Phenotypic evaluation of constitutive GPCR/G-protein signaling in zebrafish embryos and larvae
Phenotypic evaluation of constitutive GPCR/G-protein signaling in zebrafish embryos and larvae
复制标题
斑马鱼胚胎和幼鱼组成型 GPCR/G 蛋白信号传导的表型评估
DOI：
10.1016/j.bbrc.2022.02.098
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发表时间：
2022
影响因子：
3.1
通讯作者：
Inoue Asuka
中科院分区：
文献类型：
--
作者：
Shibata Takeaki;Kawakami Kouki;Kawana Hiroki;Aoki Junken;Inoue Asuka
通过G蛋白偶联受体（GPCR）的信号转导起始通常在细胞外配体结合后开始。一些致癌GPCR突变体能够在没有配体刺激的情况下诱导G蛋白信号传导，因此表现为组成型活性受体。在动物模型中评估组成型活性突变的致病能力需要数月的耗时实验，这阻碍了研究进展。在这里，使用斑马鱼胚胎瞬时表达与组成型活性突变通过mRNA显微注射，我们描述了G蛋白亚型特异性表型，可以在几天内进行评估。外源性表达的半胱氨酰白三烯受体II型（CysLT2R）与致癌的L1293.43Q突变的mRNA注射到受精胚胎诱导发育停滞期间epiboly和最终的胚胎死亡，这是抑制治疗与Gq抑制剂，YM-254890。具有组成型活性G α q突变体的胚胎表现出类似的表型。有趣的是，组成型活性G α s、G α i和G α 13突变体的表达诱导了不同的表型。因此，这些表型可以作为有用的指标，用于快速体内评价GPCR和G蛋白突变体的信号活性。
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 Gαqmutant exhibited an analogous phenotype. Interestingly, expression of constitutively active Gαs, Gαi, and Gα13mutants induced distinct phenotypes. These phenotypes may thus serve as useful indicators for rapidin vivoevaluation of signaling activity of GPCR and G-protein mutants.