scholarly journals Antibodies Against the NH2-Terminus of the GluA Subunits Affect the AMPA-Evoked Releasing Activity: The Role of Complement

2021 ◽  
Vol 12 ◽  
Author(s):  
Francesca Cisani ◽  
Guendalina Olivero ◽  
Cesare Usai ◽  
Gilles Van Camp ◽  
Stefania Maccari ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Glutamate Release ◽  
Dependent Manner ◽  
Functional Studies ◽  
Amino Terminal ◽  
Neuronal Membranes ◽  
Presynaptic Release ◽  
Amino Terminal Domain ◽  
Classic Pathway

Antibodies recognizing the amino-terminal domain of receptor subunit proteins modify the receptor efficiency to controlling transmitter release in isolated nerve endings (e.g., synaptosomes) indirectly confirming their presence in these particles but also allowing to speculate on their subunit composition. Western blot analysis and confocal microscopy unveiled the presence of the GluA1, GluA2, GluA3, and GluA4 receptor subunits in cortical synaptosomes. Functional studies confirmed the presence of presynaptic release-regulating AMPA autoreceptors in these terminals, whose activation releases [3H]D-aspartate ([3H]D-Asp, here used as a marker of glutamate) in a NBQX-dependent manner. The AMPA autoreceptors traffic in a constitutive manner, since entrapping synaptosomes with the pep2-SVKI peptide (which interferes with the GluA2-GRIP1/PICK1 interaction) amplified the AMPA-evoked releasing activity, while the inactive pep2-SVKE peptide was devoid of activity. Incubation of synaptosomes with antibodies recognizing the NH2 terminus of the GluA2 and the GluA3 subunits increased, although to a different extent, the GluA2 and 3 densities in synaptosomal membranes, also amplifying the AMPA-evoked glutamate release in a NBQX-dependent fashion. We then analyzed the releasing activity of complement (1:300) from both treated and untreated synaptosomes and found that the complement-induced overflow occurred in a DL-t-BOA-sensitive, NBQX-insensitive fashion. We hypothesized that anti-GluA/GluA complexes in neuronal membranes could trigger the classic pathway of activation of the complement, modifying its releasing activity. Accordingly, the complement-evoked release of [3H]D-Asp from antiGluA2 and anti-GluA3 antibody treated synaptosomes was significantly increased when compared to untreated terminals and facilitation was prevented by omitting the C1q component of the immunocomplex. Antibodies recognizing the NH2 terminus of the GluA1 or the GluA4 subunits failed to affect both the AMPA and the complement-evoked tritium overflow. Our results suggest the presence of GluA2/GluA3-containing release-regulating AMPA autoreceptors in cortical synaptosomes. Incubation of synaptosomes with commercial anti-GluA2 or anti-GluA3 antibodies amplifies the AMPA-evoked exocytosis of glutamate through a complement-independent pathway, involving an excessive insertion of AMPA autoreceptors in plasma membranes but also affects the complement-dependent releasing activity, by promoting the classic pathway of activation of the immunocomplex. Both events could be relevant to the development of autoimmune diseases typified by an overproduction of anti-GluA subunits.

scholarly journals Enhancer Sequences Influence the Role of the Amino-Terminal Domain of Bicoid in Transcription

2003 ◽  
Vol 23 (13) ◽  
pp. 4439-4448 ◽  
Author(s):  
Dechen Fu ◽  
Chen Zhao ◽  
Jun Ma
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Target Genes ◽  
Cooperative Binding ◽  
Dependent Manner ◽  
Enhancer Element ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Amino Terminal Domain

