STRUCTURE DEFINITION OF IGF I RECEPTOR FUNCTION DOMAINS

IGF I 受体功能域的结构定义

• 批准号: 3243819
• 负责人: SHERIDA E TOLLEFSEN
• 金额: $ 10.4万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-02-01 至 1996-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3243819
• 关键词: SDS polyacrylamide gel electrophoresis; aminoacid analyzer; autoradiography; biological signal transduction; chemical kinetics; computer assisted sequence analysis; disulfide bond; fibroblasts; growth factor receptors; immunological substance; immunoprecipitation; insulin; insulinlike growth factor; ion exchange chromatography; phosphorylation; protein kinase C; protein purification; protein sequence; protein structure function; protein tyrosine kinase; receptor binding; reversed phase chromatography; tissue /cell culture

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.

胰岛素样生长因子I（IGF I）在介导 垂体生长激素的生长影响。在响应细胞中，IGF I 在质膜上与IGF I受体（a）相互作用 由两个二硫键αβ组成的杂元糖蛋白 二聚体。 IGF I结合引起固有的跨膜激活 IGF I受体的催化活性，但是该机制 发生尚不清楚。同样，生化信息 这种互动产生的尚未阐明，尽管它是 广泛认为它们必须包括酪氨酸磷酸化 细胞质蛋白底物。在此应用中，我们建议研究 功能定义的域的结构和相互作用 IGF I受体。为此，提出了六个具体目标。第一的， 严格分析IGF I受体的关系 在存在下激活激酶活性的自磷酸化和 我将没有IGF。其次，半胱氨酸残基 有助于连接两个alpha beta二聚体的二硫键 将确定IGF I受体四聚体。第三，结合 纯化IGF I受体中IGF I，IGF II和胰岛素的域 将被比较。遵循亲和力交联和氰基溴化物 纯化的IGF I受体的处理，包括IGF I的肽， IGF II和IGF I受体中的胰岛素结合结构域将分离出来 并测序。针对表面暴露的抗肽抗体 IGF I受体的部分也将被表征。第四， IGF I受体自磷酸化的位点在体外和体内将是 确定。第五，磷酸化的功能后果 蛋白激酶C和这种修饰的位点的IGF I受体 在体外发生，并确定体内。最后，内源性 IGF I受体酪氨酸激酶活性在细胞中的底物将是 调查。在这些研究中，苯烷氧化物是三价砷 据报道可以增强内源性底物的检测 将使用在胰岛素和IGF I刺激的细胞中磷酸化的。 苯胺氧化苯二抑制IGF I刺激的生物学的能力 将检查活动，并在 将鉴定出IGF I和苯烷氧化物的存在。这是 预计这些研究将导致更好地理解 IGF I受体的信号转导及其在人类生长中的作用。