MAPPING THE IGF-II BINDING SITE OF THE IGF-II RECEPTOR

胰岛素样生长因子II（IGF-II）受体具有高亲和力， 多肽IGF-II和糖基化溶酶体的结合位点 内切酶这种独特的受体在转运溶酶体酶中的关键作用 已经很好地建立了，但是IGF-II启动信号的机制 关于胰岛素样生长因子-II受体介导的细胞内信号转导事件的研究存在争议。 为了帮助阐明这一机制，本项目的目标是绘制 IGF-II受体的ICF-II结合位点使用两个互补的 接近。 首先，纯化的125 I-IGF-II亲和标记受体将 用内切蛋白酶Glu-C消化，然后用蛋白酶的肽区消化。 将与IGF-II共价连接的受体分离用于测序。 第二，cDNA克隆编码IGF-II受体的禽类同源物， 不结合ICF-II，将通过筛选鸡cDNA文库来分离 放射性标记的哺乳动物IGF-II受体cDNA片段。序列 在这些研究中获得的映射信息以及试剂将 用于指定用于定点诱变的靶标。 哺乳动物受体的IGF-II结合位点，并设计禽/哺乳动物 受体嵌合体。野生型、突变型和嵌合受体将被 在哺乳动物细胞中表达以评估IGF-II结合参数，以及 研究IGF-II结合与 信号转导

The insulin-like growth factor II (IGF-II) receptor has high affinity binding sites for the polypeptide IGF-II and glycosylated lysosomal enzymes. This unique receptor's key role in transporting lysosomal enzymes is well established, but the mechanism by which IGF-II-initiated signal transduction events are mediated by the IGF-II receptor is controversial. To help elucidate this mechanism, the objective of this project is to map the ICF-II binding site of the IGF-II receptor using two complementary approaches. First, purified 125I-IGF-II affinity-labelled receptors will be digested with endoproteinase Glu-C, then peptide regions of the receptor covalently attached to IGF-II will be isolated for sequencing. Second, cDNA clones encoding the IGF-II receptor's avian homolog, which does not bind ICF-II, will be isolated by screening chicken cDNA libraries with radiolabelled fragments of mammalian IGF-II receptor cDNAs. Sequence and mapping information as well as reagents obtained in those studies will be used to designate targets for site-directed mutagenesis of the mammalian receptor's IGF-II binding site and to design avian/mammalian receptor chimeras. Wild-type, mutant and chimeric receptors will be expressed in mammalian cells to assess IGF-II binding parameters, and to investigate structure-function relationships linking IGF-II binding to signal transduction.