Characterization of the ligand-binding domains of glutamate receptor (Glu-R)-B and GluR-D subunits expressed in Escherichia coli as periplasmic proteins

Milla Arvola, Kari Keinänen (Corresponding Author)

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Abstract

We recently reported that a functional ligand-binding site of an alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA)-selective glutamate receptor (GluR)-D subunit can be expressed in insect cells as a soluble, N-glycosylated fusion protein consisting of two segments (S1 and S2) that are related by amino acid sequence to bacterial periplasmic binding proteins (Kuusinen, A., Arvola, M., and Keinänen, K., EMBO J. 14, 6327-6332). In an attempt to further characterize the structural determinants for ligand binding, we have now expressed the ligand-binding sites of GluR-B and GluR-D subunits in Escherichia coli as soluble periplasmic proteins. The bacterially expressed S1-S2 fusion proteins bound [3H]AMPA with a high affinity (Kd of 12 nM for GluR-B, Kd of 60 nM for GluR-D) and with a ligand pharmacology typical of native AMPA receptors, indicating that N-linked glycosylation is not required for the formation or the maintenance of the ligand-binding site. The flip and flop splice variants of the GluR-D S1-S2 fusion protein bound [3H]AMPA with equal affinities, whereas deletion of the C-terminal one-third of the S2 segment including the flip/flop sequence resulted in a loss of binding activity. Our results highlight the potential of bacterial expression for the analysis of the binding site and support a close structural similarity between glutamate receptors and bacterial proteins.
Original languageEnglish
Pages (from-to)15527-15532
Number of pages6
JournalJournal of Biological Chemistry
Volume271
Issue number26
DOIs
Publication statusPublished - 1996
MoE publication typeA1 Journal article-refereed

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Periplasmic Proteins
Escherichia coli
alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid
Ligands
Binding Sites
Bacterial Proteins
Fusion reactions
Periplasmic Binding Proteins
Glycosylation
Isoxazoles
Proteins
AMPA Receptors
Flip flop circuits
Propionates
Glutamate Receptors
Insects
Amino Acid Sequence
Maintenance
glutamate receptor type B
glutamate receptor type D

Cite this

@article{352925e81a2c4fd99a93ea050d19ff10,
title = "Characterization of the ligand-binding domains of glutamate receptor (Glu-R)-B and GluR-D subunits expressed in Escherichia coli as periplasmic proteins",
abstract = "We recently reported that a functional ligand-binding site of an alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA)-selective glutamate receptor (GluR)-D subunit can be expressed in insect cells as a soluble, N-glycosylated fusion protein consisting of two segments (S1 and S2) that are related by amino acid sequence to bacterial periplasmic binding proteins (Kuusinen, A., Arvola, M., and Kein{\"a}nen, K., EMBO J. 14, 6327-6332). In an attempt to further characterize the structural determinants for ligand binding, we have now expressed the ligand-binding sites of GluR-B and GluR-D subunits in Escherichia coli as soluble periplasmic proteins. The bacterially expressed S1-S2 fusion proteins bound [3H]AMPA with a high affinity (Kd of 12 nM for GluR-B, Kd of 60 nM for GluR-D) and with a ligand pharmacology typical of native AMPA receptors, indicating that N-linked glycosylation is not required for the formation or the maintenance of the ligand-binding site. The flip and flop splice variants of the GluR-D S1-S2 fusion protein bound [3H]AMPA with equal affinities, whereas deletion of the C-terminal one-third of the S2 segment including the flip/flop sequence resulted in a loss of binding activity. Our results highlight the potential of bacterial expression for the analysis of the binding site and support a close structural similarity between glutamate receptors and bacterial proteins.",
author = "Milla Arvola and Kari Kein{\"a}nen",
note = "Project code: BEL2031",
year = "1996",
doi = "10.1074/jbc.271.26.15527",
language = "English",
volume = "271",
pages = "15527--15532",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "26",

}

Characterization of the ligand-binding domains of glutamate receptor (Glu-R)-B and GluR-D subunits expressed in Escherichia coli as periplasmic proteins. / Arvola, Milla; Keinänen, Kari (Corresponding Author).

In: Journal of Biological Chemistry, Vol. 271, No. 26, 1996, p. 15527-15532.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Characterization of the ligand-binding domains of glutamate receptor (Glu-R)-B and GluR-D subunits expressed in Escherichia coli as periplasmic proteins

AU - Arvola, Milla

AU - Keinänen, Kari

N1 - Project code: BEL2031

PY - 1996

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N2 - We recently reported that a functional ligand-binding site of an alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA)-selective glutamate receptor (GluR)-D subunit can be expressed in insect cells as a soluble, N-glycosylated fusion protein consisting of two segments (S1 and S2) that are related by amino acid sequence to bacterial periplasmic binding proteins (Kuusinen, A., Arvola, M., and Keinänen, K., EMBO J. 14, 6327-6332). In an attempt to further characterize the structural determinants for ligand binding, we have now expressed the ligand-binding sites of GluR-B and GluR-D subunits in Escherichia coli as soluble periplasmic proteins. The bacterially expressed S1-S2 fusion proteins bound [3H]AMPA with a high affinity (Kd of 12 nM for GluR-B, Kd of 60 nM for GluR-D) and with a ligand pharmacology typical of native AMPA receptors, indicating that N-linked glycosylation is not required for the formation or the maintenance of the ligand-binding site. The flip and flop splice variants of the GluR-D S1-S2 fusion protein bound [3H]AMPA with equal affinities, whereas deletion of the C-terminal one-third of the S2 segment including the flip/flop sequence resulted in a loss of binding activity. Our results highlight the potential of bacterial expression for the analysis of the binding site and support a close structural similarity between glutamate receptors and bacterial proteins.

AB - We recently reported that a functional ligand-binding site of an alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA)-selective glutamate receptor (GluR)-D subunit can be expressed in insect cells as a soluble, N-glycosylated fusion protein consisting of two segments (S1 and S2) that are related by amino acid sequence to bacterial periplasmic binding proteins (Kuusinen, A., Arvola, M., and Keinänen, K., EMBO J. 14, 6327-6332). In an attempt to further characterize the structural determinants for ligand binding, we have now expressed the ligand-binding sites of GluR-B and GluR-D subunits in Escherichia coli as soluble periplasmic proteins. The bacterially expressed S1-S2 fusion proteins bound [3H]AMPA with a high affinity (Kd of 12 nM for GluR-B, Kd of 60 nM for GluR-D) and with a ligand pharmacology typical of native AMPA receptors, indicating that N-linked glycosylation is not required for the formation or the maintenance of the ligand-binding site. The flip and flop splice variants of the GluR-D S1-S2 fusion protein bound [3H]AMPA with equal affinities, whereas deletion of the C-terminal one-third of the S2 segment including the flip/flop sequence resulted in a loss of binding activity. Our results highlight the potential of bacterial expression for the analysis of the binding site and support a close structural similarity between glutamate receptors and bacterial proteins.

U2 - 10.1074/jbc.271.26.15527

DO - 10.1074/jbc.271.26.15527

M3 - Article

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SP - 15527

EP - 15532

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

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ER -