Surface expression of the AMPA receptor subunits GluR1, GluR2, and GluR4 in stably transfected baby hamster kidney cells

Randy A. Hall, Annette Hansen, Peter H. Andersen, Thomas Soderling

    Research output: Contribution to journalArticle

    62 Citations (Scopus)

    Abstract

    The surface expression of the α-amino-3-hydroxy-5-methylisoxazole-4- propionic acid (AMPA)-type glutamate receptor (GluR) subunits GluR1, GluR2, and GluR4 was studied in cultures of stably transfected baby hamster kidney (BHK)-570 cells. Two methods were used to quantify surface expression: cross- linking with the membrane-impermeant reagent bis(sulfosuccinimidyl)suberate (BS3) and labeling of surface receptors with the membrane-impermeant biotinylating reagent sulfosuccinimidyl 2- (biotinamido) ethyl-1,3- dithiopropionate (NHS-ss-biotin) followed by precipitation with neutravidin beads. Western blot analyses of control versus treated cultures revealed that, for all three GluR subunits examined, 25-40% of the total GluR population is located in the plasma membrane of the BHK-570 cells. This finding was corroborated by analyses of the surface expression of [3H]AMPA binding sites in the GluR-expressing BHK-570 cells performed via the biotinylation/precipitation method; these studies revealed that 30-40% of the total binding site population is found in the plasma membrane. Analyses of combinations of the subunits, both GluR1 + GluR2 and GluR2 + GluR4, revealed that heteromeric combinations of the subunits are not trafficked to the surface more efficiently than homomeric receptors. For each of the three subunits, western blots revealed two distinct bands; removal of surface receptors reduced immunoreactivity for the upper band of each subunit by >90%, whereas immunoreactivity for the lower band was reduced by only 10- 20%. Treatment of extracts from the various cell lines with glycopeptidase F resulted in the collapse of the two bands into a single band of lower molecular weight, suggesting that the two original bands represent differentially glycosylated forms of the same polypeptides. These data indicate that the majority of the stably expressed GluR subunits in these cell lines are incompletely glycosylated and that complete glycosylation is associated with trafficking of the GluR subunits to the cell surface.

    Original languageEnglish (US)
    Pages (from-to)625-630
    Number of pages6
    JournalJournal of Neurochemistry
    Volume68
    Issue number2
    StatePublished - Feb 1997

    Fingerprint

    Glutamate Receptors
    Cricetinae
    Kidney
    Cell membranes
    Western Blotting
    Binding Sites
    Cell Membrane
    Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
    Biotinylation
    Cell Line
    Cells
    Membranes
    Glycosylation
    Population
    Biotin
    bucide
    propionic acid
    Crosslinking
    Labeling
    Molecular Weight

    Keywords

    • Binding
    • Biotinylati on
    • Cross-linking
    • GluR1
    • GluR2
    • GluR4
    • Glutamate receptors
    • Glycosylation
    • Phosphorylation

    ASJC Scopus subject areas

    • Biochemistry
    • Cellular and Molecular Neuroscience

    Cite this

    Surface expression of the AMPA receptor subunits GluR1, GluR2, and GluR4 in stably transfected baby hamster kidney cells. / Hall, Randy A.; Hansen, Annette; Andersen, Peter H.; Soderling, Thomas.

    In: Journal of Neurochemistry, Vol. 68, No. 2, 02.1997, p. 625-630.

    Research output: Contribution to journalArticle

    Hall, Randy A. ; Hansen, Annette ; Andersen, Peter H. ; Soderling, Thomas. / Surface expression of the AMPA receptor subunits GluR1, GluR2, and GluR4 in stably transfected baby hamster kidney cells. In: Journal of Neurochemistry. 1997 ; Vol. 68, No. 2. pp. 625-630.
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    abstract = "The surface expression of the α-amino-3-hydroxy-5-methylisoxazole-4- propionic acid (AMPA)-type glutamate receptor (GluR) subunits GluR1, GluR2, and GluR4 was studied in cultures of stably transfected baby hamster kidney (BHK)-570 cells. Two methods were used to quantify surface expression: cross- linking with the membrane-impermeant reagent bis(sulfosuccinimidyl)suberate (BS3) and labeling of surface receptors with the membrane-impermeant biotinylating reagent sulfosuccinimidyl 2- (biotinamido) ethyl-1,3- dithiopropionate (NHS-ss-biotin) followed by precipitation with neutravidin beads. Western blot analyses of control versus treated cultures revealed that, for all three GluR subunits examined, 25-40{\%} of the total GluR population is located in the plasma membrane of the BHK-570 cells. This finding was corroborated by analyses of the surface expression of [3H]AMPA binding sites in the GluR-expressing BHK-570 cells performed via the biotinylation/precipitation method; these studies revealed that 30-40{\%} of the total binding site population is found in the plasma membrane. Analyses of combinations of the subunits, both GluR1 + GluR2 and GluR2 + GluR4, revealed that heteromeric combinations of the subunits are not trafficked to the surface more efficiently than homomeric receptors. For each of the three subunits, western blots revealed two distinct bands; removal of surface receptors reduced immunoreactivity for the upper band of each subunit by >90{\%}, whereas immunoreactivity for the lower band was reduced by only 10- 20{\%}. Treatment of extracts from the various cell lines with glycopeptidase F resulted in the collapse of the two bands into a single band of lower molecular weight, suggesting that the two original bands represent differentially glycosylated forms of the same polypeptides. These data indicate that the majority of the stably expressed GluR subunits in these cell lines are incompletely glycosylated and that complete glycosylation is associated with trafficking of the GluR subunits to the cell surface.",
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    T1 - Surface expression of the AMPA receptor subunits GluR1, GluR2, and GluR4 in stably transfected baby hamster kidney cells

