Establishment of Sf9 Transformants Constitutively Expressing PBAN Receptor Variants: Application to Functional Evaluation.
Establishment of Sf9 Transformants Constitutively Expressing PBAN Receptor Variants: Application to Functional Evaluation.
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DOI：
10.3389/fendo.2012.00056
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发表时间：
2012
影响因子：
5.2
通讯作者：
Matsumoto S
中科院分区：
文献类型：
--
作者：
Lee JM;Hull JJ;Kawai T;Tsuneizumi K;Kurihara M;Tanokura M;Nagata K;Nagasawa H;Matsumoto S
为了促进信息素生物合成激活神经肽受体（PBANR）的功能和调节的进一步评估，我们产生了培养的昆虫细胞系组成型表达绿色荧光蛋白嵌合体的最近确定的家蚕PBANR（BommoPBANR）和Pseudaletia separata PBANR（PsesePBANR）的变体。荧光嵌合体包括BommoPBANR-A、-B和-C变体以及PsesePBANR-B和-C变体。还产生了表达非嵌合BommoPBANR-B和-C变体的细胞系。使用共聚焦激光显微镜对这些转化的细胞系进行功能评价，结果显示，罗丹明红标记的PBAN衍生物（RR-C10 PBANR 2K）与所有相应的PBANR变体特异性共定位在质膜上。在所有细胞系中观察到结合后30分钟荧光RR-C10 PBANR 2K配体的几乎完全内化，除了表达BommoPBANR-A变体的那些，其中配体/受体复合物保持在质膜上。荧光Ca 2+成像进一步显示，BommoPBANR-A细胞系在多个RR-C10 PBANR 2K浓度（包括10 μM）下表现出显著不同的Ca 2+动员动力学。这些观察结果表明BommoPBANR-A变体与BommoPBANR-B和-C变体在受体调节和下游效应分子活化方面存在明显的功能差异。我们还发现，与以前的报道相反，在细胞系中稳定表达这些变体的BommoPBANR-B和BommoPBANR-C的配体诱导的内化发生在细胞外Ca 2+不存在的情况下。
To facilitate further evaluation of pheromone biosynthesis activating neuropeptide receptor (PBANR) functionality and regulation, we generated cultured insect cell lines constitutively expressing green fluorescent protein chimeras of the recently identified Bombyx mori PBANR (BommoPBANR) and Pseudaletia separata PBANR (PsesePBANR) variants. Fluorescent chimeras included the BommoPBANR-A, -B, and -C variants and the PsesePBANR-B and -C variants. Cell lines expressing non-chimeric BommoPBANR-B and -C variants were also generated. Functional evaluation of these transformed cell lines using confocal laser microscopy revealed that a Rhodamine Red-labeled PBAN derivative (RR-C10PBANR2K) specifically co-localized with all of the respective PBANR variants at the plasma membrane. Near complete internalization of the fluorescent RR-C10PBANR2K ligand 30 min after binding was observed in all cell lines except those expressing the BommoPBANR-A variant, in which the ligand/receptor complex remained at the plasma membrane. Fluorescent Ca2+ imaging further showed that the BommoPBANR-A cell line exhibited drastically different Ca2+ mobilization kinetics at a number of RR-C10PBANR2K concentrations including 10 μM. These observations demonstrate a clear functional difference between the BommoPBANR-A variant and the BommoPBANR-B and -C variants in terms of receptor regulation and activation of downstream effector molecules. We also found that, contrary to previous reports, ligand-induced internalization of BommoPBANR-B and BommoPBANR-C in cell lines stably expressing these variants occurred in the absence of extracellular Ca2+.