Ecdysone-based system for controlled inducible expression of metabotropic glutamate receptor subtypes 2, 5, and 8.
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.
Downey
Patrick M
PM
Schering-Plough Research Institute, Sun Raffaele Biomedical Science Park, Via Olgettina 58, 20132 Milano, Italy. patrick.downey@spcorp.com
Lozza
Gianluca
G
Petrò
Roberta
R
Diodato
Enrica
E
Foglia
Chiara
C
Bottazzoli
Federica
F
Brusa
Rossella
R
Asquini
Tatiana
T
Reggiani
Angelo
A
Grilli
Mariagrazia
M
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
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