CELL INTERACTIONS ORGANIZING THE INSECT SEGMENT

描述：琼·胡珀博士的这份修改后的提案解决了 刺猬信号在果蝇节段模式形成中的作用机制。Dr。 胡珀在上一次拨款期间的研究(由第一个 奖)中涉及的相关细胞间信号机制 片段极性基因的功能。这项研究，以及许多 其他实验室已经描述了两个信号中的不同基因相互作用 节段规范所需的路径，无翼和刺猬的路径。 无翅，由每个副节段后缘的细胞分泌， 刺猬，从前缘的细胞分泌，产生离散的 影响取决于响应的细胞是被其中一个还是 其他蛋白质或两者都有。这些影响变得明显起来 此外，通过在这些细胞中产生转录 积聚的因素(EN；每个副段的前缘)或松散的配对 (SLP；每个副节段的后间隔。在做这些工作的同时 通道近年来异常活跃，有很大的 在了解这些途径的分子机制方面存在差距 指定图案。许多相关基因的确切作用点 产品还不清楚，而且可能并不是所有的路径成员都是 为人所知。胡珀博士对识别受体特别感兴趣。 Hedgehog蛋白和理解细胞内对 配体-受体相互作用。 胡珀博士已经克隆并鉴定了平滑(SMO)基因，该基因具有 突变体的表型与HH突变体非常相似。以下情况需要SMO 适当的HH函数，明显作用于HH的下游和上游 中断肘(Ci)，一种似乎介导HH的转录因子 发信号。SMO的序列表明，该蛋白质是一个7- 与Frizzed相关的跨膜受体蛋白，但在相当程度上表达 明显的图案。胡珀博士假设SMO编码HH受体， 她还提出了一些实验来验证这一假设。胡珀博士还 一直在调查Ci。Ci的零表型类似于Hh，并且 在某些功能上与工作组不同。在存在空的Ci等位基因的情况下， GSB和PTC在第10期晚期和早期的外胚层中不表达 11期胚胎，这是一种模仿突变HH的效果。占主导地位的收益 -功能Ci等位基因Cid，消除了对HH信号的需要。词坛风云 蛋白质是一种序列特异性的锌指类DNA结合蛋白。 胡珀博士的团队已经证实，CI可以激活HH反应的WG 启动子在施耐德-2细胞中瞬时转染后显示 激活所需的Ci结合位点。当Ci和Hh是 在施耐德细胞中共表达，WG中Ci的活性 启动子-荧光素酶的表达增强，并与HH共表达 Patted(已知的刺猬信号抑制因子)也抑制Ci WG启动子的激活。事实证明，SMO是在水平上表达的 类似于胚胎在这个细胞系中的表达，所以如果它确实是一个 HH受体，它以足够的量存在于这一功能中。CI、PTC、 和HH的表达水平要低得多。最后，胡珀博士发现 在显性PTC表型修饰物的遗传筛选中，有两个新的 SMO的等位基因显示与PTC的等位基因特异性相互作用， 暗示在功能上和物理上有密切的互动 这两种基因产物。目前的提案集中于SMO及其 功能。具体目标是：(1)确定是否存在 用结合实验研究SMO与HH的物理相互作用 细胞与免疫共沉淀法和交联法 细胞和体内。(2)研究异三聚体G 蛋白质参与了HH信号转导。这一假设是基于 事实上，从概念上翻译的SMO的许多结构特征 异源三聚体G蛋白连锁受体共有的基因产物 蛋白质。用于G蛋白研究的一系列药理学测试将 被利用，利用细胞培养系统。(3)关系 将考察PTC和SMO之间的关系。将确定PTC是否 Smo下游的行为，使用成分激活的Ga来确定 PTC效应是否可以调制。还将确定是否 PTC可以调节HH结合位点的数量或亲和力，这一效果 会表明PTC直接对Smo采取了行动。(4)将确定 Smo是否受cAMP依赖的蛋白激酶(PKA)调节。PKA IS 已知是HH信号的负调节因子，并在或 与PTC并行。由于PTC没有假定的PKA磷酸化位点， 虽然Smo有六个一致的位点，但胡珀博士假设受体 可能受PKA依赖的磷酸化调节。这些研究将使用 Gaon突变体将PKA依赖的位置以及 测定PKA对HH结合位点数目和亲和力的影响。 还将绘制SMO上的PKA结合位点图。

DESCRIPTION: This amended proposal from Dr. Joan Hooper addresses the mechanisms of Hedgehog signalling in Drosophila segment patterning. Dr. Hooper's research during the previous grant period (supported by a FIRST award) concerned intercellular signalling mechanisms involved in the functioning of segment polarity genes. This study, and those of numerous other labs, have described various gene interactions in two signalling pathways required for segment specification, those of Wingless and Hedgehog. Wingless, secreted from cells along the posterior edge of each parasegment, and Hedgehog, secreted from cells at the anterior edge, produce discrete effects depending upon whether responding cells are contacted by one or the other protein or by both together. These effects become distinguished further by virtue of the production in these cells of the transcription factor engrailed (en; anterior edge of each parasegment) or sloppy paired (slp; posterior compartment of each parasegment. While work on these pathways has been extraordinarily active in recent years, there are large gaps in understanding the molecular mechanisms by which these pathways specify pattern. The precise point of action of many of the involved gene products is unclear, and it may be that not all members of the pathways are known. Dr. Hooper is particularly interested in identifying the receptor of the Hedgehog protein and in understanding the intracellular responses to ligand-receptor interactions. Dr. Hooper has cloned and characterized smoothened (smo), a gene whose mutant phenotype is very similar to that of hh mutants. SMO is required for proper hh function, apparently acting downstream of hh and upstream of cubitus interruptus (ci), a transcription