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Characterization of an N‐terminal secreted domain
of the type‐1 human metabotropic glutamate receptor
produced by a mammalian cell line
Author(s) -
Selkirk Julie V.,
Challiss R. A. John,
Rhodes Andrew,
McIlhinney R. A. Jeffrey
Publication year - 2002
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.0022-3042.2001.00704.x
Subject(s) - biochemistry , biology , endoglycosidase h , chinese hamster ovary cell , protein a/g , endoplasmic reticulum , microbiology and biotechnology , peptide sequence , receptor , recombinant dna , fusion protein , golgi apparatus , gene
Abstract 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 [ 3 H]quisqualic acid showed that the protein was secreted at levels of up to 2.4 pmol/mL and the secreted protein has a K d of 5.6 ± 1.8 n m compared with 10 ± 1 n m for baby hamster kidney (BHK)‐mGlu1α 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 Ca 2+ concentration.