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-couple d receptors, We utilized a vector containing a promoter with three multiple response elements, the vasoactive intestinal peptide promoter and a cAMP r esponse element controlling the transcription of the luciferase gene. An ad renergic agonist, para-aminoclonidine, inhibited forskolin-stimulated lucif erase expression when cells were cotransfected with the Gi-coupled alpha(2) -C adrenergic receptor and the MRE/CRE reporter vector. Further, we demonst rate that gastrin-releasing peptide, which activates a Gq-coupled GRP recep tor, isoproterenol, which activates a Gs-coupled beta-adrenergic receptor, calcium ionophores, and phorbol 12-myristate 13-acetate, a stimulator of pr otein kinase C, can mediate increases in luciferase expression in the prese nce 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-couple d receptors are more complex. We demonstrate that this assay is useful for pharmacological analysis of both agonists and antagonists and has the poten tial do associate orphan G-protein-coupled receptors with their correspondi ng ligands. (C) 1999 Academic Press.