Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.52 (PNGase F)
 1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The surface expression of the alpha-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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PMID:Surface expression of the AMPA receptor subunits GluR1, GluR2, and GluR4 in stably transfected baby hamster kidney cells. 900 49

Quantitative alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA) binding autoradiography was performed on frozen-thawed sections from rat brain after preincubation at 0 or 35 degrees C for 1 h. Preincubation at 35 degrees C instead of 0 degrees 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 degrees 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 (K(D) approximately 14 nM), whereas heavier organelles exhibited lower affinity for AMPA (K(D) approximately 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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PMID:High- and low-affinity alpha-[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. 960 8