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Phenotypic evaluation of constitutive GPCR/G-protein signaling in zebrafish embryos and larvae
Takeaki Shibata1, Kouki Kawakami2, Hiroki Kawana3
1Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3, Aoba, Aramaki, Aoba-ku, Sendai, Miyagi, 980-8578, Japan; Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Abstract:
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.
Insights
Zebrafish embryos enable rapid, G-protein-subtype-specific evaluation of constitutively active G-protein-coupled receptor (GPCR) mutants. This method accelerates research by identifying disease-causing mutations in days, not months.
Area of Science:
- Molecular Biology
- Developmental Biology
- Pharmacology
Background:
- G-protein-coupled receptors (GPCRs) initiate signal transduction upon ligand binding.
- Constitutively active GPCR mutants signal without ligands, contributing to diseases like cancer.
- Current methods for evaluating these mutants in animal models are lengthy and inefficient.
Purpose of the Study:
- To establish a rapid zebrafish embryo model for assessing G-protein-subtype-specific phenotypes of constitutively active GPCR and G-protein mutants.
- To demonstrate the utility of this model for evaluating oncogenic mutations and their inhibitors.
Main Methods:
- Transient expression of constitutively active GPCR (CysLT2R L129Q) and G-protein alpha subunit mutants in zebrafish embryos via mRNA microinjection.
- Observation and evaluation of G-protein-subtype-specific developmental phenotypes, including developmental arrest and embryonic lethality.
- Pharmacological inhibition using YM-254890 (Gq inhibitor) to validate observed phenotypes.
Main Results:
- Expression of oncogenic CysLT2R L129Q induced developmental arrest and lethality, phenotypically similar to constitutively active Gαq.
- These phenotypes were suppressed by the Gq inhibitor YM-254890.
- Distinct phenotypes were observed for constitutively active Gαs, Gαi, and Gα13 mutants, indicating G-protein subtype specificity.
Conclusions:
- Zebrafish embryos provide a rapid, cost-effective in vivo system for evaluating the signaling activity of GPCR and G-protein mutants.
- This model allows for G-protein subtype-specific assessment of mutant phenotypes and potential therapeutic interventions.
- The observed phenotypes serve as valuable indicators for the rapid assessment of disease-causing mutations.
