A Clathrin/Dynamin- and Mannose-6-Phosphate Receptor-Independent Pathway for Granzyme B-Induced Cell Death

• Published in: The Journal of cell biology Vol. 160; no. 2; pp. 223 - 233
• Main Authors: Trapani, Joseph A., Sutton, Vivien R., Kevin Y. T. Thia, Li, Yu Qin, Froelich, Christopher J., Jans, David A., Sandrin, Mauro S., Browne, Kylie A.
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 20.01.2003; The Rockefeller University Press
• Subjects: Animals; Apoptosis; Apoptosis - drug effects; Apoptosis - immunology; Cell death; Cell lines; Cell Membrane - drug effects; Cell Membrane - immunology; Cell Membrane - metabolism; Cell membranes; Cells; Cellular biology; Clathrin - drug effects; Clathrin - genetics; Clathrin - metabolism; Dynamins - drug effects; Dynamins - genetics; Dynamins - metabolism; Endocytosis - drug effects; Endocytosis - immunology; Female; Fluorescence; Graft Rejection - genetics; Graft Rejection - immunology; Granzymes; HeLa Cells; Humans; Killer Cells, Natural - enzymology; Killer Cells, Natural - immunology; L cells; Male; Membrane Glycoproteins - immunology; Membrane Glycoproteins - metabolism; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Knockout; Mutation - genetics; Neoplasms - immunology; Neoplasms - metabolism; Neurotransmitters; Perforin; Pore Forming Cytotoxic Proteins; Receptor, IGF Type 2 - deficiency; Receptor, IGF Type 2 - drug effects; Receptor, IGF Type 2 - genetics; Serine Endopeptidases - deficiency; Serine Endopeptidases - immunology; Serine Endopeptidases - pharmacology; T lymphocytes; T-Lymphocytes, Cytotoxic - enzymology; T-Lymphocytes, Cytotoxic - immunology; Tumors
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.200210150

The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B-mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR-null L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.