Mutational analysis of the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor. A consensus casein kinase II site followed by 2 leucines near the carboxyl terminus is important for intracellular targeting of lysosomal enzymes.
Mutational analysis of the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor. A consensus casein kinase II site followed by 2 leucines near the carboxyl terminus is important for intracellular targeting of lysosomal enzymes.
复制标题
DOI：
10.1016/s0021-9258(18)41533-5
复制
发表时间：
1993-10
期刊：
影响因子：
--
通讯作者：
H. J. Chen;J. Remmler;J. Delaney;D. Messner;P. Lobel
中科院分区：
文献类型：
--
作者：
H. J. Chen;J. Remmler;J. Delaney;D. Messner;P. Lobel
阳离子非依赖性甘露糖6-磷酸/胰岛素样生长因子II受体（CI-MPR）介导溶酶体酶的细胞内分选，结合高尔基体中的溶酶体酶并将其递送至溶酶体区室。该受体还介导细胞外配体的内吞作用。我们已经设计了一种新的方法，该方法严格测量了CI-MPR在细胞内分选中的功能，并使用它来识别分选所需的受体COOH末端附近的先前未表征的信号。我们将突变体受体稳定转染到CI-MPR缺陷小鼠L细胞中，分离表达一系列受体水平的每种突变体的同质克隆细胞系，并测定每种细胞系的受体表达水平和总磷酸化溶酶体酶的分泌水平。作为受体水平的函数的细胞的分泌表型的检查提供了每种突变受体的内在分选效率的灵敏指标。我们发现，嵌合CI-MPR含有牛的胞质外结构域和人或小鼠的跨膜和胞质结构域的功能相同的牛受体，从而证明，分选信号是保守的。对一系列截短和丙氨酸扫描突变体的分析表明，共有酪蛋白激酶II位点后接COOH末端附近的2个亮氨酸，其序列为（-10）DDSDEDLL（-3），对于溶酶体酶分选中的受体功能很重要。
The cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (CI-MPR) mediates intracellular sorting of lysosomal enzymes, binding lysosomal enzymes in the Golgi and delivering them to a lysosomal compartment. The receptor also mediates endocytosis of extracellular ligands. We have devised a new method that rigorously measures function of the CI-MPR in intracellular sorting and used it to identify a previously uncharacterized signal near the COOH terminus of the receptor that is required for sorting. We stably transfect mutant receptors into CI-MPR-deficient mouse L cells, isolate homogeneous clonal cell lines that express a range of receptor levels for each mutant, and assay each cell line for levels of receptor expression and secretion of total phosphorylated lysosomal enzymes. Examination of the secretion phenotype of the cells as a function of receptor levels provides a sensitive indicator of the intrinsic sorting efficiency of each mutant receptor. We find that chimeric CI-MPRs that contain the bovine extracytoplasmic domain and the human or mouse transmembrane and cytoplasmic domains function identically to the bovine receptor, thus demonstrating that sorting signals are conserved. Analysis of a series of truncation and alanine scanning mutants reveals that a consensus casein kinase II site followed by 2 leucines near the COOH terminus that has the sequence (-10)DDSDEDLL(-3) is important for receptor function in sorting of lysosomal enzymes.