FUNCTIONAL ANALYSIS OF RET SIGNALING IN RENAL EPITHELIAL CELLS

哺乳动物肾脏的发育需要 两个祖细胞组织，输尿管芽上皮和 后肾间充质输尿管芽发出的信号 间充质增殖和分化，从而产生大部分的 肾单位中的上皮细胞类型。相反，间充质来源 信号诱导输尿管不良上皮增生， 分支形态发生以产生集合管系统。虽然 这些信号及其受体仍然是难以捉摸的，它们的机制 作用的分子基础是了解肾 细胞生长和分化。受体酪氨酸激酶RET是 表达于输尿管芽的尖端，并且对于输尿管的 芽长出和分枝。PI实验室最近的研究表明 胶质细胞源性生长因子GDNF，表达于 后肾间充质，激活RET受体，可以刺激 肾器官培养物中的分枝形态发生。因此，GDNF和RET 一个共同的信号通路，促进输尿管芽的一部分， 增殖和分支。这条途径不仅对 早期肾脏发育和生长，但也可能在异常 输尿管芽衍生细胞的增殖，例如在膀胱囊性疾病中， 收集管道。 本提案的具体目标将涉及可再生能源技术的机制， 组织培养模型系统中的信号传导。我们建立了 转化的MDCK肾上皮中RET活性的生物测定 细胞这些细胞表现出增加的散射，细胞运动， 响应RET激活的形态学变化。我们还 鉴定了一组新的磷酸化蛋白， 激活RET。我们将确定关键的残留物， RET的细胞质结构域，是生物学反应所需的 MDCK细胞和这些潜在的第二信使的结合位点 使用一组RET突变体。RET结合蛋白将通过 免疫亲和策略或酵母双杂交系统。 此外，我们将描述候选基因的表达模式， 第二信使，并评估其在肾脏发育中的作用。这些 研究将直接解决RET信号传导的机制， 肾上皮细胞对活化RET的生物学反应。

The development of the mammalian kidney requires the inductive interactions of two progenitor tissues, the ureteric bud epithelium and the metanephric mesenchyme. Signals emanating from the ureteric bud induce the mesenchyme to proliferate and differentiate, thus generating most of the epithelial cell types in the nephron. Reciprocally, mesenchyme derived signals induce the ureteric bad epithelium to proliferate and undergo branching morphogenesis to generate the collecting duct system. Although these signals and their receptors have remained elusive, their mechanisms of action are fundamental to understanding the molecular basis of renal cell growth and differentiation. The receptor tyrosine kinase RET is expressed at the tips of the ureteric bud and is essential for ureteric bud outgrowth and branching. Recent work in the PI's lab has demonstrated that the glial cell derived growth factor GDNF, expressed in the metanephric mesenchyme, activates the RET receptor and can stimulate branching morphogenesis in kidney organ cultures. Thus, GDNF and RET are part of a common signaling pathway that promoters ureteric bud proliferation and branching. This pathway may not only be critical for early kidney development and growth, but may also function in aberrant proliferation of ureteric bud derived cells, such as in cystic diseases of the collecting duct. The specific aims of this proposal will address the mechanism of RET signaling in a tissue culture model system. We have established a biological assay for RET activity in transformed MDCK renal epithelial cells. These cells show increased scattering, cell motility, and morphological changes in response to RET activation. We have also identified a set of novel phospho-proteins that co-precipitate with activated RET in MDCK cells. We will determine critical residues in the cytoplasmic domain of RET that are required for the biological response of MDCK cells and the binding sites for these potential second messengers using a panel of RET mutants. The RET binding proteins will be cloned by either an immunoaffinity strategy or with the yeast two-hybrid system. Furthermore, we will characterize the expression patterns of candidate second messengers and assess their roles in the developing kidney. These studies will directly address the mechanism of RET signaling and the biological responses of renal epithelial cells to activated RET.