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

Synaptic Pharmaceutical Corporation, a Lundbeck Company, Paramus, NJ

Kenneth A. Jones

Synaptic Pharmaceutical Corporation, a Lundbeck Company, Paramus, NJ

Joseph Tamm

Synaptic Pharmaceutical Corporation, a Lundbeck Company, Paramus, NJ

Huailing Zhong

Synaptic Pharmaceutical Corporation, a Lundbeck Company, Paramus, NJ

Kelli E. Smith

Synaptic Pharmaceutical Corporation, a Lundbeck Company, Paramus, NJ

Christophe Gerald

Synaptic Pharmaceutical Corporation, a Lundbeck Company, Paramus, NJ

Pierre Vaysse

Synaptic Pharmaceutical Corporation, a Lundbeck Company, Paramus, NJ

Theresa A. Branchek

Synaptic Pharmaceutical Corporation, a Lundbeck Company, Paramus, NJ

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 G_q to promote promiscuous receptor interactions. Starting with a human G_q containing 5 G_z residues in the C-terminal receptor recognition domain (hG_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 G_q containing 5 or 9 C-terminal G_z residues (cG_q/z5, cG_q/z9). Signal detection was enhanced with cG_q/z5 and cG_q/z9 (yielding 25/33 and 26/33 responders, respectively). In a separate study of G_s-coupled receptors, the authors compared hG_q/s5 versus hG_q/s9, cG_q/s9, andcGq/s21 and observed optimal function with cG_q/s9. Cotransfection of an engineered G_q "cocktail" (cG_q/z5 plus cG_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.

Key Words: G-protein-coupled receptors (GPCRs) • cell-based assays • fluorescent imaging plate reader (FLIPR^TM) • Galphaq • chimera

Journal of Biomolecular Screening, Vol. 10, No. 2, 127-136 (2005)
DOI: 10.1177/1087057104272006


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