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

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 L129^3.43Q 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 Gα_q mutant exhibited an analogous phenotype. Interestingly, expression of constitutively active Gα_s, Gα_i, 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.

Keywords: Constitutive activity; CysLT(2)R; G protein; GPCR; L129Q; Zebrafish.