title: cAMP assay for GPCR ligand characterization: application of BacMam expression system.
abstract: Cyclic adenosine monophosphate (cAMP) is a second messenger of many G-protein-coupled receptors. The change in cellular cAMP level has widely been used to estimate the biological activity of various GPCR-specific agents. Förster resonance energy transfer (FRET) biosensors have been around for almost 10 years and became increasingly popular for cAMP detection. Ratiometric sensitized emission assay of a FRET biosensor can easily be implemented on fluorescence plate reader platforms. For such assays a considerable amount of cells expressing the desired biosensor is needed. A method to achieve sufficient and reproducible level of cAMP biosensor protein expression with the means of BacMam transduction system is the subject of this chapter.
authors.affiliation: Institute of Chemistry, University of Tartu, Ravila 14a, Tartu, 50411, Estonia.
authors.forename: Olga
authors.initials: O
authors.lastname: Mazina
authors.forename: Anni
authors.initials: A
authors.lastname: Allikalt
authors.forename: Annika
authors.initials: A
authors.lastname: Heinloo
authors.forename: Reet
authors.initials: R
authors.lastname: Reinart-Okugbeni
authors.forename: Sergei
authors.initials: S
authors.lastname: Kopanchuk
authors.forename: Ago
authors.initials: A
authors.lastname: Rinken
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D001681:Biological Assay; D015374:Biosensing Techniques; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000032:analysis* / Q000378:metabolism; D004298:Dopamine / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D017447:Receptors, Dopamine D1 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D015290:Second Messenger Systems; D061987:Sf9 Cells; D018411:Spodoptera; D014162:Transfection
chemical_list: D008024:Ligands; D017447:Receptors, Dopamine D1; D005576:Colforsin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D004298:Dopamine