Analysis of BMP Heterodimer formation and function

骨形态发生蛋白（BMP）在发育中起关键作用，I类（BMP 2和BMP 3）的成员在发育中起关键作用。 4)和II类（BMP 5 - 7）BMP亚家族是主要参与者。我们已经证明，I/II类 异源二聚体，而不是单独的同源二聚体，产生早期骨形成所需的大部分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BMP 7异二聚化的BMP。与出生后死亡的Bmp7无效纯合子不同，Bmp7R-GFlag 纯合子是胚胎致死的，具有广泛降低的BMP活性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的无效等位基因的复合杂合子 在胚胎发育过程中死亡，并在腹体壁闭合、眼睛和心脏发育方面表现出缺陷。因此，在本发明中， BMP 4/7和BMP 2/7异二聚体在早期胚胎发生中起关键作用。这很重要，因为类 I/II异源二聚体具有比任一同二聚体显著更高的比活性。选择一个给定的 BMP将在生物合成途径内形成同源二聚体或异源二聚体。BMP被制成 无活性的前体蛋白，其被切割以产生活性的二硫键键合配体沿着有两个 前结构域片段。在生物合成过程中，前结构域在指导二聚化和 配体的折叠。我们先前已经表明，当BMP-4与BMP-7结合时， 在非洲爪蟾胚胎中共表达，并且BMP 4的前结构域对于 异二聚体形成。在新的初步研究中，我们确定了BMP 4前结构域中的一个关键残基， 第二种是产生全功能同源二聚体所需的，第二种是同源二聚体和 异二聚体功能任何一种突变的杂合子都有先天性出生缺陷。在当前 根据这一提议，我们将检验BMP 4前结构域内的序列元件需要 与II类BMP产生功能性BMP 4同源二聚体和/或功能性异源二聚体。我们将：1）识别 BMP 4前结构域中同源二聚体和/或异源二聚体形成所需的序列元件。我们 将产生编码BMP 4的cDNA，所述BMP 4在前结构域内携带氨基酸取代， 有先天缺陷的人野生型或突变体BMP 4将单独表达或与BMP 7一起表达， 非洲爪蟾胚胎将使用功能和生物化学测定来比较比活性和折叠 野生型和突变型BMP 4同源二聚体和异源二聚体的体内表达，以及2）确定BMP 4 哺乳动物发育中的前结构域序列元件。我们将产生条件性基因敲入小鼠， Bmp4基因座上的点突变，其导致前结构域内的氨基酸取代，并且 我们将对发育进行初步分析，以收集数据 这将支持未来进一步分析的资金申请。

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.