Analysis of BMP Heterodimer Formation and Function

项目摘要 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等位基因以及Bmp2或Bmp3的无效等位基因的复合杂合子也可被认为是一种新的突变。 bmp4在胚胎发育过程中死亡，并在腹体壁闭合、眼睛和心脏发育中显示出缺陷。 因此，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 I/II类异源二聚体比同二聚体具有显著更高的比活性。选择是否 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是 作为无活性的前体蛋白，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，前结构域在引导 配体的二聚化和折叠。我们先前已经表明BMP 4优先形成异二聚体， 当在非洲爪蟾胚胎中共表达时， 并且足以形成异二聚体。在新的初步研究中，我们确定了一个关键的残留物， BMP4前结构域是产生全功能同源二聚体所需的，而第二个是产生同源二聚体所需的。 同二聚体和异二聚体都起作用。任何一种突变的杂合子都有先天性出生 缺陷在目前的建议中，我们将测试的假设，序列元件内的BMP 4 前结构域是产生功能性BMP 4同源二聚体和/或具有II类的功能性异源二聚体所必需的 BMP。我们将：1）鉴定BMP 4前结构域中同源二聚体和/或同源二聚体所需的序列元件。 异二聚体形成。我们将产生编码BMP4的cDNA，其在BMP4基因中携带氨基酸取代。 与人类先天性缺陷相关的前结构域。将表达野生型或突变型BMP 4 单独或与BMP 7一起在非洲爪蟾胚胎中。将使用功能和生化测定来比较 野生型和突变体BMP 4同源二聚体和异源二聚体的体内比活性和折叠，以及 2)确定BMP 4前结构域序列元件在哺乳动物发育中的作用。我们将产生 在Bmp4基因座携带导致氨基酸取代的点突变的条件敲入小鼠 在前结构域内，与人类先天性缺陷有关，我们将进行初步的 分析发展情况，以收集数据，支持今后的资金申请，供进一步分析。

Project Summary 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.