Growth Factor Interactions with Type II Procollagen

描述(申请人提供)：关节软骨的形成 需要软骨生成细胞、生长因子、 基质大分子，包括II型前胶原。软骨生成是 以产生类型II的交替剪接形式为特征的 氨基端含有富含半胱氨酸(CR)结构域的前胶原(IIA) (NH_2)前肽。这个外显子2编码的结构域与BMP-2结合，并且是同源的 在结合BMP的其他分泌蛋白中的多个拷贝中发现的基序 (例如，Chordin和Sog)。这些BMP结合蛋白在细胞中起着关键作用 类型确定和形态发生。我们将检验这一假设 IIA型前胶原中CR结构域的空间呈现对 与生长因子的相互作用。对IIA NH2-前肽的研究已经被 受制于从组织中分离胶原原多肽的复杂性。我们, 因此，产生了由IIA NH2-前肽组成的嵌合蛋白 连接到C00H末端的C型凝集素三聚盒以模拟 原发前胶原中IIA受体结构域的呈现。因此，我们将 使用该构造及其突变构造有三个目的：我们将：1) 确定外显子2结构域是否足以产生最佳的BMP-2 或是否需要全长前肽来正确结合 这些外显子2结构域的空间排列；2)比较生长 不同野生型和突变型IIA氨基前肽的因子结合活性 外显子2结构域的数量和分布；3)检查其功能 在体外的生物系统中构建这些结构。的结构解释 野生型前肽将使我们能够创造具有CR突变的蛋白质 结构域，以破译与生长因子结合有关的重要氨基酸。这个 这些分子的结构将用生物化学和 免疫学技术及其与BMP-2的体外相互作用将是 使用结合试验进行了研究。对于第三个目标，我们将使用高度的 测量BMP依赖的延伸率(分化)的可重复性测定 对晶状体上皮细胞的生物学活性进行监测 含有突变外显子2的前肽。总而言之，拟议的研究将 提供有关外显子2序列功能作用的重要数据 软骨的发育和修复。所获得的数据将有助于 检查过度表达选择性影响的策略的发展 转基因小鼠中的突变体。

DESCRIPTION (provided by applicant): The formation of articular cartilage requires a complex interplay between cartilage producing cells, growth factors, and matrix macromolecules, including type II procollagen. Chondrogenesis is characterized by the production of an alternatively-spliced form of type II procollagen (IIA) containing a cysteine-rich (CR) domain in the amino-terminal (NH2) propeptide. This exon 2-encoded domain binds to BMP-2 and is homologous to motifs found in multiple copies in other secreted proteins that bind BMPs (e.g., chordin and sog). These BMP-binding proteins play critical roles in cell type determination and morphogenesis. We will test the hypothesis that the spatial presentation of CR domains in type IIA procollagen is critical for interactions with growth factors. Studies of the IIA NH2-propeptide have been hampered by the complexity of isolating collagen propeptides from tissues. We, therefore, produced a chimeric protein consisting of the IIA NH2-propeptide linked to a C00H-terminal C-type lectin trimerization cassette to mimic the presentation of the IIA CR domains in native procollagen. Accordingly, we will use this construct and mutant constructs thereof for three aims: We will: 1) determine whether the exon 2 domain alone is sufficient for optimal BMP-2 binding or whether the full-length propeptide is necessary for the correct spatial arrangement of these exon 2 domains; 2) compare the growth factor-binding activity of wild-type and mutant IIA NH2-propeptides that differ in the number and distribution of exon 2 domains; and 3) examine the function of these constructs in a biological system in vitro. Structural elucidation of wild-type propeptides will allow us to create proteins with mutations in the CR domain to decipher important amino acids involved in growth factor binding. The structure of these molecules will be characterized using biochemical and immunological techniques, and their in vitro interactions with BMP-2 will be studied using binding assays. For the third aim, we will use a highly reproducible assay that measures the BMP-dependent elongation (differentiation) of lens epithelial cells to monitor the biological activity of wild-type or mutant exon 2-containing propeptides. Together, the proposed studies will provide important data about the functional roles of exon 2 sequences in cartilage development and repair. The data obtained will assist with the development of strategies to examine the effect of over-expressing selected mutants in transgenic mice.