title: Development of generic calcium imaging assay for monitoring Gi-coupled receptors and G-protein interaction.
abstract: G-protein-coupled receptors (GPCRs) are important therapeutic targets for many areas of drug research and development. Although chimeric Galpha16 proteins are valuable tools for detecting the activation of Galpha(i/o)-coupled receptors, the details of the activation process remain unclear. The authors introduce a series of chimeras that combine both Galpha16 and Galpha(i/o) (Galpha(16/o), Galpha(16/i2), and Galpha(16/i3)) into a well-established transient expression system to examine the ability of these chimeras to interact with D2 long-form (D2L) dopamine and 5-HT1A serotonin receptors. The pEC50 data obtained for known agonists were similar to results from previous studies that used other cell-based assays, thus indicating sufficient sensitivity for the assay. Moreover, quinpirole exhibited similar intrinsic activity to dopamine at the D2L receptor, whereas S-(-)-3-PPP displayed partial activity of dopamine and quinpirole in the presence of the Galpha(16/o) chimera. The potency of dopamine for D2L receptors was similar among Galpha(16/o), Galpha(16/i2), and Galpha(16/i3). In contrast, the 5-HT1A receptor exhibited a significantly preferential coupling for Galpha(16/i3) compared with Galpha(16/i2) when serotonin was used as a ligand. This finding was in close agreement with the results of previous reports. The present system could therefore be used as a rapid functional assay for high-throughput screening and deorphanization.
authors.affiliation: Department of Neurobiology and Anatomy, Graduate School of Medical Science, Nagoya City University, Nagoya, Japan. tueda@med.nagoya-cu.ac.jp
authors.forename: Takashi
authors.initials: T
authors.lastname: Ueda
authors.forename: Shinya
authors.initials: S
authors.lastname: Ugawa
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Ishida
authors.forename: Aki
authors.initials: A
authors.lastname: Hondoh
authors.forename: Shoichi
authors.initials: S
authors.lastname: Shimada
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008024:Ligands; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D011485:Protein Binding / Q000187:drug effects; D044282:Receptor, Serotonin, 5-HT1A / Q000378:metabolism; D017448:Receptors, Dopamine D2 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012701:Serotonin / Q000494:pharmacology
chemical_list: D044385:GTP-Binding Protein alpha Subunits; D008024:Ligands; D017448:Receptors, Dopamine D2; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D044282:Receptor, Serotonin, 5-HT1A; D012701:Serotonin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D002118:Calcium