scholarly journals Distinct Cell Surface Expression Patterns of N-Glycosylation Site Mutants of AMPA-Type Glutamate Receptor under the Homo-Oligomeric Expression Conditions

2020 ◽  
Vol 21 (14) ◽  
pp. 5101
Author(s):  
Jyoji Morise ◽  
Saki Yamamoto ◽  
Ryosuke Midorikawa ◽  
Kogo Takamiya ◽  
Motohiro Nonaka ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glutamate Receptor ◽  
Expression Patterns ◽  
Surface Expression ◽  
Glycosylation Site ◽  
Synaptic Activity ◽  
Cell Surface Expression ◽  
Dependent Manner ◽  
Distinct Cell

The AMPA-type glutamate receptor (AMPAR) is a homotetrameric or heterotetrameric ion channel composed of various combinations of four subunits (GluA1–4), and its abundance in the synapse determines the strength of synaptic activity. The formation of oligomers in the endoplasmatic reticulum (ER) is crucial for AMPAR subunits’ ER-exit and translocation to the cell membrane. Although N-glycosylation on different AMPAR subunits has been shown to regulate the ER-exit of hetero-oligomers, its role in the ER-exit of homo-oligomers remains unclear. In this study, we investigated the role of N-glycans at GluA1N63/N363 and GluA2N370 in ER-exit under the homo-oligomeric expression conditions, whose mutants are known to show low cell surface expressions. In contrast to the N-glycosylation site mutant GluA1N63Q, the cell surface expression levels of GluA1N363Q and GluA2N370Q increased in a time-dependent manner. Unlike wild-type (WT) GluA1, GluA2WT rescued surface GluA2N370Q expression. Additionally, the expression of GluA1N63Q reduced the cell surface expression level of GluA1WT. In conclusion, our findings suggest that these N-glycans have distinct roles in the ER-exit of GluA1 and GluA2 homo-oligomers; N-glycan at GluA1N63 is a prerequisite for GluA1 ER-exit, whereas N-glycans at GluA1N363 and GluA2N370 control the ER-exit rate.

scholarly journals Cellular Activation of MMP-2 (Gelatinase A) by MT2-MMP Occurs via a TIMP-2-independent Pathway

2001 ◽  
Vol 276 (50) ◽  
pp. 47402-47410 ◽  
Author(s):  
Charlotte J. Morrison ◽  
Georgina S. Butler ◽  
Heather F. Bigg ◽  
Clive R. Roberts ◽  
Paul D. Soloway ◽  
...  
Keyword(s):  
Cell Surface ◽  
Matrix Metalloproteinase ◽  
Surface Expression ◽  
Free Cell ◽  
Cell Surface Expression ◽  
Dependent Manner ◽  
Cellular Activation ◽  
Membrane Type ◽  
Association Rate Constants

The role of membrane-type (MT) 2-matrix metalloproteinase (MMP) in the cellular activation of MMP-2 and the tissue inhibitor of matrix metalloproteinase (TIMP) requirements for this process have not been clearly established. To address these issues a TIMP-2-free cell line derived from aTimp2−/− mouse was transfected for stable cell surface expression of hMT2-MMP. Untransfected cells did not activate endogenous or exogenous TIMP-2-free MMP-2 unless both TIMP-2 and concanavalin A (ConA) were added. Transfected cells expressing hMT2-MMP efficiently activated both endogenous and exogenous MMP-2 (within 4 h) via the 68-kDa intermediate in the absence of TIMP-2 and ConA. In contrast, activation of MMP-2 byTimp2−/− cells expressing recombinant hMT1-MMP occurred more slowly (12 h) and required the addition of 0.3–27 nmTIMP-2. Addition of TIMP-2 or TIMP-4 did not enhance MMP-2 activation by MT2-MMP at any concentration tested; furthermore, activation was inhibited by both TIMPs at concentrations >9 nm, consistent with the similar association rate constants (kon) calculated for the binding of TIMP-4 and TIMP-2 to MT2-MMP (3.56 × 105m−1s−1and 6.52 × 105m−1s−1, respectively). MT2-MMP-mediated activation involved cell surface association of the MMP-2 in a hemopexin carboxyl-terminal domain (C domain)-dependent manner: Exogenous MMP-2 hemopexin C domain blocked activation, and cells expressing hMT2-MMP did not bind or activate a truncated form of MMP-2 lacking the hemopexin C domain. These studies demonstrate the existence of an alternative TIMP-2-independent pathway for MMP-2 activation involving MT2-MMP, which may be important in mediating MMP-2 activation in specific tissues or pathologies where MT2-MMP is expressed.

scholarly journals Regulation of the Expression, Oligomerisation and Signaling of the Inhibitory Receptor CLEC12A by Cysteine Residues in the Stalk Region