ABSTRACT Bicoid (Bcd) is a Drosophila melanogaster morphogenetic gradient that controls embryonic patterning by activating target gene expression in a concentration-dependent manner. In this study we describe experiments to determine how different enhancers respond to Bcd distinctively, focusing on two natural Bcd-responsive enhancer elements, hunchback (hb) and knirps (kni). Our results show that, on the hb enhancer element, the amino-terminal domain of Bcd (residues 1 to 91) plays primarily an inhibitory role, whereas on the kni enhancer element this same Bcd domain plays a positive role at low protein concentrations. We further demonstrate that while the amino-terminal domain is largely dispensable for cooperative binding to the hb enhancer element, it is preferentially required for cooperative binding to the kni enhancer element. Alteration of the arrangement of Bcd binding sites in the kni enhancer element reduces the role of the amino-terminal domain in cooperative DNA binding but increases the effectiveness of the self-inhibitory function. In addition, elimination of symmetric pairs of Bcd binding sites in the kni enhancer element reduces both DNA binding and activation by Bcd. We propose that the amino-terminal domain of Bcd is an enhancer-specific switch that contributes to the protein's ability to activate different target genes in distinct manners.

scholarly journals Role of Amino-Terminal Domain in the Assembly Mechanism of Kainate-Subtype Glutamate Receptor Ion Channels

2014 ◽  
Vol 106 (2) ◽  
pp. 151a
Author(s):  
Sagar Chittori ◽  
Janesh Kumar ◽  
Suvendu Lomash ◽  
Huaying Zhao ◽  
Peter Schuck ◽  
...  
Keyword(s):  
Ion Channels ◽  
Glutamate Receptor ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Assembly Mechanism ◽  
Amino Terminal Domain

scholarly journals β-Glucan enhances complement-mediated hematopoietic recovery after bone marrow injury

Blood ◽  
2006 ◽  
Vol 107 (2) ◽  
pp. 835-840 ◽  
Author(s):  
Daniel E. Cramer ◽  
Daniel J. Allendorf ◽  
Jarek T. Baran ◽  
Richard Hansen ◽  
Jose Marroquin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Serum ◽  
Complement Receptor ◽  
Dependent Manner ◽  
Complement Receptor 3 ◽  
Total Body ◽  
Stimulating Factor ◽  
Sublethal Irradiation ◽  
Classic Pathway

AbstractMyelotoxic injury in the bone marrow (BM) as a consequence of total body irradiation (TBI) or granulocyte colony-stimulating factor (G-CSF) mobilization results in the deposition of iC3b on BM stroma (stroma-iC3b). In the present study, we have examined how stroma-iC3b interacts with hematopoietic progenitor cells (HPCs) and the role of complement (C) and complement receptor 3 (CR3) in BM injury/repair. We demonstrate here that stroma-iC3b tethers HPCs via the inserted (I) domain of HPC complement receptor 3 (CR3, CD11b/CD18, Mac-1). Following irradiation, stroma-iC3b was observed in the presence of purified IgM and normal mouse serum (NMS), but not serum from Rag-2-/- mice, implicating a role for antibody (Ab) and the classic pathway of C activation. Furthermore, a novel role for soluble yeast β-glucan, a ligand for the CR3 lectin-like domain (LLD), in the priming of CR3+ HPC is suggested. Soluble yeast β-glucan could enhance the proliferation of tethered HPCs, promote leukocyte recovery following sublethal irradiation, and increase the survival of lethally irradiated animals following allogeneic HPC transplantation in a CR3-dependent manner. Taken together, these observations suggest a novel role for C, CR3, and β-glucan in the restoration of hematopoiesis following injury. (Blood. 2006;107:835-840)

scholarly journals The Multiple Roles of Cyk1p in the Assembly and Function of the Actomyosin Ring in Budding Yeast

1999 ◽  
Vol 10 (2) ◽  
pp. 283-296 ◽  
Author(s):  
Katie B. Shannon ◽  
Rong Li
Keyword(s):  
Actin Filaments ◽  
Fluorescent Protein ◽  
Budding Yeast ◽  
Dependent Manner ◽  
Size Change ◽  
Direct Role ◽  
Dynamic Component ◽  
Amino Terminal ◽  
Ring Assembly

