Analysis of BMP-4 activity in Cleavage mutant mice

描述(由申请人提供)：拟议研究的长期目标是确定原蛋白的蛋白分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)前体在原结构域内的两个位置经历了连续的切割。第一位点(S1)的裂解发生在所有组织中，并释放成熟的BMP-4，它在短距离内发出信号，并迅速降解。如果原结构域随后在上游位置(S2)被切割，这就将成熟的BMP-4转化为长范围、稳定的信号分子。S2裂解的生理学相关性正在含有破坏或加速该位点裂解的突变的小鼠身上进行检验。拟议的研究将提出，组织特异性地使用S2位点是否提供了一种进化上保守的机制来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独特的成像和遗传学优势，直接可视化S2裂解对BMP蛋白梯度形成的影响，并测试一个模型，说明前结构域中的裂解如何调节配体释放后的活性。将产生携带GFP标记版本的野生型proDpp(BMP-4的同源果蝇)的转基因果蝇，或携带干扰或加速S2位点切割的突变的proDpp-GFP。GAL4-UAS系统将用于驱动DPP突变翼盘或胚胎中肠的表达，已知内源DPP分别在长距离或短距离发出信号。将分析苍蝇表型缺陷的修复、DPP靶基因的诱导和DPP-GFP蛋白的分布。最后，为了验证S2位点的切割通过引导成熟配体的内吞而不是受体介导的内吞之后的溶酶体隔室来调节信号范围的假设，野生型和切割突变的前体将在翼盘中表达，其中内吞的不同方面受到损害，并将检测对DPP梯度形成的影响。BMP-4活性的功能增强或丧失会导致结构性出生缺陷。因此，了解BMP活性在体内是如何调节的，是治疗和预防先天性异常的关键。

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.