2021 ◽  
Vol 22 (19) ◽  
pp. 10207
Author(s):  
Julien Vitry ◽  
Guillaume Paré ◽  
Andréa Murru ◽  
Xavier Charest-Morin ◽  
Halim Maaroufi ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Activation ◽  
Secretory Pathway ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Dependent Manner ◽  
Inhibitory Receptor ◽  
Lectin Receptors ◽  
Stalk Domain

CLEC12A is a myeloid inhibitory receptor that negatively regulates inflammation in mouse models of autoimmune and autoinflammatory arthritis. Reduced CLEC12A expression enhances myeloid cell activation and inflammation in CLEC12A knock-out mice with collagen antibody-induced or gout-like arthritis. Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. While it is presumed that the cysteines in the stalk domain have multimerisation properties, their role in CLEC12A expression and/or signaling remain unknown. We thus used site-directed mutagenesis to determine whether the stalk domain cysteines play a role in CLEC12A expression, internalisation, oligomerisation, and/or signaling. Mutation of C118 blocks CLEC12A transport through the secretory pathway diminishing its cell-surface expression. In contrast, mutating C130 does not affect CLEC12A cell-surface expression but increases its oligomerisation, inducing ligand-independent phosphorylation of the receptor. Moreover, we provide evidence that CLEC12A dimerisation is regulated in a redox-dependent manner. We also show that antibody-induced CLEC12A cross-linking induces flotillin oligomerisation in insoluble membrane domains in which CLEC12A signals. Taken together, these data indicate that the stalk cysteines in CLEC12A differentially modulate this inhibitory receptor’s expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation.

scholarly journals Analysis of the Role of N-Linked Glycosylation in Cell Surface Expression, Function, and Binding Properties of SARS-CoV-2 Receptor ACE2

2021 ◽  
Author(s):  
Raymond Rowland ◽  
Alberto Brandariz-Nuñez
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Receptor Interaction ◽  
Minimal Effect ◽  
Binding Properties ◽  
Virus Receptor

Understanding the role of glycosylation in the virus-receptor interaction is important for developing approaches that disrupt infection. In this study, we showed that deglycosylation of both ACE2 and S had a minimal effect on the spike-ACE2 interaction.

scholarly journals All-Trans Retinoic Acid Enhances Bacterial Flagellin-Stimulated Proinflammatory Responses in Human Monocyte THP-1 Cells by Upregulating CD14

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Thi Xoan Hoang ◽  
Jong Hyeok Jung ◽  
Jae Young Kim
Keyword(s):  
Retinoic Acid ◽  
Cell Surface ◽  
Human Monocyte ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Dependent Manner ◽  
Atra Treatment ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Proinflammatory Responses

All-trans retinoic acid (ATRA), an active form of vitamin A, exerts immunomodulatory functions. In this study, we examined the immune potentiating effect of ATRA on bacterial flagellin-induced NF-κB activation and proinflammatory cytokine production in human monocytic cell line THP-1. ATRA treatment significantly enhanced the flagellin-induced NF-κB/AP-1 activity in THP-1 via the RAR/RXR pathway. Similarly, ATRA enhanced the expression and production of TNF-α and IL-1β in THP-1 cells upon flagellin challenge. The cell surface expression of toll-like receptor 5 (TLR5), which is the receptor for bacterial flagellin, was significantly reduced by ATRA in a concentration- and time-dependent manner. To determine the mechanisms underlying the ATRA-enhanced immune response against bacterial flagellin despite the reduced cell surface expression of TLR5 in ATRA-treated THP-1, we examined the cell surface expression of CD14, which has been proposed to be a TLR co-receptor that enhances the response to microbial components. The cell surface expression of CD14 was significantly enhanced by ATRA treatment, especially in the presence of flagellin. Anti-CD14 antibody treatment prior to ATRA and flagellin treatments completely abolished ATRA-enhanced TNF-α and IL-1β production. Our results suggest that ATRA enhances flagellin-stimulated proinflammatory responses in human monocyte THP-1 cells by upregulating CD14 in a RAR/RXR-dependent manner.

scholarly journals Characterization, cell-surface expression and ligand-binding properties of different truncated N-terminal extracellular domains of the ionotropic glutamate receptor subunit GluR1

1996 ◽  
Vol 315 (1) ◽  
pp. 217-225 ◽  
Author(s):  
R. A. Jeffrey McILHINNEY ◽  
Elek MOLNÁR
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Glutamate Receptor ◽  
Cell Membranes ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Receptor Subunit ◽  
Binding Studies ◽  
Terminal Domain ◽  
Glutamate Receptor Subunit

