PMID: 11905408Mar 22, 2002Paper

Characterization of an N-terminal secreted domain of the type-1 human metabotropic glutamate receptor produced by a mammalian cell line

Journal of Neurochemistry
Julie V SelkirkR A Jeffrey McIlhinney

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 [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 ...Continue Reading

Citations

Aug 19, 2011·Molecular Biotechnology·Geraldo Santana MagalhãesAna Maria Moura da Silva
Nov 13, 2007·Journal of Neurochemistry·Rosaria RemelliR A Jeffrey McIlhinney
May 6, 2006·Biochemical and Biophysical Research Communications·Donghui Kuang, David R Hampson
May 14, 2010·Journal of Neurophysiology·Donald Beqollari, Paul J Kammermeier
Dec 30, 2008·Pharmacological Reviews·Francesco FerragutiFerdinando Nicoletti
Sep 16, 2020·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Da-Ha ParkYoung Ho Suh
Oct 16, 2004·The Biochemical Journal·Daniela DeriuMarkus G Grütter

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