CELL BIOLOGY OF TRANSMEMBRANE SIGNALING PROTEINS

跨膜信号蛋白的细胞生物学

• 批准号: 2734838
• 负责人: DOUGLAS L. FALLS
• 金额: $ 12.36万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734838
• 关键词: SDS polyacrylamide gel electrophoresis; biological signal transduction; cell biology; cell cell interaction; enzyme linked immunosorbent assay; enzyme substrate; immunocytochemistry; immunofluorescence technique; immunoprecipitation; ion exchange chromatography; laboratory rat; membrane proteins; neurons; phosphorylation; protein signal sequence; protein structure function; protein tyrosine kinase; tissue /cell culture; transcription factor; western blottings; yeast two hybrid system

The long-term goal of this research is to elucidate novel mechanisms that regulate the exchange of information between cells. Of specific interest here are the biological functions for transmembrane ligands of receptor tyrosine kinases. This proposal focuses on the neuregulins, a family of multipotent cell-to-cell signaling molecules. The neuregulins and their receptors, which are receptor tyrosinekinases, play essential roles in developmental decision-making within the nervous system and heart, and they are also implicated in the pathogenesis of breast cancer. Many of the neuregulins are synthesized as transmembrane proteins. Although it is the extracellular domain of transmembrane neuregulins that activates their cognate tyrosinekinase receptors, the cytoplasmic domain sequence is more highly conserved (87% identical between humans and chickens). This stringent conservation of sequence strongly suggests involvement of the cytoplasmic domain in functions critical to the biological actions of neuregulins. Surprisingly, the neuregulin cytoplasmic domain has been largely ignored in prior studies, so its function are currently unknown. Furthermore, little is known regarding cytoplasmic domain functions for other transmembrane ligands of receptor tyrosine kinases. Two non-receptor protein kinases that interact with the cytoplasmic domain of the neuregulins have now been identified by yeast two- hybrid screening. These new results support the idea that the neuregulin cytoplasmic domain functions in regulatory pathways central to neuregulin biology. The physical and functional interactions of the neuregulins with these identified kinases will be determined in mammalian cells to confirm this hypothesized role for the neuregulin cytoplasmic domain. Co-immunoprecipitation, immunocytochemistry, and phosphorylation assays in cultured cells will be used in these first studies of neuregulin cytoplasmic domain interactions with kinases. Immunohistochemistry will be used to determine whether neuregulins and the interacting kinases are co- expressed in developing and adult motor and sensory neurons. To determine if neuregulins themselves function as receptors, experiments will test whether events affecting the neuregulin extracellular domain regulate the interactions of neuregulin and the identified kinases. To identify additional proteins mediating cytoplasmic domain function, further two-hybrid screening will be conducted. The proposed studies are expected to produce new insights into molecular mechanisms regulating developmental and pathological processes in the nervous system, heart, and breast.

这项研究的长期目标是阐明新的 规范之间信息交换的机制 细胞。 这里特别感兴趣的是以下生物功能： 受体酪氨酸激酶的跨膜配体。 这个提议 专注于神经调节蛋白，一个多能细胞间的家族 信号分子。 神经调节蛋白及其受体， 是受体酪氨酸激酶，在发育中发挥重要作用 神经系统和心脏内的决策，它们是 也参与乳腺癌的发病机制。 许多 神经调节蛋白被合成为跨膜蛋白。 虽然它 是跨膜神经调节蛋白的胞外结构域 激活它们的同源酪氨酸激酶受体，细胞质 结构域序列更加高度保守（87% 相同） 人和鸡）。 这种严格的序列保守 强烈表明细胞质结构域参与 对神经调节蛋白的生物作用至关重要的功能。 令人惊讶的是，神经调节蛋白胞质结构域在很大程度上被 在之前的研究中被忽视，因此其功能目前尚不清楚。 此外，对于细胞质结构域知之甚少。 受体酪氨酸其他跨膜配体的功能 激酶。 两种与细胞质相互作用的非受体蛋白激酶 神经调节蛋白的结构域现已被酵母鉴定为两类： 混合筛选。 这些新结果支持了以下观点： 神经调节蛋白胞质结构域在调节途径中的功能 神经调节蛋白生物学的核心。 物理和功能 神经调节蛋白与这些已识别的激酶的相互作用将是 在哺乳动物细胞中进行测定以证实这种假设的作用 对于神经调节蛋白胞质结构域。 免疫共沉淀， 培养细胞中的免疫细胞化学和磷酸化测定 将用于神经调节蛋白胞质结构域的这些初步研究 与激酶的相互作用。 免疫组织化学将用于 确定神经调节蛋白和相互作用的激酶是否共同作用 在发育中和成年的运动和感觉神经元中表达。 到 确定神经调节蛋白本身是否起到受体的作用， 实验将测试是否影响神经调节蛋白的事件 细胞外结构域调节神经调节蛋白和 鉴定出激酶。 鉴定介导其他蛋白质 细胞质结构域功能，进一步的二杂交筛选 实施。 拟议的研究预计将产生新的 对调节发育和发育的分子机制的见解 神经系统、心脏和乳房的病理过程。