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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.

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Localization of MT1-F at the adherent edge and its internalization. (A) CHO-K1 cells were cotransfected with the plasmids for either MT1-F or ΔCP mutant and that for GFP. The cells were incubated with anti-FLAG M2 antibody, washed, and then incubated at 37°C for the indicated periods of time. The M2 antibody was detected by immunostaining with Cy3-labeled anti–mouse IgG. Signals were observed by confocal laser microscopy. The internalized antibody was stained using the cells that had been acid washed. Adherent layer; confocal section at the adherent layer, Integrated layers; integrated confocal layers. Bar, 10 μm. (B) The number of cells that retained the signals at the adherent layer were counted. Transfected cells were identified by GFP expression. At time 0, almost 100% of GFP-positive cells expressed MT1-F or MT1-F(ΔCP) at the adherent layer. Cells that retained more than 30% of the original average fluorescence intensity at the adherent layer at each time point were counted as positive and presented. 100 cells were counted independently three times and the values presented are the mean ± SD.
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fig7: Localization of MT1-F at the adherent edge and its internalization. (A) CHO-K1 cells were cotransfected with the plasmids for either MT1-F or ΔCP mutant and that for GFP. The cells were incubated with anti-FLAG M2 antibody, washed, and then incubated at 37°C for the indicated periods of time. The M2 antibody was detected by immunostaining with Cy3-labeled anti–mouse IgG. Signals were observed by confocal laser microscopy. The internalized antibody was stained using the cells that had been acid washed. Adherent layer; confocal section at the adherent layer, Integrated layers; integrated confocal layers. Bar, 10 μm. (B) The number of cells that retained the signals at the adherent layer were counted. Transfected cells were identified by GFP expression. At time 0, almost 100% of GFP-positive cells expressed MT1-F or MT1-F(ΔCP) at the adherent layer. Cells that retained more than 30% of the original average fluorescence intensity at the adherent layer at each time point were counted as positive and presented. 100 cells were counted independently three times and the values presented are the mean ± SD.

Mentions: MT1-MMP is mainly localized at the adherent edge and migration front of cells. This localization appears to be appropriate for promoting cell invasion and migration. The continuous internalization of MT1-MMP at the edge may serve to substitute inactivated molecules, including TIMP-2 inhibited molecules and processed fragments, with freshly synthesized ones during cell migration. To investigate this possibility further, we examined the internalization of MT1-F at the cell adherent edge using a confocal laser microscope. The localization of the enzyme at the adherent layer was not affected by the mutation that abolishes internalization (ΔCP) (Fig. 7 A, Adherent layer and Integrated layers, 0 min). When MT1-F–expressing cells were incubated for 30 min, the wild-type enzyme present at the cell adherent layer was drastically decreased and internalized molecules were detected (Wild Type, Integrated layers, Adherent layer, and Intracellular; 30 min). However, when MT1-F(ΔCP) was expressed, the signals at the cell adherent layer were not diminished even after a 30-min incubation (Adherent layer, ΔCP). Internalization at this point was also not observed (Intracellular, ΔCP). The number of cells that expressed MT1-F or MT1-F(ΔCP) at the adherent edge was also counted (Fig. 7 B). Thus, the mutation that disturbs the internalization of MT1-MMP clearly abolishes the active turnover of the molecule at the adherent edge. Similar results were obtained with other internalization-defective mutants (unpublished data).


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)

Localization of MT1-F at the adherent edge and its internalization. (A) CHO-K1 cells were cotransfected with the plasmids for either MT1-F or ΔCP mutant and that for GFP. The cells were incubated with anti-FLAG M2 antibody, washed, and then incubated at 37°C for the indicated periods of time. The M2 antibody was detected by immunostaining with Cy3-labeled anti–mouse IgG. Signals were observed by confocal laser microscopy. The internalized antibody was stained using the cells that had been acid washed. Adherent layer; confocal section at the adherent layer, Integrated layers; integrated confocal layers. Bar, 10 μm. (B) The number of cells that retained the signals at the adherent layer were counted. Transfected cells were identified by GFP expression. At time 0, almost 100% of GFP-positive cells expressed MT1-F or MT1-F(ΔCP) at the adherent layer. Cells that retained more than 30% of the original average fluorescence intensity at the adherent layer at each time point were counted as positive and presented. 100 cells were counted independently three times and the values presented are the mean ± SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199326&req=5

fig7: Localization of MT1-F at the adherent edge and its internalization. (A) CHO-K1 cells were cotransfected with the plasmids for either MT1-F or ΔCP mutant and that for GFP. The cells were incubated with anti-FLAG M2 antibody, washed, and then incubated at 37°C for the indicated periods of time. The M2 antibody was detected by immunostaining with Cy3-labeled anti–mouse IgG. Signals were observed by confocal laser microscopy. The internalized antibody was stained using the cells that had been acid washed. Adherent layer; confocal section at the adherent layer, Integrated layers; integrated confocal layers. Bar, 10 μm. (B) The number of cells that retained the signals at the adherent layer were counted. Transfected cells were identified by GFP expression. At time 0, almost 100% of GFP-positive cells expressed MT1-F or MT1-F(ΔCP) at the adherent layer. Cells that retained more than 30% of the original average fluorescence intensity at the adherent layer at each time point were counted as positive and presented. 100 cells were counted independently three times and the values presented are the mean ± SD.
Mentions: MT1-MMP is mainly localized at the adherent edge and migration front of cells. This localization appears to be appropriate for promoting cell invasion and migration. The continuous internalization of MT1-MMP at the edge may serve to substitute inactivated molecules, including TIMP-2 inhibited molecules and processed fragments, with freshly synthesized ones during cell migration. To investigate this possibility further, we examined the internalization of MT1-F at the cell adherent edge using a confocal laser microscope. The localization of the enzyme at the adherent layer was not affected by the mutation that abolishes internalization (ΔCP) (Fig. 7 A, Adherent layer and Integrated layers, 0 min). When MT1-F–expressing cells were incubated for 30 min, the wild-type enzyme present at the cell adherent layer was drastically decreased and internalized molecules were detected (Wild Type, Integrated layers, Adherent layer, and Intracellular; 30 min). However, when MT1-F(ΔCP) was expressed, the signals at the cell adherent layer were not diminished even after a 30-min incubation (Adherent layer, ΔCP). Internalization at this point was also not observed (Intracellular, ΔCP). The number of cells that expressed MT1-F or MT1-F(ΔCP) at the adherent edge was also counted (Fig. 7 B). Thus, the mutation that disturbs the internalization of MT1-MMP clearly abolishes the active turnover of the molecule at the adherent edge. Similar results were obtained with other internalization-defective mutants (unpublished data).

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