SORTING OF THE IGF-II RECEPTOR IN POLARIZED CELLS

极化细胞中 IGF-II 受体的分类

• 批准号: 2734112
• 负责人: Nancy M. Dahms
• 金额: $ 12.63万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734112
• 关键词: apical membrane; basolateral membrane; binding proteins; carbohydrate receptor; cell differentiation; cellular polarity; gastrointestinal epithelium; growth factor receptors; insulinlike growth factor; intracellular transport; laboratory rat; lysosomes; mannose 6 phosphate; membrane activity; phosphorylation; protein transport; receptor binding; receptor expression; site directed mutagenesis; tissue /cell culture; transfection

The insulin-like growth factor II/cation-independent mannose 6-phosphate receptor (IFG-II/CI-MPR) is a multifunctional protein that binds both IGF-II and lysosomal enzymes and, along with the cation-dependent MPR (CD-MPR), mediates the targeting of newly synthesized lysosomal enzymes to the lysosome. Our recent studies have demonstrated the polarized expression of the MPRs in the enterocyte-like cell line, Caco-2. In addition, we have found that the IGF-II/CI-MPR expressed on the apical surface of Caco-2 cells, unlike the receptor expressed on the basolateral surface, is unable to endocytose lysosomal enzymes. The objectives of the proposal are to determine: 1) the factors which regulate the intra cellular sorting of the MPRs in polarized epithelial cells, and 2) the molecular/cellular basis for the failure of the IGF-II/CI-MPR to internalize ligands from the apical surface of enterocytes. To achieve these goals, pulse-chase labeling studies on filter grown Caco-2 cells in conjunction with domain-specific cell surface labeling techniques will be performed to determine the kinetics and intracellular pathways of targeting MPRs to the apical and basolateral surfaces. The region(s) of the MPRs' cytoplasmic domain important for their intracellular targeting will be identified by transfecting mutant MPR cDNAs into Caco-2 cells and analyzing the mutant MPRs' sorting and steady state surface distribution. To probe the molecular/cellular basis for the expression of functionally distinct IGF-II/CI-MPRs on the apical and basolateral surface, the receptor from isolated apical and basolateral membranes will be characterized with respect to size, presence of an intact cytoplasmic domain, presence and type of N-linked oligosaccharides, susceptibility to proteolysis, and the ability to bind and internalize ligands in different membrane environments. Taken together, these studies will lead to a better understanding of the functional expression of the MPRs that are essential for the biogenesis of lysosomes, an organelle important for regulating protein turnover in absorptive enterocytes. These studies will also address our long-term goals of identifying: 1) the factors involved in the establishment and maintenance of polarity in enterocytes, and 2) the role of IGF-II.CI-MPR in enterocyte differentiation and proliferation.

胰岛素样生长因子II/阳离子独立于6-磷酸 受体（IFG-II/CI-MPR）是一种多功能蛋白，结合两者 IGF-II和溶酶体酶以及阳离子依赖性MPR （CD-MPR），介导新合成的溶酶体酶的靶向 到溶酶体。 我们最近的研究表明了两极分化 MPRS在肠细胞样细胞系Caco-2中的表达。 在 此外，我们发现IGF-II/CI-MPR在顶端表达 与基底外侧表达的受体不同，Caco-2细胞的表面 表面，无法内吞溶酶体酶。 目标的目标 该提议是确定：1​​）调节内部内部的因素 在极化上皮细胞中MPRS的细胞分选，2） IGF-II/CI-MPR失败的分子/细胞基础 从肠上皮细胞的顶部表面内部化配体。 实现 这些目标，脉冲马在过滤器生长的caco-2细胞上的标记研究 结合域特异性细胞表面标记技术将 进行以确定动力学和细胞内途径 将MPR靶向顶端和基底外侧表面。 区域 MPRS的细胞质结构域对于其细胞内靶向很重要 将通过将突变体MPR cDNA转染到Caco-2细胞和 分析突变体MPRS的排序和稳态表面分布。 探测分子/细胞基础的功能表达 顶端和基底外侧表面上的不同IGF-II/CI-MPR 来自孤立的顶端和基底外侧膜的受体将是 在大小，完整的细胞质的存在方面。 N连接的寡糖的域，存在和类型，易感性 蛋白水解以及结合和内在化配体的能力 不同的膜环境。 综上所述，这些研究将领导 可以更好地理解MPR的功能表达 对于溶酶体的生物发生是必不可少的，这对于细胞器而言至关重要 调节吸收性肠细胞中的蛋白质更新。 这些研究 还将解决我们识别的长期目标：1）因素 参与肠细胞极性的建立和维持， 2）IGF-ii.ci-mpr在肠细胞分化和 增殖。