Cloning and characterization of the pheromone biosynthesis activating neuropeptide receptor from the silkmoth, Bombyx mori -: Significance of the carboxyl terminus in receptor internalization
Cloning and characterization of the pheromone biosynthesis activating neuropeptide receptor from the silkmoth, Bombyx mori -: Significance of the carboxyl terminus in receptor internalization
复制标题
DOI：
10.1074/jbc.m408142200
复制
发表时间：
2004-12-03
影响因子：
4.8
通讯作者：
Matsumoto, S
中科院分区：
文献类型：
--
作者：
Hull, JJ;Ohnishi, A;Matsumoto, S
在大多数鳞翅目中，信息素生物合成是由一种称为信息素生物合成激活神经肽（PBAN）的神经肽调节的。尽管人们对 PBAN 的细胞靶标了解很多，但 PBAN 受体 (PBANR) 的识别和功能表征已被证明是难以捉摸的。鉴于 PBAN 和神经调节肽 U 的活性 C 端区域之间的序列相似性，推测它们各自的受体在结构上也可能相似（Park, Y.、Kim, Y. J. 和 Adams, M. E. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11423 - 11428）。因此，利用从昆虫神经调节肽U受体同源物的保守区域构建的引物，从家蚕信息素腺cDNA文库中扩增出编码46-kDa G蛋白偶联受体的全长2780个核苷酸的克隆。组织分布分析表明，受体转录物是信息素腺特异的，在羽化前一天它会经历显着的上调。当在 Sf9 细胞中瞬时表达时，家蚕 PBANR 通过以剂量依赖性方式动员细胞外钙来响应 PBAN。共聚焦显微镜研究证明了增强型绿色荧光蛋白标记的家蚕 PBANR 对 PBAN 的特异性，并表明 PBAN 诱导 PBANR 的内化。 PBAN 综合体。内化的快速发生是由克隆的玉米夜蛾 PBANR 中不存在的 67 个氨基酸 C 端延伸介导的，这表明该物种中的受体内化可能利用了不同的机制。从这些结果中，我们得出结论，克隆的受体基因编码 B. mori PBANR，并且它在结构和功能上都与 H. zea PBANR 不同。
In most Lepidoptera, pheromone biosynthesis is regulated by a neuropeptide termed pheromone biosynthesis activating neuropeptide ( PBAN). Although much is known about the cellular targets of PBAN, identification and functional characterization of the PBAN receptor (PBANR) has proven to be elusive. Given the sequence similarity between the active C-terminal regions of PBAN and neuromedin U, it was hypothesized that their respective receptors might also be similar in structure ( Park, Y., Kim, Y. J., and Adams, M. E. ( 2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11423 - 11428). Consequently, utilizing primers constructed from the conserved regions of insect neuromedin U receptor homologues, a full-length 2780-nucleotide clone encoding a 46-kDa G protein-coupled receptor was amplified from a Bombyx mori pheromone gland cDNA library. Tissue distribution analyses revealed that the receptor transcript is specific to the pheromone gland where it undergoes significant up-regulation in the day preceding eclosion. When transiently expressed in Sf9 cells, the B. mori PBANR responds to PBAN by mobilizing extracellular calcium in a dose-dependent manner. Confocal microscopic studies demonstrated the specificity of enhanced green fluorescent protein-tagged B. mori PBANR for PBAN and showed that PBAN induces internalization of the PBANR . PBAN complex. The rapid onset of internalization is mediated by a 67-amino acid C-terminal extension absent in the cloned Helicoverpa zea PBANR, which suggests that receptor internalization in that species likely utilizes a different mechanism. From these results, we have concluded that the cloned receptor gene encodes the B. mori PBANR and that it is both structurally and functionally distinct from the H. zea PBANR.