To identify the location of the first transmembrane segment of the GluR1 glutamate receptor subunit artificial stop codons have been introduced into the N-terminal domain at amino acid positions 442, 510 and 563, namely just before and spanning the proposed first two transmembrane regions. The resultant truncated N-terminal fragments of GluR1, termed NT1, NT2 and NT3 respectively were expressed in Cos-7 cells and their cellular distribution and cell-surface expression analysed using an N-terminal antibody to GluR1. All the fragments were fully glycosylated and were found to be associated with cell membranes but none was secreted. Differential extraction of the cell membranes indicated that both NT1 and NT2 behave as peripheral membrane proteins. In contrast NT3, like the full subunit, has integral membrane protein properties. Furthermore only NT3 is expressed at the cell surface as determined by immunofluorescence and cell-surface biotinylation. Protease protection assays indicated that only NT3 had a cytoplasmic tail. Binding studies using the selective ligand [3H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate ([3H]AMPA) demonstrated that NT3 does not bind ligand. Together these results indicate that the first transmembrane domain of the GluR1 subunit lies between residues 509 and 562, that the N-terminal domain alone cannot form a functional ligand-binding site and that this domain can be targeted to the cell surface provided that it has a transmembrane-spanning region.

scholarly journals Roles of the N- and C-termini of GLUT4 in endocytosis

2002 ◽  
Vol 115 (1) ◽  
pp. 131-140 ◽  
Author(s):  
Hadi Al-Hasani ◽  
Raghu K. Kunamneni ◽  
Kevin Dawson ◽  
Cynthia S. Hinck ◽  
Dirk Müller-Wieland ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Surface Expression ◽  
Dominant Negative ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Dileucine Motif ◽  
Intracellular Compartments ◽  
Targeting Signal

In insulin target cells, the predominantly expressed glucose transporter isoform GLUT4 recycles between distinct intracellular compartments and the plasma membrane. To characterize putative targeting signals within GLUT4 in a physiologically relevant cell type, we have analyzed the trafficking of hemagglutinin (HA)-epitope-tagged GLUT4 mutants in transiently transfected primary rat adipose cells. Mutation of the C-terminal dileucine motif (LL489/90) did not affect the cell-surface expression of HA-GLUT4. However, mutation of the N-terminal phenylalanine-based targeting sequence (F5) resulted in substantial increases, whereas deletion of 37 or 28 of the 44 C-terminal residues led to substantial decreases in cell-surface HA-GLUT4 in both the basal and insulin-stimulated states. Studies with wortmannin and coexpression of a dominant-negative dynamin GTPase mutant indicate that these effects appear to be primarily due to decreases and increases, respectively, in the rate of endocytosis. Yeast two-hybrid analyses revealed that the N-terminal phenylalanine-based targeting signal in GLUT4 constitutes a binding site for medium chain adaptins μ1, μ2, and μ3A, implicating a role of this motif in the targeting of GLUT4 to clathrin-coated vesicles.

scholarly journals Analysis of the Role of N-glycosylation in Cell-surface expression, Function and Binding Properties of SARS-CoV-2 receptor ACE2

2021 ◽  
Author(s):  
Alberto Brandariz-Nuñez ◽  
Raymond R Rowland
Keyword(s):  
Cell Surface ◽  
Viral Entry ◽  
Biological Activities ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Type I ◽  
Protein Activity ◽  
Host Cell Membrane ◽  
S Protein

Human angiotensin I-converting enzyme 2 (hACE2) is a type-I transmembrane glycoprotein that serves as the major cell entry receptor for SARS-CoV and SARS-CoV-2. The viral spike (S) protein is required for attachment to ACE2 and subsequent virus-host cell membrane fusion. Previous work has demonstrated the presence of N-linked glycans in ACE2. N-glycosylation is implicated in many biological activities, including protein folding, protein activity, and cell surface expression of biomolecules. However, the contribution of N-glycosylation to ACE2 function is poorly understood. Here, we examined the role of N-glycosylation in the activity and localization of two species with different susceptibility to SARS-CoV-2 infection, porcine ACE2 (pACE2) and hACE2. The elimination of N-glycosylation by tunicamycin (TM) treatment or mutagenesis, showed that N-glycosylation is critical for the proper cell surface expression of ACE2 but not for its carboxiprotease activity. Furthermore, nonglycosylable ACE2 localized predominantly in the endoplasmic reticulum (ER) and not at the cell surface. Our data also revealed that binding of SARS-CoV and SARS-CoV-2 S protein to porcine or human ACE2 was not affected by deglycosylation of ACE2 or S proteins, suggesting that N-glycosylation plays no role in the interaction between SARS coronaviruses and the ACE2 receptor. Impairment of hACE2 N-glycosylation decreased cell to cell fusion mediated by SARS-CoV S protein but not SARS-CoV-2 S protein. Finally, we found that hACE2 N-glycosylation is required for an efficient viral entry of SARS-CoV/SARS-CoV-2 S pseudotyped viruses, which could be the result of low cell surface expression of the deglycosylated ACE2 receptor.

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