Phenotypic evaluation of constitutive GPCR/G-protein signaling in zebrafish embryos and larvae

• Published in: Biochemical and biophysical research communications Vol. 602; pp. 70 - 76
• Main Authors: Shibata, Takeaki, Kawakami, Kouki, Kawana, Hiroki, Aoki, Junken, Inoue, Asuka
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 30.04.2022
• Subjects: Animals; Carcinogenesis; Constitutive activity; CysLT2R; G protein; GPCR; GTP-Binding Protein alpha Subunits, Gq-G11 - metabolism; L129Q; Larva - metabolism; Ligands; Phenotype; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; RNA, Messenger; Zebrafish; Zebrafish - genetics; L129Q; DMSO; Constitutive activity; Zebrafish; PKC; PLCβ; CysLT2R; MFI; PEI; GPCR; TGFα; dpf; WT; G protein; hpf; CysLTR
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2022.02.098

Signal transduction initiation by G-protein-coupled receptors (GPCRs) normally begins upon extracellular ligand binding. Some oncogenic GPCR mutants are capable of inducing G-protein signaling without ligand stimulation, thus behaving as constitutively active receptors. Evaluation of disease-causing capacity of constitutively active mutations in animal models requires months of time-consuming experiments, which hampers research progress. Here, using zebrafish embryos transiently expressing with constitutively active mutations via mRNA microinjection, we describe G-protein-subtype-specific phenotypes that can be evaluated over several days. Exogenous expression of the cysteinyl leukotriene receptor type II (CysLT2R) with an oncogenic L1293.43Q mutation by mRNA injection into a fertilized embryo induced developmental arrest during epiboly and eventual embryonic lethality, which were suppressed by treatment with the Gq inhibitor, YM-254890. Embryos with a constitutively active Gαq mutant exhibited an analogous phenotype. Interestingly, expression of constitutively active Gαs, Gαi, and Gα13 mutants induced distinct phenotypes. These phenotypes may thus serve as useful indicators for rapid in vivo evaluation of signaling activity of GPCR and G-protein mutants. •L129Q CysLT2R constitutively activates Gq but not other G proteins (Gs, Gi, and G13).•L129Q CysLT2R- and Q209L Gαq-expressing embryos die during epiboly.•The other Gα subunits with the activating QL mutation causes distinct phenotypes (failed epiboly initiation, cyclopia and heart edema in Gα13, Gαs and Gαi1, respectively).•Treatment with the Gq inhibitor YM-254890 rescues L129Q CysLT2R-induced lethality in zebrafish embryos.