Analysis of BMP-4 activity in Cleavage mutant mice

Cleavage突变小鼠BMP-4活性分析

• 批准号: 6953441
• 负责人: Jan L Christian
• 金额: $ 14.94万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6953441
• 关键词: Analysis; BMP; activity; Cleavage; mutant

DESCRIPTION (provided by applicant): The long term goal of the proposed research is to determine how proteolytic maturation of proproteins regulates the activity and range of action of cell-cell signaling molecules. The Bone morphogenetic protein-4 (BMP-4) precursor undergoes sequential cleavage at two sites within the prodomain. Cleavage at the first site (S1) occurs in all tissues and releases mature BMP-4, which signals at short range and is rapidly degraded. If the prodomain is subsequently cleaved at an upstream site (S2), this converts mature BMP-4 into a long range, stable signaling molecule. The physiologic relevance of S2 cleavage is being examined in mice harboring mutations that disrupt or accelerate cleavage at this site. The proposed studies will ask whether tissue-specific use of the S2 site provides an evolutionarily conserved mechanism for regulating the range of BMP signaling in vertebrates and Drosophila. In addition, these studies will take advantage of the unique strengths of Drosophila imaging and genetics to directly visualize the effect of S2 cleavage on formation of the BMP protein gradient and to test a model for how cleavages in the prodomain regulate the activity of the ligand after it is released. Transgenic flies will be generated that carry GFP-tagged versions of wild-type proDpp (the fly ortholog of BMP-4), or proDpp-GFP harboring mutations that disrupt or accelerate cleavage at the S2 site. The GAL4-UAS system will be used to drive expression in Dpp mutant wing discs or embryonic midgut, where endogenous Dpp is known to signal at long or short range, respectively. Flies will be analyzed for rescue of phenotypic defects, Dpp target gene induction and Dpp-GFP protein distribution. Finally, to test the hypothesis that cleavage at the S2 site regulates signaling range by directing endocytic trafficking of the mature ligand to a recycling, rather than a lysosomal compartment following receptor mediated endocytosis, wild-type and cleavage mutant precursors will be expressed in wing discs in which distinct aspects of endocytic trafficking are impaired and the effect on Dpp gradient formation will be examined. Gain or loss of function of BMP-4 activity leads to structural birth defects. Thus, understanding how BMP activity is regulated in vivo is key to treating and preventing congenital anomalies.

描述（由申请人提供）：拟议研究的长期目标是确定前蛋白的蛋白水解成熟如何调节细胞-细胞信号分子的活性和作用范围。骨形态发生蛋白-4（BMP-4）前体在前结构域内的两个位点处经历连续切割。在第一个位点（S1）的裂解发生在所有组织中，并释放成熟的BMP-4，其在短距离内发出信号并迅速降解。如果前结构域随后在上游位点（S2）被切割，则这将成熟的BMP-4转化为长距离稳定的信号分子。S2裂解的生理相关性正在检测小鼠中，这些小鼠携带破坏或加速该位点裂解的突变。拟议的研究将询问是否组织特异性使用S2位点提供了一个进化上保守的机制，调节脊椎动物和果蝇中BMP信号的范围。此外，这些研究将利用果蝇成像和遗传学的独特优势，直接可视化S2切割对BMP蛋白梯度形成的影响，并测试前结构域中的切割如何调节配体释放后的活性的模型。将产生携带GFP标记的野生型proDpp（BMP-4的果蝇直系同源物）版本或携带破坏或加速S2位点处的切割的突变的proDpp-GFP的转基因果蝇。GAL 4-UAS系统将用于驱动Dpp突变翅盘或胚胎中肠中的表达，其中已知内源性Dpp分别在长或短范围内发出信号。将分析果蝇的表型缺陷拯救、Dpp靶基因诱导和Dpp-GFP蛋白分布。最后，为了检验S2位点处的裂解通过将成熟配体的内吞运输引导至再循环而不是受体介导的内吞作用后的溶酶体区室来调节信号传导范围的假设，野生型和裂解突变体前体将在翅盘中表达，其中内吞运输的不同方面受损，并且将检查对Dpp梯度形成的影响。BMP-4活性功能的获得或丧失导致结构性出生缺陷。因此，了解BMP活性如何在体内调节是治疗和预防先天性异常的关键。