title: Cloning, expression and pharmacological characterization of a human glutamate receptor: hGluR4.
abstract: A member of the ionotropic family of glutamate receptors, hGluR4, was isolated from a human cDNA library and characterized following expression in mammalian cell lines. Human GluR4 possessed a 99% amino acid and 92% nucleotide homology to that of its rat counterpart with sequence differences restricted to the carboxy and amino terminal regions of the molecule. Transfection of simian kidney cells (COS-1) with an hGluR4 expression plasmid resulted in the transient formation of a membrane protein that possessed high specific binding for [3H](RS)-alpha-amino- 3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA) but not [3H]kainate. Competition studies yielded a displacement profile of AMPA = quisqualate > glutamate > domoate > kainate >> N-methyl-D-aspartate (NMDA) or dihydrokainate. Whole-cell, voltage-clamp recordings from a human embryonic kidney cell line (HEK 293) stably expressing hGluR4 confirmed the presence of constitutively active, ligand-gated ion channels activated by AMPA, glutamate and kainate but not N-methyl-D-aspartate. Kainate-evoked currents were reversibly attenuated by 6-cyano-7-nitro- quinoxaline-2,3-dione (CNQX) but not DL-2-amino-5- phosphonovalerate (DL-AP5). Agonist-evoked currents exhibited inward rectification and ion substitution experiments indicated that hGluR4 receptor-linked ion channels in their homomeric state are permeable to both CA2+ and Na+ ions. In the same cell line antibody to rat GluR4 immunoprecipitated a major protein band at approximately 108 kDa and a minor one at approximately 340 kDa. The immunoblot analysis of membranes chemically crosslinked with dithiobis(succinimidylpropionate) showed a broad band at 550-600 kDa suggesting that the GluR4 receptor forms a pentamer in situ. This is the first report of the cloning of hGluR4 receptor and its stable expression in a human cell line.
authors.affiliation: Allelix Biopharmaceuticals Inc., Mississauga, ON, Canada.
authors.forename: E J
authors.initials: EJ
authors.lastname: Fletcher
authors.forename: S L
authors.initials: SL
authors.lastname: Nutt
authors.forename: K H
authors.initials: KH
authors.lastname: Hoo
authors.forename: C E
authors.initials: CE
authors.lastname: Elliott
authors.forename: B
authors.initials: B
authors.lastname: Korczak
authors.forename: E A
authors.initials: EA
authors.lastname: McWhinnie
authors.forename: R K
authors.initials: RK
authors.lastname: Kamboj
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004594:Electrophysiology; D015870:Gene Expression; D018698:Glutamic Acid / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007608:Kainic Acid / Q000378:metabolism / Q000494:pharmacology; D008024:Ligands; D008969:Molecular Sequence Data; D051381:Rats; D017470:Receptors, Glutamate / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014162:Transfection; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000378:metabolism / Q000494:pharmacology
chemical_list: D018076:DNA, Complementary; D008024:Ligands; D017470:Receptors, Glutamate; D018698:Glutamic Acid; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D007608:Kainic Acid