    AU - Hall, Randy A.

    AU - Hansen, Annette

    AU - Andersen, Peter H.

    AU - Soderling, Thomas

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    N2 - The surface expression of the α-amino-3-hydroxy-5-methylisoxazole-4- propionic acid (AMPA)-type glutamate receptor (GluR) subunits GluR1, GluR2, and GluR4 was studied in cultures of stably transfected baby hamster kidney (BHK)-570 cells. Two methods were used to quantify surface expression: cross- linking with the membrane-impermeant reagent bis(sulfosuccinimidyl)suberate (BS3) and labeling of surface receptors with the membrane-impermeant biotinylating reagent sulfosuccinimidyl 2- (biotinamido) ethyl-1,3- dithiopropionate (NHS-ss-biotin) followed by precipitation with neutravidin beads. Western blot analyses of control versus treated cultures revealed that, for all three GluR subunits examined, 25-40% of the total GluR population is located in the plasma membrane of the BHK-570 cells. This finding was corroborated by analyses of the surface expression of [3H]AMPA binding sites in the GluR-expressing BHK-570 cells performed via the biotinylation/precipitation method; these studies revealed that 30-40% of the total binding site population is found in the plasma membrane. Analyses of combinations of the subunits, both GluR1 + GluR2 and GluR2 + GluR4, revealed that heteromeric combinations of the subunits are not trafficked to the surface more efficiently than homomeric receptors. For each of the three subunits, western blots revealed two distinct bands; removal of surface receptors reduced immunoreactivity for the upper band of each subunit by >90%, whereas immunoreactivity for the lower band was reduced by only 10- 20%. Treatment of extracts from the various cell lines with glycopeptidase F resulted in the collapse of the two bands into a single band of lower molecular weight, suggesting that the two original bands represent differentially glycosylated forms of the same polypeptides. These data indicate that the majority of the stably expressed GluR subunits in these cell lines are incompletely glycosylated and that complete glycosylation is associated with trafficking of the GluR subunits to the cell surface.

    AB - The surface expression of the α-amino-3-hydroxy-5-methylisoxazole-4- propionic acid (AMPA)-type glutamate receptor (GluR) subunits GluR1, GluR2, and GluR4 was studied in cultures of stably transfected baby hamster kidney (BHK)-570 cells. Two methods were used to quantify surface expression: cross- linking with the membrane-impermeant reagent bis(sulfosuccinimidyl)suberate (BS3) and labeling of surface receptors with the membrane-impermeant biotinylating reagent sulfosuccinimidyl 2- (biotinamido) ethyl-1,3- dithiopropionate (NHS-ss-biotin) followed by precipitation with neutravidin beads. Western blot analyses of control versus treated cultures revealed that, for all three GluR subunits examined, 25-40% of the total GluR population is located in the plasma membrane of the BHK-570 cells. This finding was corroborated by analyses of the surface expression of [3H]AMPA binding sites in the GluR-expressing BHK-570 cells performed via the biotinylation/precipitation method; these studies revealed that 30-40% of the total binding site population is found in the plasma membrane. Analyses of combinations of the subunits, both GluR1 + GluR2 and GluR2 + GluR4, revealed that heteromeric combinations of the subunits are not trafficked to the surface more efficiently than homomeric receptors. For each of the three subunits, western blots revealed two distinct bands; removal of surface receptors reduced immunoreactivity for the upper band of each subunit by >90%, whereas immunoreactivity for the lower band was reduced by only 10- 20%. Treatment of extracts from the various cell lines with glycopeptidase F resulted in the collapse of the two bands into a single band of lower molecular weight, suggesting that the two original bands represent differentially glycosylated forms of the same polypeptides. These data indicate that the majority of the stably expressed GluR subunits in these cell lines are incompletely glycosylated and that complete glycosylation is associated with trafficking of the GluR subunits to the cell surface.

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    KW - Cross-linking

    KW - GluR1

    KW - GluR2

    KW - GluR4

    KW - Glutamate receptors

    KW - Glycosylation

    KW - Phosphorylation

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