Genetic and Cellular Analysis of Retromer Components

描述(由申请人提供)： 膜结合受体的细胞内转运在受体的加工和信号调节中起着关键作用。酵母中由五种蛋白质(Vps5p，Vps17p)组成的逆转录复合体。Vps26p、Vps29p和Vps35p)指导膜结合的水解酶受体Vps10p在液泡和跨高尔基网络之间的运输。分类连接素1和2(SNX1和SNX2)是Vps5p的人类同源基因，也是哺乳动物逆转录复合体的成员，同样被认为在哺乳动物细胞的蛋白质运输中发挥作用。在哺乳动物细胞中，SNX1参与了表皮生长因子受体(EGFR)和凝血酶受体部分的正常转运以降解溶酶体，这是受体下调的关键步骤。最近在小鼠中产生了Snx1和Snr2的零突变，以评估这些蛋白质在体内的作用。虽然单一缺陷的动物表面上是正常的，但双缺陷的胚胎在胚胎第10.5天就会死亡。此外，小鼠逆转录成分Vps26，Hbeta58的零突变会导致E9.5的胚胎死亡。这些表型的相似性表明哺乳动物逆转录复合体在胚胎发育中具有潜在的关键作用。这项建议的广泛目标是确定SNX1、SNX2和Hbeta58在使用来自转基因胚胎的成纤维细胞的受体运输和信号转导中的作用。将对这些细胞进行形态异常、受体错误运输和受体信号异常的分析，这将有助于理解Snx1-/-、Snx2-/-和Hbeta58-/-胚胎致死性。拟议的实验解决了哺乳动物逆转录复合体的三个组成部分在生长因子受体运输和信号转导中的作用，其中的错误调控与肿瘤的发生和胚胎发育有关。

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.