Expression and characterization of the alpha 2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-selective glutamate receptor channel in a baculovirus system.
Using a baculovirus expression vector system, the alpha 2 subunit of the mouse alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-selective glutamate receptor (GluR) channel was expressed in Spodoptera frugiperda insect cells. Immunoblotting using the antibody made to the synthetic peptide corresponding to the C-terminus of GluR alpha 2 and [35S]methionine/[35S]cysteine metabolic radiolabeling revealed the major 102-kDa and the minor 98-kDa protein bands. Metabolic radiolabeling with tunicamycin suggested that the two bands correspond to glycosylated and unglycosylated forms, respectively. The recombinant GluR alpha 2 proteins expressed in insect cells were also identified by immunofluorescence staining. The results of [3H]AMPA binding assay using whole cells suggested that, in infected Sf21 cells, binding sites of the GluR alpha 2 proteins were possibly located on the extracellular side. Scatchard analysis of AMPA binding showed the following parameters: Kd = 16 nM, Bmax = 1.9 x 10(5) binding sites per cell or 1 pmol/mg protein in the total particulate fraction. The ligand binding characteristics of the receptors expressed in insect cells were examined. From the effect of various agonists on [3H]AMPA binding of the receptors expressed in insect cells, the rank order potency of agonists was quisqualate > AMPA > L-glutamate > kainate. Thus, the baculovirus-insect cell expression system provides high-efficiency expression of the receptor sufficient to permit structural and functional analyses.
Hattori
S
S
Department of Bacteriology, Yokohama City University School of Medicine, Japan.
Okuda
K
K
Hamajima
K
K
Sakimura
K
K
Mishina
M
M
Kawamoto
S
S
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D015151:Immunoblotting; D007313:Insecta / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects; D007700:Kinetics; D008024:Ligands; D008566:Membranes / Q000378:metabolism; D011869:Radioligand Assay; D018091:Receptors, AMPA / Q000096:biosynthesis* / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera / Q000235:genetics
D003062:Codon; D008024:Ligands; D018091:Receptors, AMPA; D011994:Recombinant Proteins