The budding yeast IQGAP-like protein Cyk1p/Iqg1p localizes to the mother-bud junction during anaphase and has been shown to be required for the completion of cytokinesis. In this study, video microscopy analysis of cells expressing green fluorescent protein-tagged Cyk1p/Iqg1p demonstrates that Cyk1p/Iqg1p is a dynamic component of the contractile ring during cytokinesis. Furthermore, in the absence of Cyk1p/Iqg1p, myosin II fails to undergo the contraction-like size change at the end of mitosis. To understand the mechanistic role of Cyk1p/Iqg1p in actomyosin ring assembly and dynamics, we have investigated the role of the structural domains that Cyk1p/Iqg1p shares with IQGAPs. An amino terminal portion containing the calponin homology domain binds to actin filaments and is required for the assembly of actin filaments to the ring. This result supports the hypothesis that Cyk1p/Iqg1p plays a direct role in F-actin recruitment. Deletion of the domain harboring the eight IQ motifs abolishes the localization of Cyk1p/Iqg1p to the bud neck, suggesting that Cyk1p/Iqg1p may be localized through interactions with a calmodulin-like protein. Interestingly, deletion of the COOH-terminal GTPase-activating protein-related domain does not affect Cyk1p/Iqg1p localization or actin recruitment to the ring but prevents actomyosin ring contraction. In vitro binding experiments show that Cyk1p/Iqg1p binds to calmodulin, Cmd1p, in a calcium-dependent manner, and to Tem1p, a small GTP-binding protein previously found to be required for the completion of anaphase. These results demonstrate the critical function of Cyk1p/Iqg1p in regulating various steps of actomyosin ring assembly and cytokinesis.

Functional Analysis of Human Smad1: Role of the Amino-Terminal Domain

1999 ◽  
Vol 258 (2) ◽  
pp. 366-373 ◽  
Author(s):  
Ren-He Xu ◽  
Robert J. Lechleider ◽  
Hsiu-Ming Shih ◽  
Chen-Fei Hao ◽  
Dvora Sredni ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Amino Terminal Domain

Porcine sst1 can physically interact with other somatostatin receptors, and its expression is regulated by metabolic/inflammatory sensors

2014 ◽  
Vol 306 (5) ◽  
pp. E483-E493 ◽  
Author(s):  
Manuel D. Gahete ◽  
Mario Durán-Prado ◽  
Elena Delgado-Niebla ◽  
Juan J. Garrido ◽  
Simon J. Rhodes ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Somatostatin Receptors ◽  
Amino Terminal ◽  
Mediated Effects ◽  
Relevant Role ◽  
Amino Terminal Domain ◽  
G Protein Coupled

The majority of the biological actions attributed to somatostatin (SST) are thought to be mediated by SST receptor 2 (sst2), the most ubiquitous sst, and, to a lesser extent, by sst5. However, a growing body of evidence suggests a relevant role of sst1 in mediating SST actions in (patho)physiological situations (i.e., endometriosis, type 2 diabetes). Moreover, sst1 together with sst2 and sst5 is involved in the well-known actions of SST on pituitary somatotropes in pig and primates. Here, we cloned the porcine sst1 (psst1) and performed a structural and functional characterization using both primary and heterologous models. The psst1 sequence presents the majority of signature motifs shared among G protein-coupled receptors and, specifically, among ssts and exhibits a high homology with other mammalian sst1, with only minor differences in the amino-terminal domain, reinforcing the idea of an early evolutive divergence between mammalian and nonmammalian sst1s. psst1 is functional in terms of decreasing cAMP levels in response to SST when transfected in heterologous models. The psst1 receptor is expressed in several tissues, and analyses of gene cis elements predict regulation by multiple transcription factors and metabolic stimuli. Finally, psst1 is coexpressed with other sst subtypes in various tissues, and in vitro data demonstrate that psst1 can interact with itself forming homodimers and with other ssts forming heterodimers. These data highlight the functional importance of sst1 on the SST-mediated effects and its functional interaction with different ssts, which point out the necessity of exploring the consequences of such interactions.

