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Abstract

G-protein-coupled receptors (GPCRs) activate heterotrimeric G-proteins (G_i-, G_s-, G_q-, 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 Gα_q to promote promiscuous receptor interactions. Starting with a human Gα_q containing 5 Gα_z residues in the C-terminal receptor recognition domain (hGα_q/z5), they evaluated agonist-stimulated calcium responses for 33 diverse GPCRs (G_i-, G_s-, and G_q-coupled) and found 20 of 33 responders. In parallel, they tested Caenorhabditis elegans Gα_q containing 5 or 9 C-terminal Gα_z residues (cGα_q/z5, cGα_q/z9). Signal detection was enhanced with cGα_q/z5 and cGα_q/z9 (yielding 25/33 and 26/33 responders, respectively). In a separate study of Gα_s-coupled receptors, the authors compared hGα_q/s5 versus hGα_q/s9, cGα_q/s9, andcGαq/s21 and observed optimal function with cGα_q/s9. Cotransfection of an engineered Gα_q “cocktail” (cGα_q/z5 plus cGα_q/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.

Keywords

G-protein-coupled receptors (GPCRs)cell-based assays fluorescent imaging plate reader (FLIPR™)Galphaq chimera

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Use of Caenorhabditis elegans Gαq Chimeras to Detect G-Protein-Coupled Receptor Signals

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