α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Channels Lacking the N-terminal Domain

Ionotropic glutamate receptor (iGluR) subunits contain a ∼400-residue extracellular N-terminal domain (“X domain”), which is sequence-related to bacterial amino acid-binding proteins and to class C G-protein-coupled receptors. The X domain has been implicated in the assembly, transport to the cell surface, allosteric ligand binding, and desensitization in various members of the iGluR family, but its actual role in these events is poorly characterized. We have studied the properties of homomeric α-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA)-selective GluR-D glutamate receptors carrying N-terminal deletions. Our analysis indicates that, surprisingly, transport to the cell surface, ligand binding properties, agonist-triggered channel activation, rapid desensitization, and allosteric potentiation by cyclothiazide can occur normally in the complete absence of the X domain (residues 22–402). The relatively intact ligand-gated channel function of a homomeric AMPA receptor in the absence of the X domain indirectly suggests more subtle roles for this domain in AMPA receptors,e.g.in the assembly of heteromeric receptors and in synaptic protein interactions.

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GluR2 protein-protein interactions and the regulation of AMPA receptors during synaptic plasticity

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2002.1215 ◽

2003 ◽

Vol 358 (1432) ◽

pp. 715-720 ◽

Cited By ~ 17

Author(s):

Fabrice Duprat ◽

Michael Daw ◽

Wonil Lim ◽

Graham Collingridge ◽

John Isaac

Keyword(s):

Synaptic Plasticity ◽

Ampa Receptor ◽

Protein Interactions ◽

Ampa Receptors ◽

Long Term Potentiation ◽

Synaptic Proteins ◽

Mammalian Brain ◽

Protein Protein Interactions ◽

Term Potentiation

AMPA-type glutamate receptors mediate most fast excitatory synaptic transmissions in the mammalian brain. They are critically involved in the expression of long-term potentiation and long-term depression, forms of synaptic plasticity that are thought to underlie learning and memory. A number of synaptic proteins have been identified that interact with the intracellular C-termini of AMPA receptor subunits. Here, we review recent studies and present new experimental data on the roles of these interacting proteins in regulating the AMPA receptor function during basal synaptic transmission and plasticity.

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

Biochemical Journal ◽

10.1042/bj3150217 ◽

1996 ◽

Vol 315 (1) ◽

pp. 217-225 ◽

Cited By ~ 15

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.

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The action of Con-ikot-ikot toxin on single AMPA-type glutamate receptors

10.1101/2021.02.19.432041 ◽

2021 ◽

Author(s):

Jelena Baranovic ◽

Sebastian Braunbeck ◽

Nikolai Zaki ◽

Sonja Minniberger ◽

Miriam Chebli ◽

...

Keyword(s):

Ligand Binding ◽

Glutamate Receptors ◽

Ampa Receptor ◽

Ampa Receptors ◽

Single Channel ◽

Basic Research ◽

High Specificity ◽

Direct Consequence ◽

Binding Mode ◽

Binding Domains

SummaryConotoxins are a large group of naturally occurring toxic peptides produced by the predatory sea snails of the genus Conus. Many of these toxins target ion channels, often with high specificity and affinity. As such, they have proven to be invaluable for basic research as well as acting as leads for therapeutic strategies. Con-ikot-ikot is the only conotoxin so far identified that targets AMPA-type glutamate receptors, the main mediators of excitatory neurotransmission in the vertebrate brain. Here, we describe how the toxin modifies the activity of AMPA receptors at the single-channel level. The toxin binds to the AMPA receptor with high affinity (EC50 = 5 nM) and once bound, takes minutes to wash out. As shown previously, it effectively blocks desensitization of AMPA receptors, however, compared to other desensitisation blockers, it is a poor stabiliser of the open channel because toxin-bound AMPA receptors undergo frequent, brief closures. We propose this is a direct consequence of its unique binding mode to the ligand binding domains. Unlike other blockers of desensitization, which stabilise individual dimers within an AMPA receptor tetramer, the toxin immobilizes all four ligand binding domains of the tetramer. This result further emphasises that quaternary reorganization of independent LBD dimers is essential for the full activity of AMPA receptors.

