title: Ligand binding is a critical requirement for plasma membrane expression of heteromeric kainate receptors.
abstract: Intracellular trafficking of ionotropic glutamate receptors is controlled by multiple discrete determinants in receptor subunits. Most such determinants have been localized to the cytoplasmic carboxyl-terminal domain, but other domains in the subunit proteins can play roles in modulating receptor surface expression. Here we demonstrate that formation of an intact glutamate binding site also acts as an additional quality-control check for surface expression of homomeric and heteromeric kainate receptors. A key ligand-binding residue in the KA2 subunit, threonine 675, was mutated to either alanine or glutamate, which eliminated affinity for the receptor ligands kainate and glutamate. We found that plasma membrane expression of heteromeric GluR6/KA2(T675A) or GluR6/KA2(T675E) kainate receptors was markedly reduced compared with wild-type GluR6/KA2 receptors in transfected HEK 293 and COS-7 cells and in cultured neurons. Surface expression of homomeric KA2 receptors lacking a retention/retrieval determinant (KA2-R/A) was also reduced upon mutation of Thr-675 and elimination of the ligand binding site. KA2 Thr-675 mutant subunits were able to co-assemble with GluR5 and GluR6 subunits and were degraded at the same rate as wild-type KA2 subunit protein. These results suggest that glutamate binding and associated conformational changes are prerequisites for forward trafficking of intracellular kainate receptors following multimeric assembly.
authors.affiliation: Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1031, USA.
authors.forename: Lokanatha
authors.initials: L
authors.lastname: Valluru
authors.forename: Jian
authors.initials: J
authors.lastname: Xu
authors.forename: Yongling
authors.initials: Y
authors.lastname: Zhu
authors.forename: Sheng
authors.initials: S
authors.lastname: Yan
authors.forename: Anis
authors.initials: A
authors.lastname: Contractor
authors.forename: Geoffrey T
authors.initials: GT
authors.lastname: Swanson
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D007608:Kainic Acid / Q000378:metabolism; D008024:Ligands; D009154:Mutation / Q000235:genetics; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D011869:Radioligand Assay; D051381:Rats; D018092:Kainic Acid Receptors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D007608:Kainic Acid; D008024:Ligands; D021122:Protein Subunits; D018092:Kainic Acid Receptors