P-30 THE ROLE OF THE CONSERVED AMINO-TERMINAL DOMAIN OF TIC SUPERFAMILY PROTEINS IN IGF BINDING

2006 ◽  
Vol 16 ◽  
pp. S46
Author(s):  
Sue M Firth ◽  
Xiaolang Yan ◽  
Bernard Perbal ◽  
Robert C Baxter
Keyword(s):  
Amino Terminal ◽  
Terminal Domain ◽  
Igf Binding ◽  
Amino Terminal Domain

scholarly journals Effects of an Atypical Antipsychotic, Zotepine, on Astroglial L-Glutamate Release through Hemichannels: Exploring the Mechanism of Mood-Stabilising Antipsychotic Actions and Antipsychotic-Induced Convulsion

2021 ◽  
Vol 14 (11) ◽  
pp. 1116
Author(s):  
Kouji Fukuyama ◽  
Motohiro Okada
Keyword(s):  
Plasma Membrane ◽  
Atypical Antipsychotic ◽  
Plasma Membranes ◽  
Glutamate Release ◽  
Chronic Administration ◽  
Antipsychotic Agent ◽  
Acute Administration ◽  
Dependent Manner ◽  
Akt Inhibitor ◽  
Cx43 Expression

Accumulating neuropsychopharmacological evidence has suggested that functional abnormalities of astroglial transmission and protein kinase B (Akt) contribute to the pathophysiology and/or pathomechanisms of several neuropsychiatric disorders, such as epilepsy, schizophrenia, affective disorders and antipsychotic-induced convulsions. Therefore, to explore the pathophysiology of mood-stabilising antipsychotics and the proconvulsive actions of atypical antipsychotics, the present study determined the effects of a mood-stabilising, atypical, antipsychotic agent, zotepine (ZTP), on astroglial L-glutamate release and the expression of connexin43 (Cx43) protein in cortical, primary, cultured astrocytes using ultra-high-pressure liquid chromatography and capillary immunoblotting systems. Both acute and subchronic administrations of therapeutically relevant concentrations of ZTP did not affect astroglial L-glutamate release or Cx43 expression in plasma membranes; however, chronic administration of a therapeutically relevant concentration of ZTP increased astroglial L-glutamate release and Cx43 expression in the plasma membrane. Subchronic administrations of a supratherapeutic concentration of ZTP enhanced astroglial L-glutamate release and Cx43 expression in the plasma membrane, whereas acute administration of a supratherapeutic concentration of ZTP enhanced astroglial L-glutamate release without affecting Cx43 expression. These stimulatory effects of ZTP on astroglial L-glutamate release through activated hemichannels and Cx43 trafficking to the astroglial plasma membrane were suppressed by the Akt inhibitor. These results suggest that ZTP enhances astroglial L-glutamate release in a concentration-dependent and time-dependent manner due to the enhanced function of astroglial hemichannels, probably via activation of Akt signalling. Therefore, the enhanced astroglial L-glutamatergic transmission induced by ZTP is, at least partially, involved in the mood-stabilising antipsychotic and proconvulsive actions of ZTP.

Potentiated Activation of VLA-4 and VLA-5 Accelerates Proplatelet-Like Formation In Megakaryocytes.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2585-2585
Author(s):  
Takuya Matsunaga ◽  
Fumio Fukai ◽  
Takuro Kameda ◽  
Kotaro Shide ◽  
Haruko Shimoda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Culture System ◽  
Dependent Manner ◽  
Adhesive Interaction ◽  
Hematopoietic Stem ◽  
Serum Free ◽  
Amino Terminal ◽  
Three Phase ◽  
Cytoplasmic Processes