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Architecture and subunit arrangement of native AMPA receptors elucidated by cryo-EM

Science ◽

10.1126/science.aaw8250 ◽

2019 ◽

Vol 364 (6438) ◽

pp. 355-362 ◽

Cited By ~ 32

Author(s):

Yan Zhao ◽

Shanshuang Chen ◽

Adam C. Swensen ◽

Wei-Jun Qian ◽

Eric Gouaux

Keyword(s):

Molecular Structure ◽

Electron Microscopy ◽

Signal Transduction ◽

Ligand Binding ◽

Ampa Receptor ◽

Ampa Receptors ◽

Mammalian Brain ◽

Receptor Complexes ◽

Cryo Electron Microscopy ◽

Chemical Synapses

Glutamate-gated AMPA receptors mediate the fast component of excitatory signal transduction at chemical synapses throughout all regions of the mammalian brain. AMPA receptors are tetrameric assemblies composed of four subunits, GluA1–GluA4. Despite decades of study, the subunit composition, subunit arrangement, and molecular structure of native AMPA receptors remain unknown. Here we elucidate the structures of 10 distinct native AMPA receptor complexes by single-particle cryo–electron microscopy (cryo-EM). We find that receptor subunits are arranged nonstochastically, with the GluA2 subunit preferentially occupying the B and D positions of the tetramer and with triheteromeric assemblies comprising a major population of native AMPA receptors. Cryo-EM maps define the structure for S2-M4 linkers between the ligand-binding and transmembrane domains, suggesting how neurotransmitter binding is coupled to ion channel gating.

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A Ligand-Binding Site in the Glua3 AMPA Receptor N-Terminal Domain Observed in Druggability Simulations and X-Ray Crystallography

Biophysical Journal ◽

10.1016/j.bpj.2018.11.2639 ◽

2019 ◽

Vol 116 (3) ◽

pp. 489a

Author(s):

Ji Young Lee ◽

James Krieger ◽

Beatriz Herguedas ◽

Javier García-Nafría ◽

Anindita Dutta ◽

...

Keyword(s):

Ligand Binding ◽

Binding Site ◽

Ampa Receptor ◽

Ligand Binding Site ◽

X Ray ◽

X Ray Crystallography ◽

Terminal Domain

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Calcitonin/Amylin Receptors and Ramps

The Scientific World JOURNAL ◽

10.1100/tsw.2001.442 ◽

2001 ◽

Vol 1 ◽

pp. 9-9

Author(s):

Patrick M. Sexton

Keyword(s):

Cell Surface ◽

Protein Interactions ◽

Receptor Expression ◽

Cell Surface Receptor ◽

Calcitonin Receptor ◽

Receptor Function ◽

Surface Receptor ◽

Receptor Activity Modifying Proteins ◽

G Protein Coupled ◽

Novel Protein

Our understanding of G protein-coupled receptor function has recently expanded to encompass novel protein interactions that underlie both cell surface receptor expression and the exhibited phenotype. The most notable examples are those involving receptor activity modifying proteins (RAMPs). RAMP association with the calcitonin receptor-like receptor (CRLR) traffics this receptor to the cell surface, where individual RAMPs dictate the expression of unique phenotypes.

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Endogenous thrombospondin-1 is a cell-surface ligand for regulation of integrin-dependent T-lymphocyte adhesion

Blood ◽

10.1182/blood-2006-04-016832 ◽

2006 ◽

Vol 108 (9) ◽

pp. 3112-3120 ◽

Cited By ~ 50

Author(s):

Shu Shun Li ◽

Zhiwen Liu ◽

Mehmet Uzunel ◽

Karl-Gösta Sundqvist

Keyword(s):

T Cell ◽

Cell Surface ◽

Surface Expression ◽

Lymphocyte Adhesion ◽

Surface Receptors ◽

Terminal Domain ◽

Thrombospondin 1 ◽

A Cell ◽

Surface Ligand ◽

Quiescent T Cells

Abstract Lymphocyte adhesion to cells and extracellular matrix (ECM) via integrins plays a pivotal role for the function of the immune system. We show here that endogenous thrombospondin-1 (TSP-1) is a cell-surface ligand for cis interaction of surface receptors in T lymphocytes controlled by integrins and the T-cell antigen receptor (TCR/CD3). Stimulation of CD3 triggers rapid surface expression of TSP-1 in quiescent T cells, whereas activated cells express TSP-1 constitutively. Endogenous TSP-1 is attached to lipoprotein receptor-related protein 1 (LRP1/CD91) and calreticulin (CRT) on the cell surface through its NH2-terminal domain. Adhesion via integrins to ICAM-1 or ECM components up-regulates TSP turnover dramatically from a low level in nonadherent cells, whereas CD3 stimulation inhibits TSP turnover through interference with CD91/CRT-mediated internalization. Integrin-associated protein (IAP/CD47) is essential for TSP turnover and adhesion through interaction with the C-terminal domain of TSP-1 in response to triggering signals delivered at the NH2-terminal. These results indicate that endogenous TSP-1 connects separate cell-surface receptors functionally and regulates T-cell adhesion.

