The lysosome plays an important role in eukaryotic cells as the primary site of degradation of both cellular and extracellular macromolecules. Selective delivery of proteins to the lysosome involves addition of a common address marker (the mannose 6-phosphate marker) to the proteins, and membrane-associated receptors that bind the marker and mediate translocation of the proteins to the lysosomal compartment. Recent studies suggest that under certain conditions this targeting process can be altered such that enzymes normally destined for lysosomes are secreted from the cell. This regulatory mechanism may be important physiologically since extracellular lysosomal enzymes may play important roles in tumor growth and metastasis as well as in normal cell growth. The long range goal of this proposal is to determine the molecular basis for normal and regulated trafficking of lysosomal enzymes. The proposed studies will involve molecular and cellular biological studies on MEP, a lysosomal cysteine proteinase whose trafficking is regulated by growth factors and cellular transformation, and the receptors thought to mediate lysosomal trafficking: the cation dependent mannose 6-phosphate (Man-6-P) receptor and the Man-6-P/IGF-II receptor. Site-specific mutagenesis and expression of MEP cDNA will be used to identify structural determinants on the protein responsible for addition of the Man-6-P marker. Analysis of the protein and carbohydrate portions of MEP, and examination of the effects of growth factors on the synthesis and trafficking of the two receptors, will be carried out in order to further define the basis for MEP's regulated secretion. These studies will provide information leading to determination of the recognition structure responsible for selective phosphorylation of lysosomal proteins and will help elucidate the mechanisms involved in regulated trafficking of lysosomal proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK036632-07
Application #
3235108
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1986-01-01
Project End
1995-03-31
Budget Start
1993-04-01
Budget End
1994-03-31
Support Year
7
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Tufts University
Department
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02111
Cuozzo, J W; Tao, K; Wu, Q L et al. (1995) Lysine-based structure in the proregion of procathepsin L is the recognition site for mannose phosphorylation. J Biol Chem 270:15611-9
Cuozzo, J W; Sahagian, G G (1994) Lysine is a common determinant for mannose phosphorylation of lysosomal proteins. J Biol Chem 269:14490-6
Tao, K; Stearns, N A; Dong, J et al. (1994) The proregion of cathepsin L is required for proper folding, stability, and ER exit. Arch Biochem Biophys 311:19-27
Prence, E M; Dong, J M; Sahagian, G G (1990) Modulation of the transport of a lysosomal enzyme by PDGF. J Cell Biol 110:319-26
Dong, J M; Sahagian, G G (1990) Basis for low affinity binding of a lysosomal cysteine protease to the cation-independent mannose 6-phosphate receptor. J Biol Chem 265:4210-7
Stearns, N A; Dong, J M; Pan, J X et al. (1990) Comparison of cathepsin L synthesized by normal and transformed cells at the gene, message, protein, and oligosaccharide levels. Arch Biochem Biophys 283:447-57
Kiess, W; Thomas, C L; Greenstein, L A et al. (1989) Insulin-like growth factor-II (IGF-II) inhibits both the cellular uptake of beta-galactosidase and the binding of beta-galactosidase to purified IGF-II/mannose 6-phosphate receptor. J Biol Chem 264:4710-4
Dong, J M; Prence, E M; Sahagian, G G (1989) Mechanism for selective secretion of a lysosomal protease by transformed mouse fibroblasts. J Biol Chem 264:7377-83
Jin, M; Sahagian Jr, G G; Snider, M D (1989) Transport of surface mannose 6-phosphate receptor to the Golgi complex in cultured human cells. J Biol Chem 264:7675-80
Kiess, W; Blickenstaff, G D; Sklar, M M et al. (1988) Biochemical evidence that the type II insulin-like growth factor receptor is identical to the cation-independent mannose 6-phosphate receptor. J Biol Chem 263:9339-44