CELL BIOLOGY OF TRANSMEMBRANE SIGNALING PROTEINS

这项研究的长期目标是阐明新的 管理信息交流的机制， 细胞 这里特别感兴趣的是 受体酪氨酸激酶的跨膜配体。 这项建议 重点是神经调节蛋白，一个多能细胞对细胞的家族， 信号分子 神经调节素及其受体， 是受体酪氨酸激酶，在发育过程中发挥重要作用， 神经系统和心脏的决策，他们是 也与乳腺癌的发病机制有关。 许多 神经调节蛋白作为跨膜蛋白合成。 虽然 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， 结构域序列更高度保守（87%相同， 人和鸡）。 这种严格的序列保守性 强烈表明细胞质结构域参与了 对神经调节蛋白的生物学作用至关重要。 令人惊讶的是，神经调节蛋白的胞质结构域在很大程度上被 在以前的研究中被忽略，因此其功能目前尚不清楚。 此外，关于胞质结构域知之甚少 受体酪氨酸的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二- 杂交筛选 这些新的结果支持了这样的观点， 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鉴定的激酶的相互作用将被 在哺乳动物细胞中测定，以证实这种假设的作用 神经调节蛋白胞质结构域。 免疫共沉淀， 免疫细胞化学和培养细胞中的磷酸化测定 将用于神经调节蛋白胞质结构域的这些第一次研究 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是共同的， 在发育和成年运动和感觉神经元中表达。 到 确定神经调节蛋白本身是否作为受体起作用， 实验将测试影响神经调节蛋白的事件 细胞外结构域调节神经调节素和细胞的相互作用 识别的激酶。 为了鉴定其他蛋白质， 胞质结构域功能，进一步的双杂交筛选将 进行。 预计拟议的研究将产生新的 深入了解调节发育和 神经系统、心脏和乳房的病理过程。

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.