Transmembrane Domains of Receptor Tyrosine Kinases

受体酪氨酸激酶的跨膜结构域

• 批准号: 6382533
• 负责人: DANIEL J DONOGHUE
• 金额: $ 22.54万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6382533
• 关键词: 3T3 cells; SDS polyacrylamide gel electrophoresis; autoradiography; biological signal transduction; dihydrofolate reductase; enzyme activity; fibroblast growth factor; gastrointestinal neoplasms; gene induction /repression; gene mutation; immunoprecipitation; ligands; medullary thyroid carcinoma; membrane proteins; multiple endocrine neoplasia; phosphorylation; platelet derived growth factor; protein tyrosine kinase; protooncogene; receptor binding; receptor coupling; receptor expression; western blottings

DESCRIPTION (PROVIDED BY APPLICANT): This revised application will examine the role of the transmembrane domain in regulating the activity of several important receptor tyrosine kinases (RTKs), including Neu/ErbB2, FGFR3, KIT and RET. Mutations in each of these RTKs lead to significant human developmental and neoplastic syndromes. In Aim 1, model transmembrane domains will be used to study whether constitutive dimerization via the transmembrane domain regulates RTK activation. We have previously shown that a transmembrane domain consisting of the consensus repeat [VVVEVVV]n results in Neu/ErbB2 activation. Using consensus sequence transmembrane domains to provide constitutive dimerization, we will examine rotational coupling of the transmembrane domain with the kinase domain for FGFR3, KIT and RET. In Aim 2, we will explore whether rotational coupling accompanies ligand-stimulated RTK activation. Using PDGFR-beta, chosen because it can be readily activated by its ligand, PDGF-BB, we will determine whether ligand binding to the extracellular domain induces rotational translation of the intracellular domain, coupled through the alpha-helical transmembrane domain. Using a DHFR protein fragment complementation assay, as well as conventional assays for receptor activation such as transformation and autophosphorylation, these experiments will quantitate rotational coupling between the transmembrane domain and the kinase domain that may be induced by ligand. In Aim 3, we will examine the role of the juxtamembrane domain in regulating two different RTKs, RET and JUT. Mutations in the extracellular juxtamembrane domain of RET cause multiple endocrine neoplasia (MEN) and familial medullary thyroid carcinoma (FMTC), whereas mutations in the intracellular juxtamembrane domain of KIT lead to gastrointestinal stromal tumors (GIST) and mastocytoma. These experiments will thus investigate the mechanism whereby these gain-of-function mutations in the juxtamembrane domains of RET and KIT lead to receptor activation and downstream signal transduction.

描述（由申请人提供）：本修订后的申请将审查 跨膜结构域在调节多种活性中的作用 重要的受体酪氨酸激酶 (RTK)，包括 Neu/ErbB2、FGFR3、KIT 和 RET。这些 RTK 中的每一个突变都会导致显着的人类发育 和肿瘤综合征。在目标 1 中，模型跨膜结构域将用于 研究是否通过跨膜结构域调节组成型二聚化 RTK 激活。我们之前已经证明跨膜结构域包含 一致重复序列 [VVVEVVV]n 导致 Neu/ErbB2 激活。使用 共有序列跨膜结构域以提供组成型二聚化， 我们将检查跨膜结构域与激酶的旋转耦合 FGFR3、KIT 和 RET 的结构域。 在目标 2 中，我们将探讨旋转耦合是否伴随 配体刺激的 RTK 激活。使用 PDGFR-beta，选择它是因为它可以 很容易被其配体PDGF-BB激活，我们将确定配体是否 与细胞外结构域的结合诱导细胞的旋转平移 细胞内结构域，通过α螺旋跨膜结构域耦合。 使用 DHFR 蛋白片段互补测定以及常规方法 受体激活测定，例如转化和自磷酸化， 这些实验将量化跨膜之间的旋转耦合 结构域和可由配体诱导的激酶结构域。在目标 3 中，我们将 检查近膜结构域在调节两种不同 RTK 中的作用， RET 和 JUT。 RET 细胞外近膜结构域突变 多发性内分泌肿瘤（MEN）和家族性甲状腺髓样癌 (FMTC)，而 KIT 的细胞内近膜结构域的突变导致 胃肠道间质瘤（GIST）和肥大细胞瘤。这些实验 因此，我们将研究这些功能获得性突变的机制 RET 和 KIT 的近膜结构域导致受体激活 下游信号转导。