Gates Julie, Lam Geanette, Ortiz José A, Losson Régine, Thummel Carl S
Howard Hughes Medical Institute, Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112-5331, USA.
Development. 2004 Jan;131(1):25-36. doi: 10.1242/dev.00920. Epub 2003 Nov 26.
Pulses of the steroid hormone ecdysone trigger the major developmental transitions in Drosophila, including molting and puparium formation. The ecdysone signal is transduced by the EcR/USP nuclear receptor heterodimer that binds to specific response elements in the genome and directly regulates target gene transcription. We describe a novel nuclear receptor interacting protein encoded by rigor mortis (rig) that is required for ecdysone responses during larval development. rig mutants display defects in molting, delayed larval development, larval lethality, duplicated mouth parts, and defects in puparium formation--phenotypes that resemble those seen in EcR, usp, E75A and betaFTZ-F1 mutants. Although the expression of these nuclear receptor genes is essentially normal in rig mutant larvae, the ecdysone-triggered switch in E74 isoform expression is defective. rig encodes a protein with multiple WD-40 repeats and an LXXLL motif, sequences that act as specific protein-protein interaction domains. Consistent with the presence of these elements and the lethal phenotypes of rig mutants, Rig protein interacts with several Drosophila nuclear receptors in GST pull-down experiments, including EcR, USP, DHR3, SVP and betaFTZ-F1. The ligand binding domain of betaFTZ-F1 is sufficient for this interaction, which can occur in an AF-2-independent manner. Antibody stains reveal that Rig protein is present in the brain and imaginal discs of second and third instar larvae, where it is restricted to the cytoplasm. In larval salivary gland and midgut cells, however, Rig shuttles between the cytoplasm and nucleus in a spatially and temporally regulated manner, at times that correlate with the major lethal phase of rig mutants and major switches in ecdysone-regulated gene expression. Taken together, these data indicate that rig exerts essential functions during larval development through gene-specific effects on ecdysone-regulated transcription, most likely as a cofactor for one or more nuclear receptors. Furthermore, the dynamic intracellular redistribution of Rig protein suggests that it may act to refine spatial and temporal responses to ecdysone during development.
类固醇激素蜕皮激素的脉冲触发了果蝇的主要发育转变，包括蜕皮和蛹化。蜕皮激素信号由EcR/USP核受体异二聚体转导，该异二聚体与基因组中的特定反应元件结合并直接调节靶基因转录。我们描述了一种由僵硬基因（rig）编码的新型核受体相互作用蛋白，它是幼虫发育过程中蜕皮激素反应所必需的。rig突变体在蜕皮、幼虫发育延迟、幼虫致死、口器重复以及蛹化缺陷方面表现出缺陷——这些表型与EcR、usp、E75A和betaFTZ-F1突变体中所见的相似。尽管这些核受体基因在rig突变体幼虫中的表达基本正常，但蜕皮激素触发的E74异构体表达转换存在缺陷。rig编码一种具有多个WD-40重复序列和一个LXXLL基序的蛋白质，这些序列作为特定的蛋白质-蛋白质相互作用结构域。与这些元件的存在以及rig突变体的致死表型一致，在GST下拉实验中，Rig蛋白与几种果蝇核受体相互作用，包括EcR、USP、DHR3、SVP和betaFTZ-F1。betaFTZ-F1的配体结合结合结合结合域足以实现这种相互作用，并且这种相互作用可以以不依赖AF-2的方式发生。抗体染色显示，Rig蛋白存在于二龄和三龄幼虫的大脑和成虫盘中，在那里它局限于细胞质中。然而，在幼虫唾液腺和中肠细胞中，Rig以时空调节的方式在细胞质和细胞核之间穿梭，其时间与rig突变体的主要致死阶段以及蜕皮激素调节的基因表达的主要转换相关。综上所述，这些数据表明，rig在幼虫发育过程中通过对蜕皮激素调节转录的基因特异性作用发挥重要功能，很可能作为一种或多种核受体的辅因子。此外，Rig蛋白在细胞内的动态重新分布表明，它可能在发育过程中对蜕皮激素的时空反应起到优化作用。
