Measurement of responses from Gi-, Gs-, or Gq-coupled receptors by a multiple response element/cAMP response element-directed reporter assay.
We have established a rapid, sensitive, high-throughput assay that requires one assay condition to detect agonist effects from Gi-, Gs-, and Gq-coupled receptors. We utilized a vector containing a promoter with three multiple response elements, the vasoactive intestinal peptide promoter and a cAMP response element controlling the transcription of the luciferase gene. An adrenergic agonist, para-aminoclonidine, inhibited forskolin-stimulated luciferase expression when cells were cotransfected with the Gi-coupled alpha(2)-C adrenergic receptor and the MRE/CRE reporter vector. Further, we demonstrate that gastrin-releasing peptide, which activates a Gq-coupled GRP receptor, isoproterenol, which activates a Gs-coupled beta-adrenergic receptor, calcium ionophores, and phorbol 12-myristate 13-acetate, a stimulator of protein kinase C, can mediate increases in luciferase expression in the presence of forskolin but not in its absence. The effect at Gi-coupled receptor activation correlates with the phosphorylation of the CRE binding protein (CREB); however, the mechanisms mediating the responses to Gq- and Gs-coupled receptors are more complex. We demonstrate that this assay is useful for pharmacological analysis of both agonists and antagonists and has the potential to associate orphan G-protein-coupled receptors with their corresponding ligands.
Fitzgerald
L R
LR
Department of Renal Pharmacology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, Pennsylvania 19406, USA.
Mannan
I J
IJ
Dytko
G M
GM
Wu
H L
HL
Nambi
P
P
D001483:Base Sequence; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000032:analysis* / Q000502:physiology; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000032:analysis* / Q000502:physiology; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D011869:Radioligand Assay / Q000379:methods; D020218:Response Elements / Q000235:genetics; D015398:Signal Transduction; D014162:Transfection
D017362:Cyclic AMP Response Element-Binding Protein; D000242:Cyclic AMP; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs; D002118:Calcium