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High‐ and Low‐Affinity α‐[ 3 H]Amino‐3‐Hydroxy‐5‐Methylisoxazole‐4‐Propionic Acid ([ 3 H]AMPA) Binding Sites Represent Immature and Mature Forms of AMPA Receptors and Are Composed of Differentially Glycosylated Subunits
Author(s) -
Standley Steve,
Tocco Georges,
Wagle Naveed,
Baudry Michel
Publication year - 1998
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1998.70062434.x
Subject(s) - ampa receptor , receptor , endoglycosidase h , biochemistry , glutamate receptor , chemistry , moiety , stereochemistry , protein subunit , enzyme , endoplasmic reticulum , gene , golgi apparatus
Quantitative α‐[ 3 H]amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionic acid ([ 3 H]AMPA) binding autoradiography was performed on frozen‐thawed sections from rat brain after preincubation at 0 or 35°C for 1 h. Preincubation at 35°C instead of 0°C resulted in a selective decrease of [ 3 H]AMPA binding assayed at a low concentration of [ 3 H]‐AMPA (50 n M ) and an enhancement of binding at a high concentration (500 n M ). The decrease in [ 3 H]AMPA binding after preincubation at 35°C was accompanied with the loss of the lighter organelles of P3 (microsomal) fractions. These organelles were found to contain a small subpopulation of AMPA/GluR receptors exhibiting a high affinity for [ 3 H]AMPA( K D ∼14 n M ), whereas heavier organelles exhibited lower affinity for AMPA ( K D ∼190 n M ). This small subpopulation of AMPA/GluR receptors contained almost exclusively a structurally distinct species of GluR2/3 subunits with an apparent molecular mass of 103.5 kDa (assessed with anti‐GluR2/3, C‐terminal antibodies). Experiments using two deglycosylating enzymes, N ‐glycopeptidase F and endoglycosidase H, clearly indicated that the 103.5‐kDa species represented a partially unglycosylated form of GluR2/3 subunits containing the high‐mannose type of oligosaccharide moiety, whereas receptors present in synaptosomal fractions were composed of subunits with complex oligosaccharides. A similar result was obtained by using an antibody recognizing the N‐terminal domain of GluR2(4). The same enzymatic treatment indicated that GluR1 subunits also exhibited a partially glycosylated form. These data indicate that high‐affinity [ 3 H]AMPA binding sites represent nonsynaptic, intracellular membrane‐bound AMPA receptors that differ from synaptic receptors by at least the glycosylation state of GluR2 (and GluR1) subunits. In addition, our results provide a relatively simple way of assessing changes in two spatially and structurally distinct [ 3 H]AMPA binding/GluR sites.