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 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.

Apical membrane and intracellular distribution of endogenous alpha 2A-adrenergic receptors in MDCK cells

1994 ◽  
Vol 267 (3) ◽  
pp. F347-F353 ◽  
Author(s):  
M. D. Okusa ◽  
K. R. Lynch ◽  
D. L. Rosin ◽  
L. Huang ◽  
Y. Y. Wei
Keyword(s):  
Cell Surface ◽  
De Novo ◽  
Mdck Cells ◽  
Specific Binding ◽  
Surface Expression ◽  
Surface Proteins ◽  
Cell Surface Expression ◽  
Surface Binding ◽  
Binding Studies ◽  
Apical Domain

The purpose of the current studies was to characterize the endogenous alpha 2-adrenergic receptor (AR) subtypes present in Madin-Darby canine kidney (MDCK) cells and to determine their level of expression and pattern of distribution. By saturation binding analysis with [3H]MK-912, MDCK cells expressed high levels of alpha 2-ARs with a maximum receptor density (Bmax) of 798 +/- 55 fmol/mg protein and an equilibrium dissociation constant (Kd) of 0.98 +/- 0.32 nM. Competitive binding studies using prazosin, oxymetazoline, phentolamine, and epinephrine to displace [3H]MK-912 demonstrated inhibition constant (Ki) values of 1,270 +/- 250, 5.0 +/- 0.4, 5.5 +/- 0.3, and 392 +/- 150 nM (n = 3), respectively. In Northern blot analysis we found that MDCK cells expressed transcripts encoding alpha 2A-AR and not alpha 2B-AR or alpha 2C-AR. Surface binding experiments suggested that approximately 60% of alpha 2A-ARs are distributed at the cell surface domain. Specific binding of [3H]MK-912 to soluble apical and basolateral surface proteins isolated by surface biotinylation indicated the expression of surface alpha 2A-ARs was limited to the apical domain of MDCK cells. No alpha 2A-ARs were detected on the basolateral surface. We conclude that endogenous alpha 2A-ARs are targeted to the apical domain of MDCK cells and that the intracellular compartment may contain ARs as a reservoir for de novo cell surface expression or, alternatively, may represent internalized receptors.

scholarly journals Functional Complexes of Angiotensin-Converting Enzyme 2 and Renin-Angiotensin System Receptors: Expression in Adult but Not Fetal Lung Tissue

2020 ◽  
Vol 21 (24) ◽  
pp. 9602
Author(s):  
Rafael Franco ◽  
Alejandro Lillo ◽  
Rafael Rivas-Santisteban ◽  
Ana I. Rodríguez-Pérez ◽  
Irene Reyes-Resina ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Cell Surface ◽  
Angiotensin Converting Enzyme ◽  
Ligand Binding ◽  
Renin Angiotensin System ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Converting Enzyme ◽  
Angiotensin System ◽  
Angiotensin Converting Enzyme 2

Angiotensin-converting enzyme 2 (ACE2) is a membrane peptidase and a component of the renin-angiotensin system (RAS) that has been found in cells of all organs, including the lungs. While ACE2 has been identified as the receptor for severe acute respiratory syndrome (SARS) coronaviruses, the mechanism underlying cell entry remains unknown. Human immunodeficiency virus infects target cells via CXC chemokine receptor 4 (CXCR4)-mediated endocytosis. Furthermore, CXCR4 interacts with dipeptidyl peptidase-4 (CD26/DPPIV), an enzyme that cleaves CXCL12/SDF-1, which is the chemokine that activates this receptor. By analogy, we hypothesized that ACE2 might also be capable of interactions with RAS-associated G-protein coupled receptors. Using resonance energy transfer and cAMP and mitogen-activated protein kinase signaling assays, we found that human ACE2 interacts with RAS-related receptors, namely the angiotensin II type 1 receptor (AT1R), the angiotensin II type 2 receptor (AT2R), and the MAS1 oncogene receptor (MasR). Although these interactions led to various alterations of signal transduction, but, more importantly, ligand binding to AT1R resulted in the downregulation of ACE2 cell surface expression, while ligand binding to AT2R, but not to MasR, resulted in upregulation of ACE2 cell surface expression. Proximity ligation assays performed in situ revealed macromolecular complexes containing ACE2 and AT1R, AT2R or MasR in adult but not fetal mouse lung tissue. These findings highlight the relevance of RAS in SARS-CoV-2 infection and the role of ACE2-containing complexes as potential therapeutic targets.

scholarly journals Trafficking and surface expression of the glutamate receptor subunit, KA2

2003 ◽  
Vol 310 (1) ◽  
pp. 8-13 ◽  
Author(s):  
Dayna M Hayes ◽  
Stephanie Braud ◽  
David E Hurtado ◽  
Jennifer McCallum ◽  
Steve Standley ◽  
...  
Keyword(s):  
Glutamate Receptor ◽  
Surface Expression ◽  
Receptor Subunit ◽  
Glutamate Receptor Subunit

scholarly journals N-glycosylation of the AMPA-type glutamate receptor regulates cell surface expression and tetramer formation affecting channel function

2018 ◽  
Vol 147 (6) ◽  
pp. 730-747 ◽  
Author(s):  
Munal Babu Kandel ◽  
Saki Yamamoto ◽  
Ryosuke Midorikawa ◽  
Jyoji Morise ◽  
Yoshihiko Wakazono ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glutamate Receptor ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Channel Function ◽  
Tetramer Formation
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