Measurement of responses from Gi-, Gs-, or Gq-coupled receptors by a multiple response element/cAMP response element-directed reporter assay

Anal Biochem. 1999 Nov 1;275(1):54-61. doi: 10.1006/abio.1999.4295.

Abstract

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.

MeSH terms

  • Base Sequence
  • Blotting, Western / methods
  • Calcium / metabolism
  • Cells, Cultured
  • Cyclic AMP / metabolism
  • Cyclic AMP Response Element-Binding Protein / physiology
  • GTP-Binding Protein alpha Subunits, Gi-Go / analysis*
  • GTP-Binding Protein alpha Subunits, Gi-Go / physiology
  • GTP-Binding Protein alpha Subunits, Gs / analysis*
  • GTP-Binding Protein alpha Subunits, Gs / physiology
  • Genes, Reporter*
  • Humans
  • Molecular Sequence Data
  • Radioligand Assay / methods
  • Response Elements / genetics
  • Signal Transduction
  • Transfection

Substances

  • Cyclic AMP Response Element-Binding Protein
  • Cyclic AMP
  • GTP-Binding Protein alpha Subunits, Gi-Go
  • GTP-Binding Protein alpha Subunits, Gs
  • Calcium