title: A novel cell-based assay for G-protein-coupled receptor-mediated cyclic adenosine monophosphate response element binding protein phosphorylation.
abstract: Currently, the most popular means of assessing functional activity of Gs/olf-coupled receptors is via the measurement of intracellular cyclic adenosine monophosphate (cAMP) accumulation. An additional readout is the downstream phosphorylation of cAMP response element binding protein (CREB), which gives an indication of gene transcription, the ultimate response of many G-protein-coupled receptor (GPCR) signals. Current methods of quantifying CREB phosphorylation are low throughput, and so we have designed a novel higher throughput method using the Odyssey infrared imaging system. Functional potencies of both agonists and antagonists correlate well with radioligand binding affinities determined using examples of both an endogenous (adenosine(2A) receptor in PC-12 cells) and a heterologous (human melanocortin 4 receptor in HEK-293 cells) expression system. For example, the antagonist ZM241385 demonstrates 0.23+/-0.03 nM affinity for the A(2A) receptor and has a functional potency of 0.26+/-0.04 nM determined using cAMP and 0.15+/-0.06 nM using CREB phosphorylation. These data demonstrate that this novel approach for the measurement of CREB phosphorylation is a useful tool for the assessment of GPCR activity in whole cells and is more amenable to the throughput required for the purposes of drug discovery.
authors.affiliation: Department of Neuroscience, Neurocrine Biosciences Inc., San Diego, CA 92130, USA. jselkirk@neurocrine.com
authors.forename: Julie V
authors.initials: JV
authors.lastname: Selkirk
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Nottebaum
authors.forename: Ian C
authors.initials: IC
authors.lastname: Ford
authors.forename: Mark
authors.initials: M
authors.lastname: Santos
authors.forename: Siobhan
authors.initials: S
authors.lastname: Malany
authors.forename: Alan C
authors.initials: AC
authors.lastname: Foster
authors.forename: Sandra M
authors.initials: SM
authors.lastname: Lechner
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D058908:Adenosine A2 Receptor Agonists; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006801:Humans; D066298:In Vitro Techniques; D007259:Infrared Rays; D016716:PC12 Cells; D010627:Phenethylamines / Q000494:pharmacology; D010766:Phosphorylation; D011869:Radioligand Assay; D051381:Rats; D044105:Receptor, Melanocortin, Type 4 / Q000378:metabolism; D043683:Receptors, Adenosine A2 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D058908:Adenosine A2 Receptor Agonists; D017362:Cyclic AMP Response Element-Binding Protein; C470072:MC4R protein, human; D010627:Phenethylamines; D044105:Receptor, Melanocortin, Type 4; D043683:Receptors, Adenosine A2; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; D000242:Cyclic AMP; D000241:Adenosine