Analysis of BMP Heterodimer formation and function

骨形态发生蛋白(BMPs)在发育过程中起着关键作用，其成员有I类(BMP2和BMPs 4)和II类(BMP5-7)BMP亚家族是主要参与者。我们已经展示了I/II类 异二聚体，而不是单个同源二聚体，产生了早期所需的大部分BMP活性 发展。我们产生了携带突变(Bmp7R-GFlag)的敲入小鼠，该突变消除了所有 与BMP7异源二聚的BMP。与出生后死亡的Bmp7缺失纯合子不同，Bmp7R-GFlag 纯合子是胚胎致命的，具有广泛的BMP活性降低，并在多个器官中表现出缺陷。 此外，携带Bmp7R-GFLag等位基因和BMP2或Bmp4零等位基因的复合杂合子 在胚胎发育过程中死亡，并在腹侧体壁关闭、眼睛和心脏发育方面表现出缺陷。因此， BMP4/7和BMP2/7异源二聚体在早期胚胎发育中起关键作用。这一点很重要，因为 I/II异二聚体的比活性明显高于同二聚体。选择是否给出了一个 BMP将在生物合成途径中形成同源二聚体或异源二聚体。BMP按如下方式制造 非活性前体蛋白被切割以产生活性的二硫键配体和两个 原结构域片段。在生物合成过程中，原结构域在引导二聚和 配体的折叠。我们先前已经证明，当BMP4优先与BMP7形成异二聚体时 在非洲爪哇胚胎中共表达，BMP4的原结构域对于 杂二聚体的形成。在新的初步研究中，我们确定了BMP4原结构域中的一个关键残基 是产生全功能同源二聚体所必需的，第二个是同源二聚体和 异二聚体功能。任何一种突变的杂合子人类都有先天性出生缺陷。在当前 提案中，我们将检验BMP4前结构域中的序列元件需要 产生功能性BMP4同源二聚体，和/或具有II类BMPs的功能性异源二聚体。我们将：1)确定 同源二聚体和/或异源二聚体形成所需的BMP4前结构域中的序列元件。我们 将产生编码BMP4的cDNA，携带原结构域内的氨基酸替换 有先天缺陷的人类。野生型或突变型BMP4将单独或与BMP7一起在 非洲爪哇的胚胎。功能和生化分析将用来比较比活性和折叠 体内野生型和突变型BMP4同源二聚体和异源二聚体的研究，以及2)确定BMP4的作用 哺乳动物发育中的前域序列元件。我们将产生有条件的敲入老鼠，携带 Bmp4基因座的点突变导致原结构域内的氨基酸替换，这是 与人类先天缺陷相关的我们将对发育进行初步分析以收集数据 这将支持未来的资金申请，以便进行进一步分析。

Bone morphogenetic proteins (BMPs) play critical roles in development, with members of the class I (BMP2 and 4) and class II (BMP5-7) BMP subfamilies being the dominant players. We have shown that class I/II heterodimers, rather than individual homodimers, generate most of the BMP activity that is required for early development. We generated knock-in mice carrying a mutation (Bmp7R-GFlag) that eliminates the function of all BMPs that heterodimerize with BMP7. Unlike Bmp7 null homozygotes, which die after birth, Bmp7R-GFlag homozygotes are embryonic lethal, have broadly reduced BMP activity and exhibit defects in multiple organs. Furthermore, compound heterozygotes carrying the Bmp7R-GFlag allele together with a null allele of Bmp2 or Bmp4 die during embryogenesis and show defects in ventral body wall closure, eye and heart development. Thus, BMP4/7 and BMP2/7 heterodimers play critical roles in early embryogenesis. This is important because class I/II heterodimers have significantly higher specific activity than either homodimer. The choice of whether a given BMP will form a homodimer or a heterodimer is made within the biosynthetic pathway. BMPs are made as inactive precursor proteins that are cleaved to generate the active, disulfide-bonded ligand along with two prodomain fragments. During biosynthesis the prodomain plays essential roles in guiding dimerization and folding of the ligand. We have previously shown that BMP4 preferentially forms heterodimers with BMP7 when co-expressed in Xenopus embryos, and that the prodomain of BMP4 is both necessary and sufficient for heterodimer formation. In new preliminary studies, we identified a key residue within the BMP4 prodomain that is required to generate fully functional homodimers and a second that is required for both homodimer and heterodimer function. Humans heterozygous for either mutation have congenital birth defects. In the current proposal, we will test the hypothesis that sequence elements within the BMP4 prodomain are required to generate functional BMP4 homodimers, and/or functional heterodimers with class II BMPs. We will: 1) Identify sequence elements in the BMP4 prodomain that are required for homodimer and/or heterodimer formation. We will generate cDNAs encoding BMP4 carrying amino acid substitutions within the prodomain that are associated with congenital defects in humans. Wild type or mutant BMP4 will be expressed alone or together with BMP7 in Xenopus embryos. Functional and biochemical assays will be used to compare the specific activity and folding of wild type and mutant BMP4 homodimers and heterodimers in vivo, and 2) Determine the role of BMP4 prodomain sequence elements in mammalian development. We will generate conditional knock-in mice carrying point mutations at the Bmp4 locus that lead to amino acid substitutions within the prodomain, and that are associated with congenital defects in humans We will perform preliminary analysis of development to collect data that will support future applications for funding for further analysis.