Use of Caenorhabditis elegans Gαq chimeras to detect G-protein-coupled receptor signals

Abstract

G-protein-coupled receptors (GPCRs) activate heterotrimeric G-proteins (Gi-, Gs-, Gq-, or G12-like) to generate specific intracellular responses, depending on the receptor/G-protein coupling. The aim was to enable a majority of GPCRs to generate a predetermined output by signaling through a single G-protein-supported pathway. The authors focused on calcium responses as the output, then engineered Gαq to promote promiscuous receptor interactions. Starting with a human Gαq containing 5 Gαz residues in the C-terminal receptor recognition domain (hGαq/z5), they evaluated agonist-stimulated calcium responses for 33 diverse GPCRs (G i-, Gs-, and Gq-coupled) and found 20 of 33 responders. In parallel, they tested Caenorhabditis elegans Gαq containing 5 or 9 C-terminal Gαz residues (cGα q/z5, cGαq/z9). Signal detection was enhanced with cGαq/z5 and cGαq/z9 (yielding 25/33 and 26/33 responders, respectively). In a separate study of Gαs-coupled receptors, the authors compared hGαq/s5 versus hGαq/s9, cGαq/s9, and cGα q/s21 and observed optimal function with cGαq/s9. Cotransfection of an engineered Gαq "cocktail" (cGαq/s5 plus cGαq/s9) provided a powerful and efficient screening platform. When the chimeras included N-terminal myristoylation sites (to promote membrane localization), calcium responses were sustained or improved, depending on the receptor. This approach toward a "universal functional assay" is particularly useful for orphan GPCRs whose signaling pathways are unknown. © 2005 The Society for Biomolecular Screening.