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过程中细胞-细胞信号分子的活性和作用范围。ProBMP-4最初在靠近成熟配体结构域的一致furin基序上被切割，这允许随后在原结构域的上游非一致furin基序上进行切割。在非洲爪蟾胚胎中，BMP-4由异位前体合成，其中上游位点不可切割，最初被降解并在较短的范围内发出信号。而从两个位点同时裂解的前体合成的BMP-4比从天然前体裂解的BMP-4更活跃，信号范围更广。提出的研究将验证以下假设：1)proBMP-4的顺序切割对于内源性BMP-4活性和正常胚胎模式的适当调节至关重要；2)上游位点的组织特异性切割为BMP-4信号传导范围的组织特异性调节提供了一种机制；3)它部分地通过调节成熟BMP-4与细胞表面或细胞外基质的粘附来实现。为此，我们将产生携带靶向点突变的小鼠，这些突变会破坏或加速上游位点的裂解，而不会改变释放成熟腺体的初级裂解。通过测定BMP-4靶基因和免疫反应性磷酸smadl的表达水平和表达模式，以及将突变体与smad1反应元件- lacz报告小鼠杂交，比较野生型和突变体在不同组织中的BMP-4活性和信号传导范围。ProBMP-4的加工过程将在野生型和突变型小鼠的各种组织中进行分析，以直接测定上游位点的组织特异性使用。还将分析从furin， PACE4或PC6B突变小鼠分离的组织中Pro-BMP-4的切割情况，以确定在每个位点切割的转化酶。最后，比较成熟BMP-4在野生型和突变型小鼠中的细胞表面附着。适当调节BMP-4的活性对于正常的胚胎模式和成人的组织稳态是必不可少的。了解BMP活性调控的分子机制是理解、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.