Surface clustering of metabotropic glutamate receptor 1 induced by long Homer proteins

BACKGROUND: Metabotropic glutamate receptors (mGluRs) regulate neuronal excitability and synaptic strength. The group I mGluRs, mGluR1 and 5, are widespread in the brain and localize to post-synaptic sites. The Homer protein family regulates group I mGluR function and distribution. Constitutively expressed 'long' Homer proteins (Homer 1b, 1c, 2 and 3) induce dendritic localization of group I mGluRs and receptor clustering, either internally or on the plasma membrane. Short Homer proteins (Homer 1a, Ania-3) exhibit regulated expression and act as dominant negatives, producing effects on mGluR distribution and function that oppose those of the long Homer proteins. There remains some controversy over whether long Homer proteins induce receptor internalization by inducing retention in the endoplasmic reticulum, or induce mGluR clustering on the plasma membrane. Further, an exhaustive study of the effects of each long Homer isoform on mGluR distribution has not been published. RESULTS: The distribution of a GFP-tagged group I mGluR, mGluR1-GFP, was examined in the absence of Homer proteins and in the presence of several Homer isoforms expressed in sympathetic neurons from the rat superior cervical ganglion (SCG) using total internal reflection fluorescence (TIRF-M) and confocal microscopy. Quantitative analysis of mGluR1-GFP fluorescence using TIRF-M revealed that expression of each long Homer isoform tested (Homer 1b, 1c, 2b and 3) induced a significant degree of surface clustering. Using confocal imaging, Homer-induced mGluR clusters were observed intra-cellularly as well as on the plasma membrane. Further, in approximately 40% of neurons co-expressing mGluR1-GFP and Homer 1b, intracellular inclusions were observed, but plasma membrane clusters were also documented in some Homer 1b coexpressing cells. CONCLUSION: All long Homer proteins examined (Homer 1b, 1c, 2b and 3) induced a significant degree of mGluR1-GFP clustering on the plasma membrane compared to cells expressing mGluR1-GFP alone. Clusters induced by long Homers appeared on the plasma membrane and intracellularly, suggesting that clusters form prior to plasma membrane insertion and/or persist after internalization. Finally, while Homer 1b induced surface clustering of mGluR1 in some cells, under some conditions intracellular retention may occur

A role for Seven in Absentia Homolog (Siah1a) in metabotropic glutamate receptor signaling

BACKGROUND: The mammalian homologue of Seven in Absentia (Siah) can act in the ubiquitin/proteasome pathway. Recent work has shown that Siah can bind group I metabotropic glutamate receptors (mGluRs), but the functional consequences of this interaction are unknown. RESULTS: The effects of coexpression of Siah on group I mGluR signaling were examined using heterologous expression in rat sympathetic, superior cervical ganglion neurons. Siah1a attenuated heterologously expressed group I mGluR-mediated calcium current inhibition, but was without effect on group II mGluR- or NE-mediated calcium current modulation via heterologously expressed mGluR2 or native a2 adrenergic receptors, respectively, indicating that the effect of Siah was specific for group I mGluRs. Surface expression and subcellular distribution of group I mGluRs were not detectably altered in the presence of Siah1a as assessed by immunoflourescence experiments with epitope tagged receptors and imaging of a GFP/mGluR fusion construct. In addition, an N-terminal Siah deletion construct, which cannot function in the proteolysis pathway, displayed effects similar to the wild type Siah1a. Finally, coexpression of calmodulin, which competes with Siah1a for binding to the C-terminal tail of group I mGluRs, reversed the effect of Siah1a on mGluR-mediated signaling. CONCLUSIONS: These data supported the conclusion that the attenuation of mGluR signaling induced by Siah1a expression was likely a direct consequence of Siah/mGluR association rather than a result of targeting of the receptors to the proteosome. In addition, the data suggest that the binding of CaM and Siah may play an important role in the regulation of group I mGluR function

