Effects of human type 1alpha metabotropic glutamate receptor expression level on phosphoinositide and Ca2+ signalling in an inducible cell expression system.
Stable expression of the human type 1alpha metabotropic glutamate (mGlu 1alpha) receptor was achieved in Chinese hamster ovary cells using an isopropyl-beta-D-thiogalactoside (IPTG)-repressible expression system. Treatment of the cells with IPTG resulted in a time- and concentration-dependent induction of receptor expression. Maximal expression was obtained after treatment of the cells with 100 microM IPTG for 20 h, leading to a marked increase in receptor immunoreactivity detected by western blot, >30-fold stimulation of 3H-labelled inositol phosphate (3H-InsP) production, and a robust increase in intracellular calcium concentration in single cells after stimulation with 20 microM quisqualate. The basal level of 3H-InsP accumulation in cells induced with IPTG was increased by two- to threefold as compared with control cells; however, this basal activity was found to be dependent on glutamate released by the cells into the incubation medium. Following IPTG treatment, stable expression of the mGlu 1alpha receptor was maintained for at least 1 week. Taken together, these results clearly indicate the advantages of working with an inducible expression system when studying the biochemical and pharmacological properties of the human mGlu 1alpha receptor in transfected cells.
Hermans
E
E
Department of Cell Physiology and Pharmacology, University of Leicester, England, UK.
Young
K W
KW
Challiss
R A
RA
Nahorski
S R
SR
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D006224:Cricetinae; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D009994:Osmolar Concentration; D010716:Phosphatidylinositols / Q000502:physiology*; D016318:Quisqualic Acid / Q000494:pharmacology; D018094:Receptors, Metabotropic Glutamate / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors
D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D018094:Receptors, Metabotropic Glutamate; D007544:Isopropyl Thiogalactoside; D016318:Quisqualic Acid; D002118:Calcium