Expression and purification of the extracellular ligand binding region of metabotropic glutamate receptor subtype 1.
Each metabotropic glutamate receptor possesses a large extracellular domain that consists of a sequence homologous to the bacterial periplasmic binding proteins and a cysteine-rich region. Previous experiments have proposed that the extracellular domain is responsible for ligand binding. However, it is currently unknown whether the extracellular ligand binding site can bind ligands without other domains of the receptor. We began by obtaining a sufficient amount of receptor protein on a baculovirus expression system. In addition to the transfer vector that encodes the entire coding region, transfer vectors that encode portions of the extracellular domain were designed. Here, we report a soluble metabotropic glutamate receptor that encodes only the extracellular domain and retains a ligand binding characteristic similar to that of the full-length receptor. The soluble receptor secreted into culture medium showed a dimerized form. Furthermore, we have succeeded in purifying it to homogeneity. Dose-response curves of agonists for the purified soluble receptor were examined. The effective concentration for half-maximal inhibition (IC50) of quisqualate for the soluble receptor was 3.8 x 10(-8) M, which was comparable to that for the full-length receptor. The rank order of inhibition of the agonists was quisqualate >> ibotenate >/= L-glutamate approximately (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid. These data demonstrate that a ligand binding event in metabotropic glutamate receptors can be dissociated from the membrane domain.
Okamoto
T
T
Department of Molecular Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565, Japan.
Sekiyama
N
N
Otsu
M
M
Shimada
Y
Y
Sato
A
A
Nakanishi
S
S
Jingami
H
H
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers; D004305:Dose-Response Relationship, Drug; D018690:Excitatory Amino Acid Agonists / Q000378:metabolism*; D018691:Excitatory Amino Acid Antagonists / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D018094:Receptors, Metabotropic Glutamate / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
D017931:DNA Primers; D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D008024:Ligands; D018094:Receptors, Metabotropic Glutamate; D011994:Recombinant Proteins