STRUCTURE DEFINITION OF IGF I RECEPTOR FUNCTION DOMAINS

胰岛素样生长因子-I(IGF-I)在血管紧张素转换酶的调节中起着关键作用。 脑下垂体生长激素的生长效应。在反应细胞中，IGF I 在质膜上与IGF I受体相互作用，a 由两个二硫键连接的α-β组成的异四聚体糖蛋白 二聚体。IGF I结合导致内源性的跨膜激活 IGF I受体的催化活性，但其作用机制 发生的原因还不是很清楚。同样，生化信息 这种相互作用产生的影响尚未阐明，尽管它是 普遍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 的功能定义域的结构和相互作用 IGF I受体。为了实现这一目标，提出了六个具体目标。第一, 胰岛素样生长因子I受体关系的严谨分析 在存在和存在的情况下，自动磷酸化以激活激酶活性 IGF I的缺席将被执行。第二，半胱氨基残基 有助于连接两个α-β二聚体的二硫键(S) 胰岛素样生长因子I受体四聚体将被鉴定。第三，约束性 纯化的IGF-I受体中IGF-I、IGF-II和胰岛素的结构域(S) 将会被比较。亲和交联剂和溴化氰 纯化的胰岛素样生长因子I受体，含有胰岛素样生长因子I的多肽， 胰岛素样生长因子II，以及胰岛素样生长因子I受体中的胰岛素结合结构域将被分离 并进行了测序。针对表面暴露的抗肽抗体 IGFI受体的一部分也将被描述。第四， 胰岛素样生长因子I受体在体外和体内的自磷酸化位点将是 已确认身份。第五，磷酸化的功能后果 蛋白激酶C介导的IGF I受体及其修饰部位 在体外和体内发生的情况将被确定。最后，内生性 细胞中IGF I受体酪氨酸激酶活性的底物将是 调查过了。在这些研究中，苯基砷氧化物，一种三价砷 据报道，它可以增强内源底物的检测 在胰岛素和胰岛素样生长因子I刺激的细胞中被磷酸化，将被使用。 氧化苯砷抑制胰岛素样生长因子I刺激的生物效应 将检查活性，以及在细胞内积累的磷蛋白 胰岛素样生长因子I和苯基氧化砷的存在将被鉴定。它是 预计这些研究将有助于更好地了解 胰岛素样生长因子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.