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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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Effect of deleting the cytoplasmic domain on MT1-MMP internalization. (A) Schematic representation of the MT1-MMP cytoplasmic domain deletion mutants. (B) Transiently expressed products in CHO-K1 cells were analyzed by Western blotting using anti-FLAG M2 antibody. Amount of cell surface MT1-F and mutant proteins was calculated from the bound 125I-labeled anti-FLAG M2 antibody. (C) Internalization of MT1-F and its mutant proteins after a 30-min incubation was analyzed as in Fig. 1. Values in B and C are the mean ± SD of three experiments. The asterisks (*) indicate statistically significant differences (P < 0.001) between MT1-F and the mutant.
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fig3: Effect of deleting the cytoplasmic domain on MT1-MMP internalization. (A) Schematic representation of the MT1-MMP cytoplasmic domain deletion mutants. (B) Transiently expressed products in CHO-K1 cells were analyzed by Western blotting using anti-FLAG M2 antibody. Amount of cell surface MT1-F and mutant proteins was calculated from the bound 125I-labeled anti-FLAG M2 antibody. (C) Internalization of MT1-F and its mutant proteins after a 30-min incubation was analyzed as in Fig. 1. Values in B and C are the mean ± SD of three experiments. The asterisks (*) indicate statistically significant differences (P < 0.001) between MT1-F and the mutant.

Mentions: To delineate the sequences that are essential for MT1-MMP internalization, a series of deletions were introduced into the MT1-MMP cytoplasmic tail (Fig. 3 A). After confirming the expression levels and the cell surface amounts of each mutant (Fig. 3 B), which did not differ from the wild-type protein, the internalization of each mutant was measured (Fig. 3 C). Deletion of four (Δ579) and eight (Δ575) amino acids from the COOH terminus resulted in a slight decrease (20%) in internalization, whereas a significant reduction (60%) was observed with the Δ571 mutant that lacks 12 amino acids. Further deletion did not reduce the internalization significantly. Thus, it appears that the last four amino acids, LLYC574, of the Δ575 mutant constitute an important internalization signal. Consistent with this observation, internal deletion of the LLYC574 amino acids (Δ571–574) reduced internalization by 50%.


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)

Effect of deleting the cytoplasmic domain on MT1-MMP internalization. (A) Schematic representation of the MT1-MMP cytoplasmic domain deletion mutants. (B) Transiently expressed products in CHO-K1 cells were analyzed by Western blotting using anti-FLAG M2 antibody. Amount of cell surface MT1-F and mutant proteins was calculated from the bound 125I-labeled anti-FLAG M2 antibody. (C) Internalization of MT1-F and its mutant proteins after a 30-min incubation was analyzed as in Fig. 1. Values in B and C are the mean ± SD of three experiments. The asterisks (*) indicate statistically significant differences (P < 0.001) between MT1-F and the mutant.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Effect of deleting the cytoplasmic domain on MT1-MMP internalization. (A) Schematic representation of the MT1-MMP cytoplasmic domain deletion mutants. (B) Transiently expressed products in CHO-K1 cells were analyzed by Western blotting using anti-FLAG M2 antibody. Amount of cell surface MT1-F and mutant proteins was calculated from the bound 125I-labeled anti-FLAG M2 antibody. (C) Internalization of MT1-F and its mutant proteins after a 30-min incubation was analyzed as in Fig. 1. Values in B and C are the mean ± SD of three experiments. The asterisks (*) indicate statistically significant differences (P < 0.001) between MT1-F and the mutant.
Mentions: To delineate the sequences that are essential for MT1-MMP internalization, a series of deletions were introduced into the MT1-MMP cytoplasmic tail (Fig. 3 A). After confirming the expression levels and the cell surface amounts of each mutant (Fig. 3 B), which did not differ from the wild-type protein, the internalization of each mutant was measured (Fig. 3 C). Deletion of four (Δ579) and eight (Δ575) amino acids from the COOH terminus resulted in a slight decrease (20%) in internalization, whereas a significant reduction (60%) was observed with the Δ571 mutant that lacks 12 amino acids. Further deletion did not reduce the internalization significantly. Thus, it appears that the last four amino acids, LLYC574, of the Δ575 mutant constitute an important internalization signal. Consistent with this observation, internal deletion of the LLYC574 amino acids (Δ571–574) reduced internalization by 50%.

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