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.
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.
Chen
H J
HJ
Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854.
Remmler
J
J
Delaney
J C
JC
Messner
D J
DJ
Lobel
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D047390:Casein Kinase II; D002402:Cathepsin D / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002999:Clone Cells; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D007739:L Cells; D008247:Lysosomes / Q000201:enzymology*; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D017527:Receptor, IGF Type 2 / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D002402:Cathepsin D