Dynamic Regulation of Homer Binding to Group I Metabotropic Glutamate Receptors by Preso1 and Converging Kinase Cascades

ABSTRACT In rat sympathetic neurons from the superior cervical ganglia (SCG) expressing metabotropic glutamate receptor mGluR1 or mGluR5, overexpression of scaffolding Homer proteins, which bind to a Homer ligand in their C termini, cause receptor clustering and uncoupling from ion channel modulation. In the absence of recombinant Homer protein overexpression, uncoupling of mGluRs from voltage-dependent channels can be induced by expression of Preso1, an adaptor of prolinedirected kinases that phosphorylates the Homer ligand and recruits binding of endogenous Homer proteins. Here we show that in SCG neurons expressing mGluR1 and the tyrosine receptor kinase B, treatment with brain-derived neurotrophic factor (BDNF) produces a similar uncoupling of the receptors from calcium channels. We investigated the pathways that mediate this uncoupling and compared it with uncoupling observed with Preso1 expression. Both BDNF-and Preso1-induced uncoupling require residues T1151 and S1154 in the mGluR1 Homer ligand (TPPSPF). Uncoupling via Preso1 but not BDNF was prevented by expression of a dominant negative Cdk5, suggesting that endogenous Cdk5 mediates Preso1-dependent phosphorylation of mGluR1. Dominant negative Cdk5 did not block the BDNF effect but this was sensitive to inhibitors of the mitogen-activated protein kinase kinase/ extracellular signal-regulated kinase cascade. Interestingly, the BDNF pathway appeared to require native Preso1 binding to mGluR, because overexpression of the Preso1 FERM domain, which mediates the Preso1-mGluR interaction, prevented BDNF-induced uncoupling. These data suggest that the BDNF/tyrosine receptor kinase B and Cdk5 pathways converge at the level of mGluR to similarly induce Homer ligand phosphorylation, recruit Homer binding, and uncouple mGluRs from channel regulation

Insulin-induced recruitment of glucose transporter 4 (GLUT4) and GLUT1 in isolated rat cardiac myocytes. Evidence of the existence of different intracellular GLUT4 vesicle populations

Using isolated rat cardiomyocytes we have examined: 1) the effect of insulin on the cellular distribution of glucose transporter 4 (GLUT4) and GLUT1, 2) the total amount of these transporters, and 3) the co-localization of GLUT4, GLUT1, and secretory carrier membrane proteins (SCAMPs) in intracellular membranes. Insulin induced 5.7- and 2.7-fold increases in GLUT4 and GLUT1 at the cell surface, respectively, as determined by the nonpermeant photoaffinity label [3H]2-N-[4(1-azi-2,2,2-trifluoroethyl)benzoyl]-1, 3-bis-(D-mannos-4-yloxy)propyl-2-amine. The total amount of GLUT1, as determined by quantitative Western blot analysis of cell homogenates, was found to represent a substantial fraction ( approximately 30%) of the total glucose transporter content. Intracellular GLUT4-containing vesicles were immunoisolated from low density microsomes by using monoclonal anti-GLUT4 (1F8) or anti-SCAMP antibodies (3F8) coupled to either agarose or acrylamide. With these different immunoisolation conditions two GLUT4 membrane pools were found in nonstimulated cells: one pool with a high proportion of GLUT4 and a low content in GLUT1 and SCAMP 39 (pool 1) and a second GLUT4 pool with a high content of GLUT1 and SCAMP 39 (pool 2). The existence of pool 1 was confirmed by immunotitration of intracellular GLUT4 membranes with 1F8-acrylamide. Acute insulin treatment caused the depletion of GLUT4 in both pools and of GLUT1 and SCAMP 39 in pool 2. In conclusion: 1) GLUT4 is the major glucose transporter to be recruited to the surface of cardiomyocytes in response to insulin; 2) these cells express a high level of GLUT1; and 3) intracellular GLUT4-containing vesicles consist of at least two populations, which is compatible with recently proposed models of GLUT4 trafficking in adipocytes

