients with emergency responder resuscitation attempts from 1 January 2005 to 31 December 2009 in Japan. The BLS TOR rule has 3 criteria whereas the ALS TOR rule includes 2 additional criteria. We extracted OHCA patients meeting all criteria for each TOR rule, and calculated the specificity and positive predictive value (PPV) of each TOR rule for identifying OHCA patients who did not have neurologically favorable one-month survival. RESULTS: During the study-period, 151,152 cases were available to evaluate the BLS TOR rule, and 137,986 cases to evaluate the ALS TOR rule. Of 113,140 patients that satisfied all three criteria for the BLS TOR rule, 193 (0.2%) had a neurologically favorable one-month survival. The specificity of BLS TOR rule was 0.968 (95% CI: 0.963-0.972), and the PPV was 0.998 (95% CI: 0.998-0.999) for predicting lack of neurologically favorable one-month survival. Of 41,030 patients that satisfied all five criteria for the ALS TOR rule, just 37 (0.1%) had a neurologically favorable one-month survival. The specificity of ALS TOR rule was 0.981 (95% CI: 0.973-0.986), and the PPV was 0.999 (95% CI: 0.998-0.999) for predicting lack of neurologically favorable one-month survival. CONCLUSIONS: The prehospital BLS and ALS TOR rules performed well in Japan with high specificity and PPV for predicting lack of neurologically favorable one-month survival in Japan. However, the specificity and PPV were not 1000 and we have to develop more specific TOR rules.

Comparison of supraglottic airway versus endotracheal intubation for the pre-hospital treatment of out-of-hospital cardiac arrest.

INTRODUCTION: Both supraglottic airway devices (SGA) and endotracheal intubation (ETI) have been used by emergency life-saving technicians (ELST) in Japan to treat out-of-hospital cardiac arrests (OHCAs). Despite traditional emphasis on airway management during cardiac arrest, its impact on survival from OHCA and time dependent effectiveness remains unclear. METHODS: All adults with witnessed, non-traumatic OHCA, from 1 January 2005 to 31 December 2008, treated by the emergency medical services (EMS) with an advanced airway in Osaka, Japan were studied in a prospective Utstein-style population cohort database. The primary outcome measure was one-month survival with neurologically favorable outcome. The association between type of advanced airway (ETI/SGA), timing of device placement and neurological outcome was assessed by multiple logistic regression. RESULTS: Of 7,517 witnessed non-traumatic OHCAs, 5,377 cases were treated with advanced airways. Of these, 1,679 were ETI while 3,698 were SGA. Favorable neurological outcome was similar between ETI and SGA (3.6% versus 3.6%, P = 0.95). The time interval from collapse to ETI placement was significantly longer than for SGA (17.2 minutes versus 15.8 minutes, P < 0.001). From multivariate analysis, early placement of an advanced airway was significantly associated with better neurological outcome (Adjusted Odds Ratio (AOR) for one minute delay, 0.91, 95% confidence interval (CI) 0.88 to 0.95). ETI was not a significant predictor (AOR 0.71, 95% CI 0.39 to 1.30) but the presence of an ETI certified ELST (AOR, 1.86, 95% CI 1.04 to 3.34) was a significant predictor for favorable neurological outcome. CONCLUSIONS: There was no difference in neurologically favorable outcome from witnessed OHCA for ETI versus SGA. Early airway management with advanced airway regardless of type and rhythm was associated with improved outcomes.

Impact of transport to critical care medical centers on outcomes after out-of-hospital cardiac arrest.

BACKGROUND: Post-resuscitation care has emerged as an important predictor of survival from out-of-hospital cardiac arrest (OHCA). In Japan, selected hospitals are certified as Critical Care Medical Centers (CCMCs) based on their ability and expertise. HYPOTHESIS: Outcome after OHCA is better in patients transported to a CCMC compared a non-critical care hospital (NCCH). MATERIALS AND METHODS: Adults with OHCA of presumed cardiac etiology, treated by emergency medical services systems, and transported in Osaka from January 1, 2005 to December 31, 2007 were registered using a prospective Utstein style population cohort database. Primary outcome measure was 1 month neurologically favorable survival (CPC< or =2). Outcomes of patients transported to CCMC were compared with patients transported to NCCH using multiple logistic regressions and stratified on the basis of stratified field ROSC. RESULTS: 10,383 cases were transported. Of these, 2881 were transported to CCMC and 7502 to NCCH. Neurologically favorable 1-month survival was greater in the CCMC group [6.7% versus 2.8%, P<0.001]. Among patients who were transported to hospital without field ROSC, neurologically favorable outcome was greater in the CCMC group than the NCCH group [1.7% versus 0.5%; adjusted odds ratio (OR), 3.39; 95% confidence interval (CI), 2.17-5.29; P<0.001]. In the presence of field ROSC, survival was similar between the groups [43% versus 41%; adjusted OR, 1.09; 95% CI, 0.82-1.45; P=0.554]. CONCLUSIONS: Survival after OHCA of presumed cardiac etiology transported to CCMCs was better than those transported to NCCHs. For OHCA patients without field ROSC, transport to a CCMC was an independent predictor for a good neurological outcome.

Axon or dendrite? cell biology and molecular pathways for neuronal cell asymmetry.

Young neurons polarize by specializing axons and dendrites from immature neurites. After synapse formations, they transmit electrical activity along the axon-dendrite axis, thereby working as functional units of the neural circuits. This axon-dendrite asymmetry is referred to as neuronal polarity. Although a great number of cell biological studies in vitro had been performed, little was known about the molecular events that establish the polarity. In the last several years, rapid advancement in molecular and genetic studies has unraveled the multiple signaling pathways. This paper summarizes current perspectives on the cell and molecular biological mechanisms of the neuronal polarization, to clarify future directions in this growing research field.

Role of the agonist binding site in up-regulation of neuronal nicotinic alpha4beta2 receptors.

Long-term exposure to nicotinic ligands results in up-regulation of neuronal nicotinic alpha4beta2 receptors in the brain and in heterologous expression systems. Up-regulation can be produced by a variety of drugs, including nicotinic agonists and competitive antagonists, but previous studies have indicated that the signal for up-regulation does not reflect a traditional nicotinic pharmacology, and the initial steps in signal transduction are not clear. We examined the signal for up-regulation and the possible involvement of the nicotine binding site in signal reception using mutated subunits transiently expressed in human embryonic kidney 293 cells. The data indicate that receptor activation and desensitization are not part of the signaling pathway. However, mutations to residues in the binding site can affect up-regulation. These results provide evidence that the binding site is directly involved in receiving the signal for up-regulation.

LL5beta: a regulator of postsynaptic differentiation identified in a screen for synaptically enriched transcripts at the neuromuscular junction.

In both neurons and muscle fibers, specific mRNAs are concentrated beneath and locally translated at synaptic sites. At the skeletal neuromuscular junction, all synaptic RNAs identified to date encode synaptic components. Using microarrays, we compared RNAs in synapse-rich and -free regions of muscles, thereby identifying transcripts that are enriched near synapses and that encode soluble membrane and nuclear proteins. One gene product, LL5beta, binds to both phosphoinositides and a cytoskeletal protein, filamin, one form of which is concentrated at synaptic sites. LL5beta is itself associated with the cytoplasmic face of the postsynaptic membrane; its highest levels border regions of highest acetylcholine receptor (AChR) density, which suggests a role in "corraling" AChRs. Consistent with this idea, perturbing LL5beta expression in myotubes inhibits AChR aggregation. Thus, a strategy designed to identify novel synaptic components led to identification of a protein required for assembly of the postsynaptic apparatus.