EGF SIGNALING IN THE INNER MEDULLARY COLLECTING DUCT

内髓收集管中的 EGF 信号传导

• 批准号: 2143156
• 负责人: ISAAC TEITELBAUM
• 金额: $ 16.19万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 1996-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2143156
• 关键词: G protein; SDS polyacrylamide gel electrophoresis; adenylate cyclase; alpha adrenergic agent; arginine vasopressin; biological signal transduction; cyclic AMP; enzyme activity; enzyme inhibitors; enzyme substrate; epidermal growth factor; glycerol; growth factor receptors; guanine nucleotide binding protein; guanosinetriphosphatases; hydrolysis; immunoprecipitation; inositol phosphates; iodine; laboratory rat; pertussis toxin; phosphatidylinositols; phospholipase C; phosphorylation; prostaglandin E; protein kinase A; protein kinase C; protein tyrosine kinase; radiotracer; receptor binding; renal tubule; site directed mutagenesis; threonine; tissue /cell culture; transfection; western blottings

The inner medullary collecting duct of the mammalian nephron is responsible for the final concentration of urine via the stimulation of adenylyl cyclase (AC) in response to arginine vasopressin (AVP). This nephron segment also exhibits phospholipase C (PLC) activity when stimulated by epidermal growth factor (EGF). The studies proposed herein are aimed at further elucidating the mechanism of EGF signalling and examining the interactions between EGF and AVP signalling systems in cultured rat inner medullary collecting tubule (RIMCT) cells. Studies will be performed to examine the roles of EGF receptor autophosphorylation, the Ras:GAP complex, and of tyrosine phosphorylation of Gi in EGF-stimulated PIP2 hydrolysis. Substrates of the EGF receptor tyrosine kinase will be identified by immunoprecipitation with anti-phosphotyrosine followed by immunoblotting with substrate-specific antibodies. The effect of inhibition of EGF-RTK on 125I-EGF binding, 35S-GTPgammaS binding and EGF-stimulated GTPase activity will be examined. The mechanism of inhibition of EGF-stimulated PIP2 hydrolysis by protein kinase C (PKC) will be explored by examining 125I-EGF binding, 35S-GTPgammaS binding and EGF-stimulated GTPase activity in the absence or presence of activation of PKC. These studies will be performed in parallel in cells expressing the T654A mutant of the EGF receptor to assess the role of threonine 654 phosphorylation in PKC-mediated inhibition of EGF-stimulated PIP2 hydrolysis. Substrates of PKC will be identified by labeling cells with 32P-H3PO4, stimulating the kinase, and subjecting the tissue to immunoprecipitation and/or immunoblotting with appropriate antibodies. Similar studies will be performed to identify substrates of cAMP-dependent protein kinase (A-kinase) and to examine the effects of A- kinase activation on 125I-EGF binding, G-protein function and the activity of PLC. To determine the mechanism of inhibition of AVP-stimulated AC by PKC, the kinase will be stimulated directly by dioctanoylglycerol and potential effects of PKC on 125I-AVP receptor binding and AVP-stimulated GTPase activity will be examined. Finally, by infecting RIMCT cells with virus containing cDNAs coding for constitutive active and dominant negative mutants of pertussis toxin- (PT) insensitive G-alpha chains, I will perform studies aimed at determining which of these PT-insensitive alpha chains transduces alpha2-adrenergic inhibition of PGE2-stimulated AC activity in RIMCT cells.

哺乳动物肾单位的内部髓质收集导管是造成的 尿液的最终浓度通过刺激 响应精氨酸加压素（AVP）的循环酶（AC）。 这个肾单位 当通过 表皮生长因子（EGF）。 本文提出的研究针对 进一步阐明EGF信号的机制并检查 培养大鼠内部EGF和AVP信号系统之间的相互作用 髓质收集小管（RIMCT）细胞。 研究将进行 检查EGF受体自磷酸化的作用，RAS：GAP复合物， GI在EGF刺激的PIP2水解中的酪氨酸磷酸化。 EGF受体酪氨酸激酶的底物将通过 用抗磷酸酪氨酸进行免疫沉淀，然后进行免疫印迹 与底物特异性抗体。 抑制EGF-RTK对 125-EGF结合，35S-GTPGAMMAS结合和EGF刺激的GTPase活性 将被检查。 抑制EGF刺激的PIP2的机制 蛋白激酶C（PKC）的水解将通过检查125I-EGF探索 结合，35S-GTPGAMMAS结合和EGF刺激的GTPase活性 PKC的不存在或存在激活。 这些研究将进行 在表达EGF受体的T654a突变体的细胞中并联 评估苏氨酸654磷酸化在PKC介导的抑制作用中的作用 EGF刺激的PIP2水解。 PKC的底物将由 用32P-H3PO4标记细胞，刺激激酶，并对 通过适当的组织进行免疫沉淀和/或免疫印迹 抗体。 将进行类似的研究以确定 cAMP依赖性蛋白激酶（A-激酶），并检查A-的作用 125I-EGF结合，G蛋白功能和活性的激酶激活 Plc的 确定抑制AVP刺激的AC的机制 PKC，激酶将直接通过二耐糖酰甘油和 PKC对125-AVP受体结合和AVP刺激的潜在影响 将检查GTPase活性。 最后，通过感染了带有RIMCT细胞的 含有编码构成活性和显性阴性的cDNA的病毒 百日咳毒素（PT）不敏感的G-α链的突变体，我将执行 旨在确定这些不敏感的α链中的哪一个 转导α2-肾上腺素能抑制PGE2刺激的AC活性在 rimct细胞。