Hull J Joe, Lee Jae Min, Kajigaya Ryosuke, Matsumoto Shogo
Molecular Entomology Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
J Biol Chem. 2009 Nov 6;284(45):31200-13. doi: 10.1074/jbc.M109.044198. Epub 2009 Sep 9.
Sex pheromone production in the pheromone gland (PG) of the silkmoth, Bombyx mori, is mediated by store-operated channels (SOCs) acting downstream of pheromone biosynthesis activating neuropeptide (PBAN) binding. Although recent studies have implicated STIM1 and Orai1 as essential components of SOCs, little is known about the molecular nature of the SOCs involved in sex pheromone production. In this study we cloned silkmoth homologs of STIM1 and Orai1 and sought to determine whether they comprise the PG SOC pathway. BmSTIM1 is expressed in multiple tissues and, in the PG, is encoded by two transcripts of differing size. BmOrai1A and BmOrai1B, which are identical except for a 37-residue N-terminal truncation in BmOrai1B, arise from alternative splicing of the bmorai1 locus and are expressed as independent transcripts in various tissues. In the PG, only BmOrai1B is actively transcribed. Fluorescent chimeras demonstrated that BmSTIM1 expression is restricted to the endoplasmic reticulum, whereas both BmOrai1A and BmOrai1B localize to the cell surface. In Ca(2+)-free medium, thapsigargin-mediated depletion of endoplasmic reticulum Ca(2+) stores resulted in redistribution of BmSTIM1 to the plasma membrane, but only when the BmOrai1 homologs were also overexpressed. Translocation was dependent on the BmSTIM1 C terminus "CRAC activation domain." Ala mutation of Lys(380), Lys(383), Lys(384), Arg(382), and Arg(385) suggests that translocation involves electrostatic interactions. Translocation was also seen following PBAN stimulation in cells co-expressing BmSTIM1, BmOrai1B, and the PBAN receptor. In vivo RNA interference-mediated knockdown of BmSTIM1 and BmOrai1 significantly reduced sex pheromone production without affecting cell viability.
家蚕性信息素腺体（PG）中的性信息素产生是由位于性信息素生物合成激活神经肽（PBAN）结合下游的储存操纵通道（SOCs）介导的。尽管最近的研究表明基质相互作用分子1（STIM1）和Orai1是SOCs的重要组成部分，但对于参与性信息素产生的SOCs的分子本质知之甚少。在本研究中，我们克隆了家蚕STIM1和Orai1的同源物，并试图确定它们是否构成PG的SOC途径。BmSTIM1在多个组织中表达，在PG中，由两个大小不同的转录本编码。BmOrai1A和BmOrai1B除了BmOrai1B的N端有37个氨基酸的截短外完全相同，它们来自bmorai1基因座的可变剪接，并在各种组织中作为独立的转录本表达。在PG中，只有BmOrai1B被积极转录。荧光嵌合体表明BmSTIM1的表达局限于内质网，而BmOrai1A和BmOrai1B都定位于细胞表面。在无钙培养基中，毒胡萝卜素介导的内质网钙库耗竭导致BmSTIM1重新分布到质膜，但只有在家蚕Orai1同源物也过表达时才会发生。转位依赖于BmSTIM1的C端“CRAC激活结构域”。赖氨酸（380）、赖氨酸（383）、赖氨酸（384）、精氨酸（382）和精氨酸（385）的丙氨酸突变表明转位涉及静电相互作用。在共表达BmSTIM1、BmOrai1B和PBAN受体的细胞中，PBAN刺激后也观察到了转位。体内RNA干扰介导的BmSTIM1和BmOrai1敲低显著降低了性信息素的产生，而不影响细胞活力。
