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与糖基化溶酶体的结合部位 酵素。这种独特的受体在运输溶酶体酶中的关键作用 是公认的，但IGF-II启动信号的机制 IGF-II受体介导的信号转导事件一直存在争议。 为了帮助阐明这一机制，该项目的目标是绘制 两个互补的IGF-II受体ICF-II结合部位的研究 接近了。首先，纯化的125I-IGF-II亲和标记受体将 用内切酶Glu-C消化，然后将多肽区 与IGF-II共价结合的受体将被分离出来进行测序。 第二，编码IGF-II受体的禽类同源基因的cDNA克隆 不结合ICF-II，将通过筛选鸡的cDNA文库来分离 用哺乳动物IGF-II受体cDNA的放射性标记片段。数列 在这些研究中获得的测绘信息和试剂将 用于指定定点突变的靶点 哺乳动物IGF-II受体结合部位及禽类/哺乳类动物的设计 受体嵌合体。野生型、突变型和嵌合型受体将 在哺乳动物细胞中表达，以评估IGF-II结合参数，并 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.