Мотивационное управление персоналом ( на примере Муниципального бюджетного дошкольного образовательного учреждения МБДОУ «Рыбинский детский сад «Колобок» село Рыбное, Красноярский край, Рыбинский район)

Объектом исследования является Муниципальное бюджетное дошкольное образовательное учреждение МБДОУ «Рыбинский детский сад «Колобок» село Рыбное, Красноярского края. Целью данной работы является изучение мотивации в управлении персоналом организации, на примере Муниципального бюджетного дошкольного образовательного учреждения МБДОУ «Рыбинский детский сад «Колобок» село Рыбное, Красноярского края. Проведённые исследования показывают, что система мотивации образовательной организации «Рыбинский детский сад «Колобок» сейчас малоэффективна и просто не сформирована. Это требует доработки такого документа, как Положение о доплатах и надбавках сотрудников, которое регулирует систему мотивации в образовательной организации «Рыбинский детский сад «Колобок».The object of study is the Municipal budget preschool educational institution MBDOU "Rybinsk kindergarten" Gingerbread Man "Fish village, the Krasnoyarsk Territory". The subject of this study is - motivational staff management. The aim of this work is the study of motivation in the management staff of the organization, by the example of the Municipal budget preschool educational institution MBDOU "Rybinsk kindergarten" Gingerbread Man "Fish village, the Krasnoyarsk Territory". Studies have shown that the system of motivation of educational organization "Rybinsk kindergarten" Gingerbread Man "is now ineffective and simply formed. This requires a revision of the document, as the Regulations on bonuses and allowances of employees, which regulates the system of motivation in the educational organization "Rybinsk kindergarten" Gingerbread Man "

Agammaglobulinaemia despite terminal B-cell differentiation in a patient with a novel LRBA mutation

Mutations in lipopolysaccharide-responsive vesicle trafficking, beach and anchor-containing protein (LRBA) cause immune deficiency and inflammation. Here, we are reporting a novel homozygous mutation in LRBA allele in 7-year-old Omani boy, born to consanguineous parents. He presented with type 1 diabetes, autoimmune haematological cytopenia, recurrent chest infections and lymphocytic interstitial lung disease. The patient was treated with CTLA4-Ig (abatacept) with good outcome every 2 weeks for a period of 3 months. He developed complete IgG deficiency, but remarkably, histological examination revealed germinal centres and plasma cells in lymphoid and inflamed lung tissue. Further charatecterisation showed these cells to express IgM but not IgG. This ex vivo analysis suggests that LRBA mutation confers a defect in class switching despite plasma cell formation

Inflammatory bowel disease clinical service recovery during the COVID-19 pandemic

© 2021 The Authors. Published by BMJ. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: http://dx.doi.org/10.1136/flgastro-2021-101805Published versio

Synaptic Depression Via Mglur1 Positive Allosteric Modulation Suppresses Cue-Induced Cocaine Craving

Cue-induced cocaine craving is a major cause of relapse in abstinent addicts. In rats, cue-induced craving progressively intensifies (incubates) during withdrawal from extended-access cocaine self-administration. After ~1 month of withdrawal, incubated craving is mediated by Ca(2+)-permeable AMPA receptors (CP-AMPARs) that accumulate in the nucleus accumbens (NAc). We found that decreased mGluR1 surface expression in the NAc preceded and enabled CP-AMPAR accumulation. Thus, restoring mGluR1 transmission by administering repeated injections of an mGluR1 positive allosteric modulator (PAM) prevented CP-AMPAR accumulation and incubation, whereas blocking mGluR1 transmission at even earlier withdrawal times accelerated CP-AMPAR accumulation. In studies conducted after prolonged withdrawal, when CP-AMPAR levels and cue-induced craving are high, we found that systemic administration of an mGluR1 PAM attenuated the expression of incubated craving by reducing CP-AMPAR transmission in the NAc to control levels. These results suggest a strategy in which recovering addicts could use a systemically active compound to protect against cue-induced relapse