title: A real-time method for measuring cAMP production modulated by Gαi/o-coupled metabotropic glutamate receptors.
abstract: Group II and group III metabotropic glutamate (mGlu) receptors are G protein-coupled receptors (GPCRs) that inhibit adenylyl cyclase via activation of Gαi/o. The purpose of this study was to design a universal method that overcomes previous challenges in consistently measuring group II and group III mGlu-receptor (mGluR) activation in stably transfected systems. In Chinese hamster ovary (CHO) cells stably transfected with the GloSensor cAMP biosensor, we optimized conditions for simple and highly reproducible (<5% S.E.M.) measurements of cAMP in real time. The GloSensor cAMP biosensor is a recombinant firefly luciferase conjugated to a cAMP-binding domain, where cAMP binding promotes a conformational shift within the GloSensor protein, inducing luciferase activity; cAMP levels are positively correlated with light output resulting from the luciferase-mediated breakdown of d-luciferin. Each group II and group III mGluR was then stably transfected into the CHO-GloSensor cell line, and experimental conditions were optimized for each receptor. During assay optimization, we observed ion sensitivity of several receptors and inverse agonist activity of the antagonist, LY341495 [2-[(1S,2S)-2-carboxycyclopropyl]-3-(9H-xanthen-9-yl)-d-alanine]. Although these phenomena have been previously reported, they remain poorly understood, emphasizing the GloSensor assay as an important tool with which to study group II and group III mGlu receptors. Our results highlight many advantages of using the GloSensor method for measuring activation of group II and group III mGlu receptors, and they further suggest that corresponding methods designed to measure activation of any Gαi/o- or Gαs-coupled GPCR will be similarly advantageous.
authors.affiliation: Department of Pharmacology and Physiology, Georgetown University, Washington, DC (J.O.D., H.A.H., E.G., B.W., B.B.W., J.T.W.); and Department of Chemistry, Emory University, Atlanta, Georgia (J.O.D., E.J.M., D.C.L.).
authors.forename: John O
authors.initials: JO
authors.lastname: DiRaddo
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Miller
authors.forename: Hannah A
authors.initials: HA
authors.lastname: Hathaway
authors.forename: Ewa
authors.initials: E
authors.lastname: Grajkowska
authors.forename: Barbara
authors.initials: B
authors.lastname: Wroblewska
authors.forename: Barry B
authors.initials: BB
authors.lastname: Wolfe
authors.forename: Dennis C
authors.initials: DC
authors.lastname: Liotta
authors.forename: Jarda T
authors.initials: JT
authors.lastname: Wroblewski
mesh_terms: D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002021:Buffers; D016466:CHO Cells; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000819:agonists / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000502:physiology*; D018698:Glutamic Acid / Q000494:pharmacology; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D011869:Radioligand Assay; D018094:Receptors, Metabotropic Glutamate / Q000235:genetics / Q000502:physiology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014162:Transfection; D014966:Xanthenes / Q000494:pharmacology
chemical_list: D000596:Amino Acids; D002021:Buffers; C114624:LY 341495; D018094:Receptors, Metabotropic Glutamate; D014966:Xanthenes; D005576:Colforsin; D018698:Glutamic Acid; D000242:Cyclic AMP; D049409:Luciferases, Firefly; D019206:GTP-Binding Protein alpha Subunits, Gi-Go