Abstract Abstract 2585 Several lines of reports have suggested that mature magakaryocytes (MKs) form long cytoplasmic processes containing platelets (PLT) organelles from which PLT break off due to blood flow pressures in bone marrow (BM). These cytoplasmic processes were termed ‘proplatelet'. MKs differentiated from hematopoietic stem cells by in vitro culture also develop similar processes, referred to as ‘proplatelet-like formation (PPF)'. It has been already reported that fibronectin (FN) and phorbol 12-myristate 13-acetate (PMA) are essential for inducing PPF in MKs using CHRF-288 human megakaryoblastic cell line (Jiang F et al. Blood 99, 2002). FN plays important roles in megakaryocytopoiesis through the FN-receptors. The role of adhesive interactions with FN in BM stroma and FN-receptor beta1-integrins has been reported in proliferation, differentiation and maintenance of megakaryocytic lineage cells. However, the substantial role of these FN-receptors and their functional assignment in PPF are not yet fully understood. We first investigated the effects of beta1-integrins on PPF using CHRF-288 cells, which express alpha4beta1-integrin (VLA-4) and alpha5beta1-integrin (VLA-5) as FN-receptors. When the cells were cultured on FN for 3 days, PMA prompted PPF in a dose-dependent manner. While nearly 15% of the cells displayed PPF with PMA (100 ng/mL), no cells cultured with FN alone or PMA alone exhibited PPF. PPF induced by FN plus PMA combination (FN/PMA) was abrogated by addition of anti-alpha4-integrin monoclonal antibodies (mAb) plus anti-alpha5-integrin mAb combination, but not by the addition of anti-alpha4-integrin mAb alone or anti-alpha5-integrin mAb alone. Thus, the adhesive interaction with FN via VLA-4 and VLA-5 were responsible for PPF. We next investigated the effect of TNIIIA2, which enhances the adhesive interaction between FN and beta1-integrins, in PPF induced by FN/PMA. TNIIIA2 (RSTDLPGLKAATHYTITIRGVC) is a 22-mer peptide derived from the 14th FN type III-like (FNIII) repeat in tenascin (TN)-C molecule which we found recently, and it induces the conformational change necessary for functional activation of beta1-integrins (Fukai F et al. J Biol Chem 282, 2007; J Biol Chem 284, 2009). The PPF induced by FN/PMA was highly accelerated when CHRF-288 cells were enforced adhering to FN by treatment with TNIIIA2 (25 microg/mL). More than 45% of the cells displayed PPF with FN/PMA plus TNIIIA2 combination (FN/PMA/TNIIIA2). Blocking experiments using anti-beta1-integrin mAbs indicated that adhesive interaction with FN via VLA-4 and VLA-5 was also responsible for acceleration of PPF induced by FN/PMA/TNIIIA2. On the other hand, control peptide, TNIIIA2mutant (RSTDLPGLKAATHYTATARGVC) did not accelerate PPF induced by PMA/FN. The calculated yield of the cells with PPF induced by FN/PMA/TNIIIA2 was 2.5-fold more than that induced by FN/PMA. We have previously established ‘a three-phase serum-free culture system' to generate large amount of PLT from human cord blood CD34+ cells (Matsunaga T et al. Stem cells 24, 2006). A study on the effect of TNIIIA2 on our ‘three-phase serum-free culture system' is now underway. Finally, we investigated signal transduction pathways responsible for PPF induced by FN/PMA. While FN/PMA induced activation of extracellular signal-regulated protein kinase 1 (ERK1/2), FN alone or PMA alone did not induce ERK1/2 activation. The results was in accordance with the data previously reported by Jiang et at (Blood 99, 2002). TNIIIA2 strongly enhanced activation of ERK1/2 by FN/PMA. However, c-Jun amino-terminal kinase 1 (JNK1), p38 and phosphoinositide-3 kinase (PI3K)/Akt were not stimulated by FN/PMA even in the presence of TNIIIA2. Thus, enhanced activation of ERK1/2 by FN/PMA/TNIIIA2 was responsible for acceleration of PPF by FN/PMA. Disclosures: No relevant conflicts of interest to declare.

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