Molecular Mechanisms Regulating Intercellular Transit of TGF-beta

描述(申请人提供)：发育生物学的一个主要挑战是理解细胞或组织之间如何恰当地传递定量信息。转化生长因子-β超家族成员(转化生长因子-β)是重要的、进化上保守的细胞通讯信使。我们正在使用秀丽线虫可用的遗传工具来研究细胞间转化生长因子-β梯度形成中的几个悬而未决的问题。这项研究的长期目标是了解利用线虫在细胞之间运输转化生长因子-2分子的分子和细胞基础。我们的中心假设是，转化生长因子-β在细胞之间的转运是由转化生长因子-β、特定的胞外蛋白(包括蛋白多糖)、受体细胞的内吞机制和其他信号通路之间的相互作用网络所介导的。为了验证这一假设，我们将回答以下问题：1)Glypcan蛋白是如何隔离转化生长因子-β配体的？Glypicans是一种蛋白多糖，是一种保守的细胞外转化生长因子-β调节剂。Glypcan核心蛋白的改变与发育缺陷和癌症有关，我们已经证明Glypcan核心蛋白限制了转化生长因子-β的活性。我们假设，Glypcan核心蛋白中的特定区域在特定位置隔离转化生长因子-2，以防止转化生长因子-2激活细胞表面受体。我们将首先通过突变Glypcan的区域来确定Glypcan蛋白中影响转化生长因子-β活性的位点(S)，并询问改变后的Glypcan是否在我们的体内生物检测中保留功能。然后，我们将确定Glypcan活性所需的改变位置本身是否足以抑制转化生长因子-β的活性。此外，我们还将证实Glypcan突变功能与其结合转化生长因子-β的能力有关。最后，我们将确定转化生长因子-β与Glypcan核心蛋白相互作用的区域。2)其他细胞外的转化生长因子-β调节因子如何影响转化生长因子-β信号传导？我们假设，其他蛋白多糖、蛋白多糖修饰酶、蛋白酶、细胞外基质成分、定向内吞作用和其他信号通路决定了转化生长因子-2的寿命和信号范围。我们将描述我们在致敏筛选中确定的新型调节因子的作用，包括细胞外蛋白、内吞机制和另一种细胞信号通路受体。我们还将使用敏化的RNAi筛选来识别新的转化生长因子-2本地化调节因子。我们的研究采用了转基因和生物化学相结合的方法来回答这些问题。这种方法是创新的，因为我们可以直接观察调节器对我们产生的荧光标记的转化生长因子-β的定位的影响，我们已经使用这个工具来筛选基因组RNAi文库并确定候选基因。这项拟议的研究意义重大，因为它有望对转化生长因子-β配体在细胞之间传递时如何调节产生更深入和更广泛的理解。这一贡献将对细胞信号领域产生积极影响，因为它为了解分级生长因子分布在发育过程中是如何实现的，以及在包括癌症在内的疾病状态下如何改变提供了广泛的基础。

DESCRIPTION (provided by applicant): A major challenge in developmental biology is to understand how quantitative information is properly relayed between cells or tissues. Transforming growth factor- beta superfamily members (TGF-betas) are critical, evolutionarily conserved messengers for cellular communication. We are using the genetic tools available in Caenorhabditis elegans to investigate several outstanding questions in intercellular TGF-beta gradient formation. The long-term objective of this study is to understand the molecular and cellular basis for the trafficking of TGF-2 molecules between cells using C. elegans. Our central hypothesis is that transport of TGF- beta between cells is mediated by a network of interactions among TGF-betas, specific extracellular proteins including proteoglycans, endocytosis machinery in the receiving cells, and other signaling pathways. To test this hypothesis, we will answer the following questions: 1) How does glypican protein sequester TGF-beta ligand? Glypicans are proteoglycans that are conserved extracellular TGF-beta regulators. Alteration of the glypican protein core is associated with developmental defects and cancers, and we have shown that glypican core protein restricts TGF-beta activity. We hypothesize that a specific region within the glypican core protein sequesters TGF-2 at a specific site to prevent TGF-2 from activating cell surface receptors. We will first identify the site(s) within the glypican protein that affect TGF-beta activity by mutating regions of the glypican and asking if the altered glypican retains function in our in vivo bioassays. We will then determine if an altered site that is required for glypican activity is sufficient by itself to inhibit TGF-beta activity. In addition, we will confirm that glypican mutant function is correlated with its ability to bind TGF-beta. Last, we will identify the region of TGF-beta that interacts with the glypican core protein. 2) How is TGF-beta signaling affected by other extracellular TGF-beta regulators? We hypothesize that other proteoglycans, proteoglycan-modifying enzymes, proteases, extracellular matrix constituents, directed endocytosis, and other signaling pathways define TGF-2 longevity and signaling range. We will characterize the roles of novel regulators we have identified in a sensitized screen, including extracellular proteins, endocytosis machinery, and another cell signaling pathway receptor. We will also 3) Identify new regulators of TGF-2 localization using a sensitized RNAi screen. Our studies to answer these questions employ a combination of transgenic and biochemical methods. The approach is innovative because we can directly observe effects of regulators on the localization of a fluorescently tagged TGF-beta we generated, and we have used this tool to screen a genomic RNAi library and identify candidates. The proposed research is significant because it is expected to produce a deeper and substantially expanded understanding of how TGF-beta ligands are regulated as they travel between cells. This contribution will have a positive impact on the cell signaling field because it provides a broad foundation from which to understand how graded growth factor distribution is achieved during development and is altered in disease states including cancers.