Although several assays already exist for monitoring G protein-coupled receptor (GPCR) functions and for pharmacologically characterizing their ligands, the development of new non-radioactive technologies will undoubtedly be an advantage in drug discovery. In this work, an innovative and non-radioactive functional cAMP assay was validated at GPR17, a dual uracil nucleotide/cysteinyl leukotrine receptor. The new assay monitors GPCR activity through a change in the intracellular cAMP concentration using a genetically modified form of firefly luciferase containing a cAMP-binding protein moiety. Binding of cAMP induces a conformational change leading to increased light output, which allows evaluating the activity of ligands at the receptor under study. The results, expressed as EC50 or IC50 values for agonists and antagonists, respectively, showed a strong correlation with those obtained with [35S]GTPγS binding assay, thus confirming the validity of this approach in the study of new ligands for GPR17. Moreover, this method allowed confirming that GPR17 is coupled with a Gαi.

Innovative functional cAMP assay: application to the pharmacological characterization of GPR17

MARUCCI, Gabriella;BUCCIONI, Michela;DAL BEN, DIEGO;LAMBERTUCCI, Catia;THOMAS, AJIROGHENE;VOLPINI, Rosaria;CRISTALLI, Gloria
2012

Abstract

Although several assays already exist for monitoring G protein-coupled receptor (GPCR) functions and for pharmacologically characterizing their ligands, the development of new non-radioactive technologies will undoubtedly be an advantage in drug discovery. In this work, an innovative and non-radioactive functional cAMP assay was validated at GPR17, a dual uracil nucleotide/cysteinyl leukotrine receptor. The new assay monitors GPCR activity through a change in the intracellular cAMP concentration using a genetically modified form of firefly luciferase containing a cAMP-binding protein moiety. Binding of cAMP induces a conformational change leading to increased light output, which allows evaluating the activity of ligands at the receptor under study. The results, expressed as EC50 or IC50 values for agonists and antagonists, respectively, showed a strong correlation with those obtained with [35S]GTPγS binding assay, thus confirming the validity of this approach in the study of new ligands for GPR17. Moreover, this method allowed confirming that GPR17 is coupled with a Gαi.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11581/250585
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