Limits...
Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Uekita T, Itoh Y, Yana I, Ohno H, Seiki M - J. Cell Biol. (2001)

Bottom Line: Di-leucine (Leu571-572 and Leu578-579) and tyrosine573 residues are important for the internalization, and the mu2 subunit of adaptor protein 2, a component of clathrin-coated pits for membrane protein internalization, was found to bind to the LLY573 sequence.MT1-MMP was internalized predominantly at the adherent edge and was found to colocalize with clathrin-coated vesicles.Interestingly, whereas expression of MT1-MMP enhances cell migration and invasion, the internalization-defective mutants failed to promote either activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan.

ABSTRACT
Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that degrades the pericellular extracellular matrix (ECM) and is expressed in many migratory cells, including invasive cancer cells. MT1-MMP has been shown to localize at the migration edge and to promote cell migration; however, it is not clear how the enzyme is regulated during the migration process. Here, we report that MT1-MMP is internalized from the surface and that this event depends on the sequence of its cytoplasmic tail. Di-leucine (Leu571-572 and Leu578-579) and tyrosine573 residues are important for the internalization, and the mu2 subunit of adaptor protein 2, a component of clathrin-coated pits for membrane protein internalization, was found to bind to the LLY573 sequence. MT1-MMP was internalized predominantly at the adherent edge and was found to colocalize with clathrin-coated vesicles. The mutations that disturb internalization caused accumulation of the enzyme at the adherent edge, though the net proteolytic activity was not affected much. Interestingly, whereas expression of MT1-MMP enhances cell migration and invasion, the internalization-defective mutants failed to promote either activity. These data indicate that dynamic turnover of MT1-MMP at the migration edge by internalization is important for proper enzyme function during cell migration and invasion.

Show MeSH

Related in: MedlinePlus

Internalization of wild-type MT1-MMP in CHO-K1 cells. (A) CHO-K1 cells were transfected with expression plasmids for MT1-F or hTfnR. 48 h later, the internalization of cell surface–expressed MT1-F or hTfnR was examined using 125I-labeled anti-FLAG M2 antibody or 125I-labeled hTfn as tracer ligands. The radioactivity relative to the amount of tracers bound to the surface at time 0 is plotted. ▪, radioactivity spontaneously released into the culture medium (Medium); ♦, radioactivity released by acid wash (Cell surface); ○, acid wash–resistant radioactivity (Internalize). Mean values of three independent experiments are shown (mean ± SD). Transfected cells were also analyzed by Western blotting using anti-FLAG M2 or anti-hTfnR antibodies (inset). (B) CHO-K1 cells expressing MT1-F or mock-transfected cells were incubated with anti-FLAG M2 antibody, after which the cells were incubated at 37°C for the indicated periods of time. The cell surface and internalized MT1-F molecules were analyzed by immunolocalization using AlexaTM488-conjugated anti–mouse IgG. Internalized MT1-F molecules were observed by washing the cells with acid solution before fixation and permeabilization. Signals were observed by confocal laser microscopy. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199326&req=5

fig1: Internalization of wild-type MT1-MMP in CHO-K1 cells. (A) CHO-K1 cells were transfected with expression plasmids for MT1-F or hTfnR. 48 h later, the internalization of cell surface–expressed MT1-F or hTfnR was examined using 125I-labeled anti-FLAG M2 antibody or 125I-labeled hTfn as tracer ligands. The radioactivity relative to the amount of tracers bound to the surface at time 0 is plotted. ▪, radioactivity spontaneously released into the culture medium (Medium); ♦, radioactivity released by acid wash (Cell surface); ○, acid wash–resistant radioactivity (Internalize). Mean values of three independent experiments are shown (mean ± SD). Transfected cells were also analyzed by Western blotting using anti-FLAG M2 or anti-hTfnR antibodies (inset). (B) CHO-K1 cells expressing MT1-F or mock-transfected cells were incubated with anti-FLAG M2 antibody, after which the cells were incubated at 37°C for the indicated periods of time. The cell surface and internalized MT1-F molecules were analyzed by immunolocalization using AlexaTM488-conjugated anti–mouse IgG. Internalized MT1-F molecules were observed by washing the cells with acid solution before fixation and permeabilization. Signals were observed by confocal laser microscopy. Bar, 10 μm.

Mentions: To study the internalization of MT1-MMP, we expressed human MT1-MMP having a FLAG tag at the NH2 terminus (MT1-F) in CHO-K1 cells, and monitored the fate of MT1-F on the cell surface. Human transferrin receptor (hTfnR), a well-characterized marker of endocytosis (Zuk and Elferink, 1999), served as a positive control. The expression of MT1-F or hTfnR by the transfected cells was confirmed by Western blotting (Fig. 1 A, inset). To monitor the fate of the cell surface molecules, either 125I-labeled anti-FLAG M2 antibody (125I-M2) or 125I-labeled human transferrin (125I-hTfn) was used as a tracer. These ligands bound specifically to the cells expressing MT1-F and hTfnR, respectively (unpublished data; Fig. 1 B). The cell surface ligands could be removed almost completely by washing the cells with acid solution (Fig. 1 A). Thus, the internalized tracer molecules could be measured as the radioactivity that was resistant to the acid wash.


Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Uekita T, Itoh Y, Yana I, Ohno H, Seiki M - J. Cell Biol. (2001)

Internalization of wild-type MT1-MMP in CHO-K1 cells. (A) CHO-K1 cells were transfected with expression plasmids for MT1-F or hTfnR. 48 h later, the internalization of cell surface–expressed MT1-F or hTfnR was examined using 125I-labeled anti-FLAG M2 antibody or 125I-labeled hTfn as tracer ligands. The radioactivity relative to the amount of tracers bound to the surface at time 0 is plotted. ▪, radioactivity spontaneously released into the culture medium (Medium); ♦, radioactivity released by acid wash (Cell surface); ○, acid wash–resistant radioactivity (Internalize). Mean values of three independent experiments are shown (mean ± SD). Transfected cells were also analyzed by Western blotting using anti-FLAG M2 or anti-hTfnR antibodies (inset). (B) CHO-K1 cells expressing MT1-F or mock-transfected cells were incubated with anti-FLAG M2 antibody, after which the cells were incubated at 37°C for the indicated periods of time. The cell surface and internalized MT1-F molecules were analyzed by immunolocalization using AlexaTM488-conjugated anti–mouse IgG. Internalized MT1-F molecules were observed by washing the cells with acid solution before fixation and permeabilization. Signals were observed by confocal laser microscopy. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199326&req=5

fig1: Internalization of wild-type MT1-MMP in CHO-K1 cells. (A) CHO-K1 cells were transfected with expression plasmids for MT1-F or hTfnR. 48 h later, the internalization of cell surface–expressed MT1-F or hTfnR was examined using 125I-labeled anti-FLAG M2 antibody or 125I-labeled hTfn as tracer ligands. The radioactivity relative to the amount of tracers bound to the surface at time 0 is plotted. ▪, radioactivity spontaneously released into the culture medium (Medium); ♦, radioactivity released by acid wash (Cell surface); ○, acid wash–resistant radioactivity (Internalize). Mean values of three independent experiments are shown (mean ± SD). Transfected cells were also analyzed by Western blotting using anti-FLAG M2 or anti-hTfnR antibodies (inset). (B) CHO-K1 cells expressing MT1-F or mock-transfected cells were incubated with anti-FLAG M2 antibody, after which the cells were incubated at 37°C for the indicated periods of time. The cell surface and internalized MT1-F molecules were analyzed by immunolocalization using AlexaTM488-conjugated anti–mouse IgG. Internalized MT1-F molecules were observed by washing the cells with acid solution before fixation and permeabilization. Signals were observed by confocal laser microscopy. Bar, 10 μm.
Mentions: To study the internalization of MT1-MMP, we expressed human MT1-MMP having a FLAG tag at the NH2 terminus (MT1-F) in CHO-K1 cells, and monitored the fate of MT1-F on the cell surface. Human transferrin receptor (hTfnR), a well-characterized marker of endocytosis (Zuk and Elferink, 1999), served as a positive control. The expression of MT1-F or hTfnR by the transfected cells was confirmed by Western blotting (Fig. 1 A, inset). To monitor the fate of the cell surface molecules, either 125I-labeled anti-FLAG M2 antibody (125I-M2) or 125I-labeled human transferrin (125I-hTfn) was used as a tracer. These ligands bound specifically to the cells expressing MT1-F and hTfnR, respectively (unpublished data; Fig. 1 B). The cell surface ligands could be removed almost completely by washing the cells with acid solution (Fig. 1 A). Thus, the internalized tracer molecules could be measured as the radioactivity that was resistant to the acid wash.

Bottom Line: Di-leucine (Leu571-572 and Leu578-579) and tyrosine573 residues are important for the internalization, and the mu2 subunit of adaptor protein 2, a component of clathrin-coated pits for membrane protein internalization, was found to bind to the LLY573 sequence.MT1-MMP was internalized predominantly at the adherent edge and was found to colocalize with clathrin-coated vesicles.Interestingly, whereas expression of MT1-MMP enhances cell migration and invasion, the internalization-defective mutants failed to promote either activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan.

ABSTRACT
Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that degrades the pericellular extracellular matrix (ECM) and is expressed in many migratory cells, including invasive cancer cells. MT1-MMP has been shown to localize at the migration edge and to promote cell migration; however, it is not clear how the enzyme is regulated during the migration process. Here, we report that MT1-MMP is internalized from the surface and that this event depends on the sequence of its cytoplasmic tail. Di-leucine (Leu571-572 and Leu578-579) and tyrosine573 residues are important for the internalization, and the mu2 subunit of adaptor protein 2, a component of clathrin-coated pits for membrane protein internalization, was found to bind to the LLY573 sequence. MT1-MMP was internalized predominantly at the adherent edge and was found to colocalize with clathrin-coated vesicles. The mutations that disturb internalization caused accumulation of the enzyme at the adherent edge, though the net proteolytic activity was not affected much. Interestingly, whereas expression of MT1-MMP enhances cell migration and invasion, the internalization-defective mutants failed to promote either activity. These data indicate that dynamic turnover of MT1-MMP at the migration edge by internalization is important for proper enzyme function during cell migration and invasion.

Show MeSH
Related in: MedlinePlus