Expression of a truncated secreted form of the mGluR3 subtype of metabotropic glutamate receptor.
In this study, 10 truncated constructs encompassing all or part of the extracellular ligand binding domain of the mGluR3 subtype of metabotropic glutamate receptor were generated, expressed in human embryonic kidney cells, and tested for secretion and binding of the high affinity agonist [(3)H]DCG-IV. The effect of inserting epitope tags into the N or C termini on cell secretion and radioligand binding was also examined. Secretion into the cell culture media was observed for 8 of the 10 truncated receptors and all secreted forms displayed high affinity agonist binding. The highest level of binding was observed in the C-terminal polyhistidine-tagged receptor truncated at serine 507. Reduction and enzymatic deglycosylation of the serine 507 truncated receptor using endoglycosidase H and PNGase F showed that the secreted receptor was a disulfide-linked dimer containing complex oligosaccharides. Pharmacological characterization demonstrated that the truncated receptor showed the same rank order of potency of agonist binding, a relatively small 2-fold decrease in agonist affinity, and a larger 10-fold decrease in affinity for the antagonist LY341495 compared to the full-length membrane-bound receptor. These results define the essential requirements for ligand binding to the extracellular domain of mGluR3 and highlight parameters important for the optimization of receptor expression in mammalian cells.
Yao
Yi
Y
Department of Pharmaceutical Sciences, Institute for Drug Research, University of Toronto, Toronto, Ont., Canada.
Koo
Joseph C P
JC
Wells
James W
JW
Hampson
David R
DR
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D006031:Glycosylation; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D018094:Receptors, Metabotropic Glutamate / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D008024:Ligands; D018094:Receptors, Metabotropic Glutamate; C102718:metabotropic glutamate receptor 3