Characterization of an N-terminal secreted domain of the type-1 human metabotropic glutamate receptor produced by a mammalian cell line.
A Chinese hamster ovary cell line has been established which secretes the N-terminal domain of human mGlu1 receptor. The secreted protein has been modified to contain a C-terminal hexa-histidine tag and can be purified by metal-chelate chromatography to yield a protein with an apparent molecular weight of 130 kDa. Following treatment with dithiothreitol the apparent molecular weight is reduced to 75 kDa showing that the protein is a disulphide-bonded dimer. N-terminal protein sequencing of both the reduced and unreduced forms of the protein yielded identical sequences, confirming that they were derived from the same protein, and identifying the site of signal-peptide cleavage of the receptor as residue 32 in the predicted amino acid sequence. Endoglycosidase treatment of the secreted and intracellular forms of the protein showed that the latter was present as an endoglycosidase H-sensitive dimer, indicating that dimerization is taking place in the endoplasmic reticulum. Characterization of the binding of [3H]quisqualic acid showed that the protein was secreted at levels of up to 2.4 pmol/mL and the secreted protein has a Kd of 5.6 +/- 1.8 nm compared with 10 +/- 1 nm for baby hamster kidney (BHK)-mGlu1alpha receptor-expressing cell membranes. The secreted protein maintained a pharmacological profile similar to that of the native receptor and the binding of glutamate and quisqualate were unaffected by changes in Ca2+ concentration.
Selkirk
Julie V
JV
Department of Cell Physiology and Pharmacology, University of Leicester, UK.
Challiss
R A John
RA
Rhodes
Andrew
A
McIlhinney
R A Jeffrey
RA
D000595:Amino Acid Sequence; D000818:Animals; D001565:Benzoates; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D019281:Dimerization; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016318:Quisqualic Acid / Q000494:pharmacology; D018094:Receptors, Metabotropic Glutamate / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014316:Tritium
D001565:Benzoates; D018691:Excitatory Amino Acid Antagonists; D010446:Peptide Fragments; D018094:Receptors, Metabotropic Glutamate; C104077:metabotropic glutamate receptor type 1; D014316:Tritium; C079694:alpha-methyl-4-carboxyphenylglycine; D016318:Quisqualic Acid; D006026:Glycoside Hydrolases; D019204:GTP-Binding Proteins; D005998:Glycine