Functional Signaling Domains of Notch Ligands

罗伯特·弗莱明……9727951在多细胞生物的发育过程中，单细胞受体通常在多个发育时期使用。Notch基因家族是一组高度保守的基因，其功能在线虫和人类等多种生物中都是必需的。Notch和Notch相关基因产物作为细胞间信号事件的细胞膜受体，在发育过程中协调各种组织的细胞命运决定。Notch活性由膜结合配体调节，在果蝇中，由Serrate （SER）和Delta （DL）基因的产物代表。SER-like和DL-like分子也被发现在Notch家族基因发现的同一物种中是保守的。这些配体表现出不同的时间和空间表达模式，表明它们各自在特定的过程中调节NOTCH。有趣的是，SER和DL都可以启动NOTCH信号，并且在某些过程中具有相同的能力，而在其他过程中则具有不同的功能。由于SER和DL可以引起单个NOTCH受体的不同反应，并且由于NOTCH是许多细胞命运决定的核心，因此这些研究将探讨每种配体在与NOTCH结合时产生特异性的机制。拟议的实验将检查SER和DL中特定蛋白质结构域的作用，这些结构域有助于配体在不同细胞环境中激活NOTCH的能力。通过构建每个配体的细胞外或细胞内（IC）结构域的突变并在体内表达突变分子，将确定配体功能的机制。通过分析这些嵌合分子相对于正常SER和DL的特性，将确定单个蛋白质结构域产生的特定功能特性。最初，研究将集中于NOTCH配体和TGF样分子之间可能的平行机制，这些分子需要IC结构域来实现适当的细胞外功能。这些领域的初步研究表明，SER分子协同作用，可能在NOTCH信号传导的某些方面以二聚体或其他聚集形式激活NOTCH。这些发现提示了其他实验领域，包括SER切割产物的分子检测和细胞外区域突变与IC结构域之间的功能相互作用。其他研究领域包括表征独特的富含半胱氨酸的结构域，仅在SER配体中发现，而不在DL中发现。该结构域的性质将通过将其从SER中删除和/或插入到DL中来表征，并评估这些修饰的配体在特定细胞环境中与NOTCH受体相互作用和/或激活NOTCH受体的能力。综上所述，这些研究有望扩大对NOTCH信号调控的理解，并进一步阐明受体在被盗窃识别的配体激活时通常可能发送多个离散信号的机制。

Fleming, Robert J.. 9727951 During the development of multicellular organisms, single cellular receptors are often used at multiple developmental periods. The Notch gene family represents a highly conserved group of genes whose functions are required in organisms as diverse as nematodes and humans. Notch and Notch-related gene products function as cell membrane receptors for intercellular signaling events to coordinate cell fate decisions in a variety of tissues during development. Notch activity is regulated by membrane-bound ligands which, in Drosophila, are represented by the products of the genes Serrate (SER) and Delta (DL). SER-like and DL-like molecules have also been found to be conserved in the same species where Notch family genes are found. These ligands demonstrate distinct temporal and spatial expression patterns suggesting that they each regulate NOTCH in particular processes. Interestingly, both SER and DL can act to initiate NOTCH signaling and appear to have equivalent capabilities during some processes yet function distinctly in others. Because SER and DL can elicit distinct responses from the single NOTCH receptor and since NOTCH is central to many cell fate decisions, these studies will investigate the mechanisms by which specificity is generated by each ligand upon association with NOTCH. The proposed experimentation will examine the roles of specific protein domains within SER and DL that contribute to the ability of that ligand to activate NOTCH in different cellular environments. Mechanisms of ligand function will be determined by constructing mutations of extracellular or intracellular (IC) domains of each ligand and expressing the mutant molecules in vivo. By analyzing the properties of these chimeric molecules relative to normal SER and DL, specific functional properties produced by individual protein domains will be determined. Initially, studies will focus on possible parallel mechanisms between NOTCH ligands and TGF(-like molecules that require IC domains f or proper extracellular function. Preliminary studies in these areas suggest that SER molecules function cooperatively and may activate NOTCH as a dimer or other aggregated form during some aspects of NOTCH signaling. These findings suggest other areas of experimentation including molecular examination for SER cleavage products and functional interactions between extracellular region mutations and the IC domain. Other areas of investigation include the characterization of a unique cysteine-rich domain found only within the SER ligand and not in DL. The properties of this domain will be characterized by deleting it from SER and/or inserting it into DL and assessing the ability of these modified ligands to interact with and/or activate the NOTCH receptor in specific cellular environments. Taken together, these studies are expected to expand the understanding of NOTCH signal regulation and further clarify mechanisms by which receptors in general may serve to send multiple, discrete signals upon activation by theft identified ligands.