title: Use of Caenorhabditis elegans G{alpha}q chimeras to detect G-protein-coupled receptor signals.
abstract: G-protein-coupled receptors (GPCRs) activate heterotrimeric G-proteins (G(i)-, G(s)-, G(q)-, or G(12)-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 Galpha(q) to promote promiscuous receptor interactions. Starting with a human Galpha(q) containing 5 Galpha(z) residues in the C-terminal receptor recognition domain (hGalpha(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 Galpha(q) containing 5 or 9 C-terminal Galpha(z) residues (cGalpha(q/z5), cGalpha(q/z9)). Signal detection was enhanced with cGalpha(q/z5) and cGalpha(q/z9) (yielding 25/33 and 26/33 responders, respectively). In a separate study of Galpha(s)-coupled receptors, the authors compared hGalpha(q/s5) versus hGalpha(q/s9), cGalpha(q/s9), andcGalphaq/s21 and observed optimal function with cGalpha(q/s9). Cotransfection of an engineered Galpha(q) "cocktail" (cGalpha(q/z5) plus cGalpha(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.
authors.affiliation: Synaptic Pharmaceutical Corporation, a Lundbeck Company, Paramus, NJ 07652, USA. mmw@lundbeck.com
authors.forename: Mary W
authors.initials: MW
authors.lastname: Walker
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Jones
authors.forename: Joseph
authors.initials: J
authors.lastname: Tamm
authors.forename: Huailing
authors.initials: H
authors.lastname: Zhong
authors.forename: Kelli E
authors.initials: KE
authors.lastname: Smith
authors.forename: Christophe
authors.initials: C
authors.lastname: Gerald
authors.forename: Pierre
authors.initials: P
authors.lastname: Vaysse
authors.forename: Theresa A
authors.initials: TA
authors.lastname: Branchek
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D017173:Caenorhabditis elegans; D065101:Chloride Channel Agonists; D018118:Chloride Channels / Q000378:metabolism; D002522:Chlorocebus aethiops; D004594:Electrophysiology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D015398:Signal Transduction; D014162:Transfection; D014982:Xenopus laevis
chemical_list: D065101:Chloride Channel Agonists; D018118:Chloride Channels; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11