Genetic and Cellular Analysis of Retromer Components

描述（由申请人提供）： 膜结合受体的细胞内运输在受体加工和信号调节中起着关键作用。在酵母中，由五种蛋白质（Vps 5 p，Vps 17 p，Vps 17 p）组成的逆转录复合物。Vps 26 p、Vps 29 p和Vps 35 p）指导膜结合水解酶受体Vps 10 p在液泡和高尔基体网络之间的运输。分选连接蛋白1和2（SNX 1和SNX 2），人类直系同源的Vps 5 p和一个假定的哺乳动物retromer复合物的成员，也被认为是在哺乳动物细胞中的蛋白质运输的作用。SNX 1参与表皮生长因子受体（EGFR）和凝血酶受体PARI向溶酶体的正常运输，以在哺乳动物细胞中降解，这是受体下调的关键步骤。最近在小鼠中产生了Snx 1和Snr 2的突变，以评估蛋白质的体内作用。虽然单缺陷动物外表正常，但双缺陷胚胎在胚胎第10.5天死亡。此外，逆转录组分Vps 26的小鼠直向同源物中的无效突变，即Hb 58，导致E9.5的胚胎致死。这些表型的相似性表明哺乳动物retromer复合物在胚胎发育中可能起关键作用。该提案的主要目的是使用来自转基因胚胎的成纤维细胞来确定SNX 1、SNX 2和Hbeta 58在受体运输和信号传导中的作用。将分析细胞的形态异常、受体误运输和受体信号传导失调，这将有助于了解Snx 1-/-; Snx 2-/-和Hbeta 58-/-胚胎致死率。拟议的实验地址的作用，一个假定的哺乳动物retromer复合物在生长因子受体的贩运和信号，其中的失调与肿瘤发生，并在胚胎发育的三个组件。

DESCRIPTION (provided by applicant): Intracellular trafficking of membrane-bound receptors plays a critical role in receptor processing and signal regulation. In yeast, a retromer complex consisting of five proteins (Vps5p, Vps17p. Vps26p, Vps29p, and Vps35p) directs trafficking of the membrane-bound hydrolase receptor Vps10p between the vacuole and trans-Golgi network. Sorting nexins 1 and 2 (SNX1 and SNX2), the human orthologs of Vps5p and members of a putative mammalian retromer complex, have likewise been attributed a role in protein trafficking in mammalian cells. SNX1 has been implicated in the normal trafficking of the epidermal growth factor receptor (EGFR) and the thrombin receptor PARI to lysosomes for degradation in mammalian cells, a critical step in receptor down-regulation. Null mutations in Snx1 and Snr2 were recently generated in mice in order to assess the in vivo roles of the proteins. While the singly-deficient animals are outwardly normal, the doubly-deficient embryos die by embryonic day 10.5. Furthermore, a null mutation in the mouse ortholog of the retromer component Vps26, Hbeta58, results in embryonic lethality by E9.5. The similarity of these phenotypes suggests a potentially critical role for a mammalian retromer complex in embryonic development. The broad objective of this proposal is to define the roles of SNX1, SNX2, and Hbeta58 in receptor trafficking and signaling using fibroblasts derived from genetically modified embryos. The cells will be analyzed for morphological abnormalities, receptor mis-trafficking, and dysregulated receptor signaling that would contribute to an understanding of Snx1-/-;Snx2-/- and Hbeta58-/- embryonic lethality. The proposed experiments address the roles of three components of a putative mammalian retromer complex in growth factor receptor trafficking and signaling, the misregulation of which is associated with tumorigenesis, and in embryonic development.