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Crystal structure of the glutamate receptor GluA1 N-terminal domain

Biochemical Journal ◽

10.1042/bj20110801 ◽

2011 ◽

Vol 438 (2) ◽

pp. 255-263 ◽

Cited By ~ 14

Author(s):

Guorui Yao ◽

Yinong Zong ◽

Shenyan Gu ◽

Jie Zhou ◽

Huaxi Xu ◽

...

Keyword(s):

Crystal Structure ◽

Protein Interactions ◽

Ampa Receptors ◽

Neurological Diseases ◽

Protein Protein Interactions ◽

Terminal Domain ◽

The Central Nervous System ◽

In Trans ◽

Excitatory Neurotransmission ◽

Active Channels

The AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) subfamily of iGluRs (ionotropic glutamate receptors) is essential for fast excitatory neurotransmission in the central nervous system. The malfunction of AMPARs (AMPA receptors) has been implicated in many neurological diseases, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. The active channels of AMPARs and other iGluR subfamilies are tetramers formed exclusively by assembly of subunits within the same subfamily. It has been proposed that the assembly process is controlled mainly by the extracellular ATD (N-terminal domain) of iGluR. In addition, ATD has also been implicated in synaptogenesis, iGluR trafficking and trans-synaptic signalling, through unknown mechanisms. We report in the present study a 2.5 Å (1 Å=0.1 nm) resolution crystal structure of the ATD of GluA1. Comparative analyses of the structure of GluA1-ATD and other subunits sheds light on our understanding of how ATD drives subfamily-specific assembly of AMPARs. In addition, analysis of the crystal lattice of GluA1-ATD suggests a novel mechanism by which the ATD might participate in inter-tetramer AMPAR clustering, as well as in trans-synaptic protein–protein interactions.

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NanoBiT Complementation to Monitor Agonist-Induced Adenosine A1 Receptor Internalization

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555219880475 ◽

2019 ◽

Vol 25 (2) ◽

pp. 186-194 ◽

Cited By ~ 3

Author(s):

Mark Soave ◽

Barrie Kellam ◽

Jeanette Woolard ◽

Stephen J. Briddon ◽

Stephen J. Hill

Keyword(s):

Ligand Binding ◽

Protein Interactions ◽

Receptor Internalization ◽

Hek293 Cells ◽

Binding Assays ◽

Nanoluc Luciferase ◽

Important Regulator ◽

Surface Luminescence ◽

A1 Receptor ◽

G Protein Coupled

Receptor internalization in response to prolonged agonist treatment is an important regulator of G protein–coupled receptor (GPCR) function. The adenosine A1 receptor (A1AR) is one of the adenosine receptor family of GPCRs, and evidence for its agonist-induced internalization is equivocal. The recently developed NanoBiT technology uses split NanoLuc Luciferase to monitor changes in protein interactions. We have modified the human A1AR on the N-terminus with the small high-affinity HiBiT tag. In the presence of the large NanoLuc subunit (LgBiT), complementation occurs, reconstituting a full-length functional NanoLuc Luciferase. Here, we have used complemented luminescence to monitor the internalization of the A1AR in living HEK293 cells. Agonist treatment resulted in a robust decrease in cell-surface luminescence, indicating an increase in A1AR internalization. These responses were inhibited by the A1AR-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), with an antagonist affinity that closely matched that measured using ligand binding with a fluorescent A1 receptor antagonist (CA200645). The agonist potencies for inducing A1AR internalization were very similar to the affinities previously determined by ligand binding, suggesting little or no amplification of the internalization response. By complementing the HiBiT tag to exogenous purified LgBiT, it was also possible to perform NanoBRET ligand-binding experiments using HiBiT–A1AR. This study demonstrates the use of NanoBiT technology to monitor internalization of the A1AR and offers the potential to combine these experiments with NanoBRET ligand-binding assays.

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