Properties of a recombinant kainate receptor expressed in baculovirus-infected insect cells.
GluR6 is a glutamate receptor of the kainate subtype that is expressed in the mammalian central nervous system. The cDNA coding for the rat brain receptor was subcloned into a baculovirus expression vector and the purified recombinant virus was used to infect Spodoptera frugiperda (Sf9) insect cells. The pharmacological profile and the status of several post-translational modifications of the GluR6 protein were analyzed. Saturation analyses of [3H]kainic acid binding demonstrated that GluR6 expressed in Sf9 cell membranes bound [3H]kainic acid at a single high affinity site with a dissociation constant of 12 nM. Competition studies indicated the inhibitory potencies of various excitatory amino acids, including the potent neurotoxin domoic acid, were comparable to those observed in mammalian brain tissue. Immunoblots of infected Sf9 cell membranes using an anti-GluR6 antibody revealed two immunoreactive bands. Enzymatic deglycosylation indicated that the higher molecular weight form corresponded to the glycosylated receptor whereas the lower molecular weight form corresponded to the unglycosylated protein. The phosphorylation of GluR6 by cyclic AMP-dependent and cyclic GMP-dependent protein kinase was examined in partially purified preparations of the receptor. GluR6 was phosphorylated by cyclic AMP but not by cyclic GMP-dependent protein kinase in vitro. These results indicate that GluR6 expressed in Sf9 cells has similar pharmacological properties and is processed post-translationally in a manner similar to GluR6 expressed in mammalian cells and tissues. The ease of production and the high levels of expression in baculovirus-infected insect cells relative to other expression systems should facilitate further biochemical and pharmacological characterization of this receptor.
Taverna
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FA
Faculty of Pharmacy, University of Toronto, Ontario, Canada.
Hampson
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DR
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001921:Brain / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005822:Genetic Vectors; D015151:Immunoblotting; D009036:Moths / Q000235:genetics; D010766:Phosphorylation; D011869:Radioligand Assay; D051381:Rats; D018092:Kainic Acid Receptors / Q000235:genetics / Q000502:physiology*; D011995:Recombination, Genetic
D018076:DNA, Complementary; D018092:Kainic Acid Receptors