Molecular characterization of two G protein-coupled receptor splice variants as FLP2 receptors in Caenorhabditis elegans
Molecular characterization of two G protein-coupled receptor splice variants as FLP2 receptors in Caenorhabditis elegans
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DOI：
10.1016/j.bbrc.2005.03.071
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发表时间：
2005-05-13
影响因子：
3.1
通讯作者：
Schoofs, L
中科院分区：
文献类型：
--
作者：
Mertens, I;Meeusen, T;Schoofs, L
两个选择性剪接秀丽隐杆线虫G蛋白偶联受体，T19F4.1a和T19F4.1b，克隆和功能的特点。T19F4.1b受体蛋白比T19F4.1a长30个氨基酸，氨基酸组成的差异仅限于细胞内C-末端区域，表明G蛋白偶联特异性的潜在差异。将受体cDNA克隆到pcDNA 3载体中并稳定或瞬时转染中国仓鼠卵巢细胞后，用水母发光蛋白生物发光/Ca 2+测定来研究受体活化。这是第一个稳定表达线虫神经肽受体的细胞系的构建报告。我们的实验将这两种受体鉴定为flp-2前体编码的两种FMRFamide相关肽的同源受体：SPREPIRFamide（FLP 2-A）和LRGEPIRFamide（FLP 2-B）。使用截短形式的FLP 2-A和FLP 2-B进行的药理学分析表明，两种肽的活性核心均为EPIRFamide。筛选由其他flps编码的肽并没有导致受体的显著激活。与在异源表达系统中测试的其他秀丽线虫受体相反，T19F4.1a和T19F4.1b的功能激活不是温度依赖性的。在缺乏混杂G 16的细胞中筛选表明T19 F4.1 a和B都与G（q）途径相关。(c)2005年爱思唯尔公司All rights reserved.
Two alternatively spliced Caenorhabditis elegans G protein-coupled receptors, T19F4.1a and T19F4.1b, were cloned and functionally characterized. The T19F4.1b receptor protein is 30 amino acids longer than T19F4.1a, and the difference in amino acid constitution is exclusively conferred to the intracellular C-terminal region, suggesting a potential difference in G protein-coupling specificity. Following cloning of the receptor cDNAs into the pcDNA3 vector and stable or transient transfection into Chinese hamster ovary cells, the aequorin bioluminescence/Ca2+ assay was used to investigate receptor activation. This is the first report of the construction of a cell line stably expressing a C elegans neuropeptide receptor. Our experiments identified both receptors as being cognate receptors for two FMRFamide-related peptides encoded by the flp-2 precursor: SPREPIRFamide (FLP2-A) and LRGEPIRFamide (FLP2-B). Pharmacological profiling using truncated forms of FLP2-A and -B revealed that the active core of both peptides is EPIRFamide. Screening of peptides encoded by other flps did not result in a significant activation of the receptor. In contrast to other C elegans receptors tested in heterologous expression systems, the functional activation of both T19F4.1a and T19F4.1b was not temperature-dependent. Screening in cells lacking the promiscuous G,16 suggests that T19F4.1a and b are both linked to the G(q) pathway. (c) 2005 Elsevier Inc. All rights reserved.