title: Ecdysone-based system for controlled inducible expression of metabotropic glutamate receptor subtypes 2, 5, and 8.
abstract: Stable and inducible expression of human metabotropic glutamate receptor types 2, 5, and 8 was achieved in HEK293 cells using the ecdysone inducible system. Treatment of the respective cell lines with ponasterone A resulted in time and concentration-dependent induction of receptor expression. In all cases, the functional activation of receptors was determined by measuring increases in intracellular calcium. The physiologically GalphaI-coupled receptors mGluR2 and mGluR8 were successfully coupled to phospholipase C activation using the chimeric G protein Galphaq/o. The pharmacological properties of recombinant receptors were characterized and proved to be similar to native receptors. Our data suggest that the ecdysone system has a number of characteristics that make it well suited for expressing mGluRs and that the combined use of this system and chimeric G proteins allows receptors to be characterized using a rapid and straightforward Ca2+ assay.
authors.affiliation: Schering-Plough Research Institute, Sun Raffaele Biomedical Science Park, Via Olgettina 58, 20132 Milano, Italy. patrick.downey@spcorp.com
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Downey
authors.forename: Gianluca
authors.initials: G
authors.lastname: Lozza
authors.forename: Roberta
authors.initials: R
authors.lastname: Petrò
authors.forename: Enrica
authors.initials: E
authors.lastname: Diodato
authors.forename: Chiara
authors.initials: C
authors.lastname: Foglia
authors.forename: Federica
authors.initials: F
authors.lastname: Bottazzoli
authors.forename: Rossella
authors.initials: R
authors.lastname: Brusa
authors.forename: Tatiana
authors.initials: T
authors.lastname: Asquini
authors.forename: Angelo
authors.initials: A
authors.lastname: Reggiani
authors.forename: Mariagrazia
authors.initials: M
authors.lastname: Grilli
mesh_terms: D002118:Calcium / Q000378:metabolism; D002460:Cell Line / Q000378:metabolism; D003001:Cloning, Molecular; D004440:Ecdysone / Q000494:pharmacology*; D004441:Ecdysterone / Q000031:analogs & derivatives / Q000494:pharmacology; D044385:GTP-Binding Protein alpha Subunits / Q000502:physiology; D015870:Gene Expression; D006801:Humans; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate / Q000096:biosynthesis*; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D044385:GTP-Binding Protein alpha Subunits; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate; C108822:metabotropic glutamate receptor 2; C111435:metabotropic glutamate receptor 8; D004440:Ecdysone; D004441:Ecdysterone; C008999:ponasterone A; D002118:Calcium