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/阳离子非依赖性甘露糖 6-磷酸 受体 (IFG-II/CI-MPR) 是一种多功能蛋白，可结合 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，极化 MPR 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表达于根尖 Caco-2 细胞表面，与基底外侧表达的受体不同 表面，无法内吞溶酶体酶。 的目标 该提案旨在确定： 1) 调节内部的因素 极化上皮细胞中 MPR 的细胞分选，以及 2) IGF-II/CI-MPR 失败的分子/细胞基础 内化肠上皮细胞顶端表面的配体。 达到 这些目标，对过滤培养的 Caco-2 细胞进行脉冲追踪标记研究 结合特定领域的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 定位到顶面和基底外侧表面。 的地区 MPR 的胞质结构域对其细胞内靶向很重要 将通过将突变型 MPR cDNA 转染到 Caco-2 细胞中来鉴定 分析突变体 MPR 的排序和稳态表面分布。 探究功能表达的分子/细胞基础 顶端和基底外侧表面有不同的 IGF-II/CI-MPR， 来自分离的顶膜和基底外侧膜的受体将是 特征在于大小、完整细胞质的存在 N-连接寡糖的结构域、存在和类型、敏感性 蛋白水解，以及结合和内化配体的能力 不同的膜环境。 总而言之，这些研究将导致 更好地理解 MPR 的功能表达 对于溶酶体的生物发生至关重要，溶酶体是一种重要的细胞器 调节吸收性肠上皮细胞中的蛋白质周转。 这些研究 还将解决我们确定的长期目标：1）因素 参与肠上皮细胞极性的建立和维持， 2) IGF-II.CI-MPR 在肠上皮细胞分化中的作用和 增殖。