STRUCTURE DEFINITION OF IGF I RECEPTOR FUNCTION DOMAINS

胰岛素样生长因子I（IGF I）在介导胰岛素抵抗中起关键作用。 垂体生长激素的作用在反应细胞中，IGF I 在质膜上与IGF I受体相互作用， 由两个二硫键连接的α β组成的异源四聚体糖蛋白 二聚体。IGF I结合引起内源性胰岛素受体的跨膜激活。 IGF I受体的催化活性，但这一机制 目前还没有很好的理解。同样地， 这种相互作用产生的结果尚未阐明，尽管 广泛认为它们必须包括酪氨酸磷酸化， 细胞质蛋白质底物。在本申请中，我们建议研究 结构和相互作用的功能定义的结构域的 IGF I受体。为此，提出了六个具体目标。第一、 对IGF I受体与胰岛素抵抗的关系进行了严格的分析， 自磷酸化激活激酶活性， 将进行IGF I缺失。第二，半胱氨酸残基 有助于二硫键连接的两个α β二聚体 将鉴定IGF I受体四聚体。三、约束力 纯化的IGF I受体中IGF I、IGF II和胰岛素的结构域 将被比较。经过亲和交联和溴化氰 处理纯化的IGF I受体，包含IGF I的肽， IGF II和IGF I受体中的胰岛素结合结构域将被分离 和序列测定针对表面暴露的 IGF I受体的部分也将被表征。四是 在体外和体内IGF I受体自磷酸化的位点将是 鉴定第五，磷酸化的功能后果， IGF I受体通过蛋白激酶C和该修饰的位点 将确定在体外和体内发生。最后，内源性 细胞中IGF I受体酪氨酸激酶活性的底物将是 研究了在这些研究中，氧化苯胂，一种三价砷化物， 据报道，其增强了内源性底物的检测 将使用在胰岛素和IGF I刺激的细胞中磷酸化的蛋白质。 氧化苯胂抑制IGF I刺激的生物活性的能力 活动将被检查，和磷蛋白，积累在 将鉴定IGF I和氧化苯胂的存在。是 预计这些研究将有助于更好地了解 IGF I受体的信号转导及其在人类生长中的作用。

Insulin-like growth factor I (IGF I) plays a key role in mediating the growth effects of pituitary growth hormone. In responsive cells, IGF I interacts at the plasma membrane with the IGF I receptor, a heterotetrameric glycoprotein composed of two disulfide-linked alpha beta dimers. IGF I binding causes the transmembrane activation of the intrinsic catalytic activity of the IGF I receptor, but the mechanism by which this occurs is not yet well understood. Similarly, the biochemical messages generated by this interaction have not been elucidated, although it is widely assumed that they must include tyrosine phosphorylation of cytoplasmic protein substrates. In this application, we propose to study the structure and interaction of the functionally-defined domains of the IGF I receptor. To accomplish this, six specific aims are proposed. First, a rigorous analysis of the relationship of IGF I receptor autophosphorylation to activation of kinase activity in the presence and absence of IGF I will be performed. Second, the cysteinyl residues contributing to the disulfide bond(s) linking the two alpha beta dimers of the IGF I receptor tetramer will be identified. Third, the binding domain(s) for IGF I, IGF II, and insulin in the purified IGF I receptor will be compared. Following affinity cross-linking and cyanogen bromide treatment of the purified IGF I receptor, peptides comprising the IGF I, IGF II, and insulin binding domains in the IGF I receptor will be isolated and sequenced. Anti-peptide antibodies directed against surface-exposed portions of the IGF I receptor will also be characterized. Fourth, the sites of IGF I receptor autophosphorylation in vitro and in vivo will be identified. Fifth, the functional consequences of phosphorylation of the IGF I receptor by protein kinase C and the sites at which this modification occurs in vitro and in vivo will be determined. Finally, endogenous substrates of IGF I receptor tyrosine kinase activity in cells will be investigated. In these studies, phenylarsine oxide, a trivalent arsenical which has been reported to enhance the detection of endogenous substrates phosphorylated in insulin- and IGF I-stimulated cells, will be employed. The ability of phenylarsine oxide to inhibit IGF I-stimulated biological activities will be examined, and phosphoproteins that accumulate in the presence of IGF I and phenylarsine oxide will be identified. It is anticipated that these studies will lead to a better understanding of signal transduction by the IGF I receptor and its role in human growth.