High- and Low-Affinity α-[3H]Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid ([3H]AMPA) Binding Sites Represent Immature and Mature Forms of AMPA Receptors and Are Composed of Differentially Glycosylated Subunits |
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Authors: | Steve Standley Georges Tocco Naveed Wagle Michel Baudry |
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Institution: | Neuroscience Program, University of Southern California, Los Angeles, California, U.S.A. |
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Abstract: | Abstract: Quantitative α-3H]amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (3H]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 3H]AMPA binding assayed at a low concentration of 3H]-AMPA (50 nM) and an enhancement of binding at a high concentration (500 nM). The decrease in 3H]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 3H]AMPA(KD~14 nM), whereas heavier organelles exhibited lower affinity for AMPA (KD~190 nM). 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 3H]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 3H]AMPA binding/GluR sites. |
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Keywords: | Receptors α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid Glutamate Glycosylation Subcellular Plasticity |
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