factor which appears to mediate Hh signalling. The sequence of smo suggests that the protein is a seven - transmembrane receptor protein related to Frizzled, but expressed in a quite distinct pattern. Dr. Hooper hypothesized that smo encodes the hh receptor, and she proposes experiments to test this hypothesis. Dr. Hooper has also been investigating ci. The null phenotype of ci is like that of hh, and distinct in some features from wg. In the presence of a null ci allele, wg, gsb, and ptc are not expressed in the ectoderm in late stage 10 and early stage 11 embryos, an effect which mimics mutant hh. A dominant gain-of -function ci allele, ciD, obviates the need for Hh signaling. The Ci protein is a sequence specific DNA binding protein of the zinc finger class. Dr. Hooper's group has established that Ci can activate the Hh-reponsive wg promoter in Schneider-2 cells after transient transfection and has shown that Ci binding sites are required for the activation. When Ci and Hh are co-expressed in Schneider cells, the activity on Ci in wg promoter-luciferase expression is enhanced, and co-expression of Hh with Patched (a known repressor of Hedgehog signalling) also represses Ci activation of the wg promoter. It turns out that smo is expressed at levels comparable to embryonic expression in this cell line, so if it is indeed a Hh receptor, it is present in adequate amounts for this function. ci, ptc, and hh are expressed at much lower levels. Finally, Dr. Hooper identified in genetic screens for modifiers of a dominant ptc phenotype, two new alleles of smo which show allele specific interactions with ptc , which suggests a close functional and, possibly a physical interaction between these two gene products. The current proposal focuses on smo and its functions. The specific aims are: (1) To determine whether there is a physical interaction between Smo and Hh, using binding assays in cultured cells and co-immunoprecipitation and cross-linking experiments in cultured cells and in vivo. (2) To investigate the possibility that heterotrimeric G proteins are involved in Hh signalling. This hypothesis is based on the fact that many of the structural features of the conceptually translated smo gene product are common to heterotrimeric G protein- linked receptor proteins. A series of pharmacological tests used in G protein studies will be used, taking advantage of the cell culture system. (3) The relationship between ptc and smo will be examined. It will be determined whether Ptc acts downstream of Smo, using constitutively activated Ga to determine whether Ptc effects can be modulated. It will also be determined whether Ptc can modulate the number or affinity of Hh binding sites, an effect which would suggest that Ptc acted directly on Smo. (4) It will be determined whether Smo is regulated by cAMP-dependent protein kinase (PKA). PKA is known to be a negative regulator of Hh signaling and to act upstream of or in parallel to Ptc. Since Ptc has no putative PKA phosphorylation sites, while Smo has six consensus sites, Dr. Hooper hypothesizes that the receptor might be regulated by PKA-dependent phosphorylation. These studies will use the GaON mutants to place the site of PKA dependence, as well as determination of PKA effects on the number and affinity of Hh binding site. PKA binding sites on Smo will be mapped also.