title: Glutamate receptor trafficking: endoplasmic reticulum quality control involves ligand binding and receptor function.
abstract: The glutamate receptor (GluR) agonist-binding site consists of amino acid residues in the extracellular S1 and S2 domains in the N-terminal and M3-M4 loop regions, respectively. In the present study, we sought to confirm that the conserved ligand-binding residues identified in the AMPA receptor S1S2 domains also participate in ligand binding of GluR6 kainate receptors. Amino acid substitutions were made in the GluR6 parent at R523, T690, and E738 to alter their potential interactions with ligand. Mutant receptors were expressed in human embryonic kidney 293 cells, confirmed by Western blot analysis, and tested by [3H]kainate binding and patch-clamp recording. Each of the binding site mutations was sufficient to reduce [3H]kainate binding to undetectable levels and eliminate functional responses to glutamate or kainate. As with our studies of other nonfunctional mutants (Fleck et al., 2003), immunocytochemical staining and cell-surface biotinylation studies showed that the mutant receptors were retained intracellularly and did not traffic to the cell surface. Endoglycosidase-H digests and colocalization with endoplasmic reticulum (ER) markers demonstrated that the mutant receptors are immaturely glycosylated and retained in the ER. Immunoprecipitation, native PAGE, and functional studies confirmed that the GluR6-binding site mutants are capable of multimeric assembly, indicating their retention in the ER does not result from a gross protein folding error. Together, these results confirm the role of R523, T690, and E738 directly in ligand binding to GluR6 and further support our previous report that nonfunctional GluRs are retained intracellularly by a functional checkpoint in ER quality control.
authors.affiliation: Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York 12208, USA.
authors.forename: Stephanie J
authors.initials: SJ
authors.lastname: Mah
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Cornell
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Mitchell
authors.forename: Mark W
authors.initials: MW
authors.lastname: Fleck
mesh_terms: D019943:Amino Acid Substitution / Q000502:physiology; D001665:Binding Sites / Q000502:physiology; D019921:Biotinylation / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004558:Electric Stimulation / Q000379:methods; D004721:Endoplasmic Reticulum / Q000502:physiology*; D015870:Gene Expression / Q000235:genetics; D018698:Glutamic Acid / Q000494:pharmacology; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods; D007608:Kainic Acid / Q000493:pharmacokinetics; D008024:Ligands; D008164:Luminescent Proteins; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000494:pharmacology; D008564:Membrane Potentials / Q000235:genetics / Q000528:radiation effects; D018613:Microscopy, Confocal / Q000379:methods; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000379:methods; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000494:pharmacology; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D021381:Protein Transport / Q000502:physiology*; D011869:Radioligand Assay / Q000379:methods; D018091:Receptors, AMPA / Q000737:chemistry / Q000378:metabolism; D018092:Kainic Acid Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment / Q000379:methods; D013329:Structure-Activity Relationship; D014162:Transfection / Q000379:methods; D014316:Tritium / Q000493:pharmacokinetics; D000097806:GluK2 Kainate Receptor
chemical_list: D018698:Glutamic Acid; D007608:Kainic Acid; D008024:Ligands; D008164:Luminescent Proteins; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D018091:Receptors, AMPA; D018092:Kainic Acid Receptors; D014316:Tritium; D000097806:GluK2 Kainate Receptor; C104722:glutamate receptor ionotropic, AMPA 2
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