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前体蛋白的蛋白水解成熟如何调节哺乳动物发育期间细胞-细胞信号分子的活性和作用范围。ProBMP-4最初在与成熟配体结构域相邻的共有弗林蛋白酶基序处被切割，这允许随后在前结构域内的上游非共有弗林蛋白酶基序处被切割。在非洲爪蟾胚胎中，由上游位点不可切割的异位前体合成的BMP-4最初被降解，并在较短范围内发出信号，而由在两个位点同时裂解的前体合成的BMP-4比由天然前体裂解的BMP-4更有活性，并在更大范围内发出信号。拟议的研究将测试以下假设：1）proBMP-4的顺序切割对于适当调节内源性BMP-4活性和正常胚胎模式至关重要，2）上游位点的组织特异性切割提供了组织特异性调节的机制BMP-4信号范围和3）部分地做到了这一点，通过调节成熟BMP-4与细胞表面或细胞外基质的附着。为此，我们将产生携带靶向点突变的小鼠，这些突变破坏或加速上游位点的切割，而不改变释放成熟tigand的初级切割。通过测量BMP-4靶基因和免疫反应性磷酸化Smad 1的表达水平和模式，并通过将突变体与Smad 1-反应元件-LacZ报告小鼠杂交，将在野生型和突变体同窝仔的各种组织中比较BMP-4活性和信号传导范围。将在野生型和突变小鼠的各种组织中分析ProBMP-4加工，以直接测定上游位点的组织特异性使用。还将在分离自弗林蛋白酶、PACE 4或PC6 B突变小鼠的组织中分析Pro-BMP-4切割，以鉴定在每个位点切割的转化酶。 最后，将在野生型和突变小鼠中比较成熟BMP-4的细胞表面附着。BMP-4活性的适当调节对于正常的胚胎形成和成人的组织稳态是必不可少的。了解BMP活性调节的分子机制是理解、治疗和预防先天性异常和疾病的关键。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to determine how proteolytic maturation of precursor proteins regulates the activity and range of action of cell-celt signaling molecules during mammalian development. ProBMP-4 is initially cleaved at a consensus furin motif adjacent to the mature ligand domain and this allows for subsequent cleavage at an upstream nonconsensus furin motif within the prodomain, in Xenopus embryos, BMP-4 synthesized from ectopic precursor in which the upstream site is non-cleavable is initially degraded and signals at shorter range, while that synthesized from a precursor that is simultaneously cleaved at both sites is more active and signals at greater range than does BMP-4 cleaved from native precursor. The proposed studies will test the hypotheses that 1) sequential cleavage of proBMP-4 is essential for proper regulation of endogenous BMP-4 activity and for normal embryonic patterning, 2) tissue-specific cleavage at the upstream site provides a mechanism for tissue-specific regulation of BMP-4 signaling range and 3) it does so, in part, by modulating attachment of mature BMP-4 to the cell surface or extracellular matrix. To de so, we will generate mice carrying targeted point mutations that disrupt or accelerate cleavage at the upstream site without altering the primary cleavage that releases the mature tigand. BMP-4 activity and signaling range wilt be compared in various tissues of wild type and mutant littermates by measuring levels and pattern of expression of BMP-4 target genes and immunoreactive phosphoSmadl, and by crossing mutants with Smad1-response element-LacZ reporter mice. ProBMP-4 processing will be analyzed in various tissues of wild type and mutant mice to directly assay for tissue-specific use of the upstream site. Pro-BMP-4 cleavage will also be analyzed in tissues isolated from furin, PACE4 or PC6B mutant mice to identify convertases that cleave at each site. Finally, cell surface attachment of mature BMP-4 will be compared in wild type and mutant mice. Proper regulation of BMP-4 activity is essential for normal embryonic patterning and for tissue homeostasis in adults. Understanding the molecular mechanisms by which BMP activity is regulated is key to understanding, treating and preventing congenital anomalies and diseases.