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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 site-directed mutations on MT1-MMP internalization. (A) Schematic representation of the MT1-MMP cytoplasmic sequence and mutations. (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. Values indicate the mean ± SD of three experiments. (C) Internalization of MT1-F and its mutant proteins after a 30-min incubation was analyzed as in Fig. 1. Values 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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fig4: Effect of site-directed mutations on MT1-MMP internalization. (A) Schematic representation of the MT1-MMP cytoplasmic sequence and mutations. (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. Values indicate the mean ± SD of three experiments. (C) Internalization of MT1-F and its mutant proteins after a 30-min incubation was analyzed as in Fig. 1. Values are the mean ± SD of three experiments. The asterisks (*) indicate statistically significant differences (P < 0.001) between MT1-F and the mutant.

Mentions: The tyrosine (Y573) and di-leucine (LL572, referred to as L1) residues in the deleted sequence are found in the internalization motifs that have been reported previously (Heilker et al., 1999). Another di-leucine sequence (LL579, referred to as L2) was also found in the tail sequence. To determine which of these motifs participates in the MT1-MMP internalization, targeted mutations were introduced into one or more of the three motifs (Fig. 4 A). Mutation of either of the two di-leucine sequences to alanines (L1/A or L2/A) reduced internalization by 53 and 33%, respectively (Fig. 4 C). Mutation of both di-leucines showed a slightly additive effect (55% decrease). Mutation of tyrosine573 to alanine (Y/A) also reduced internalization by 40% and combining this mutation with L1/A (L1Y/A) inhibited it further (53%). Combined mutations in all three sites (L1YL2/A) reduced internalization to levels comparable with the cytoplasmic deletion mutant (ΔCP; 63%). In contrast, mutating either of the other two amino acids, i.e., serine577 (S/A) and valine582 (V/A), to alanine had no effect on internalization. The expression of the mutant MT1-MMPs and their cell surface amounts were confirmed and did not differ from the wild-type protein (Fig. 4 B).


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 site-directed mutations on MT1-MMP internalization. (A) Schematic representation of the MT1-MMP cytoplasmic sequence and mutations. (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. Values indicate the mean ± SD of three experiments. (C) Internalization of MT1-F and its mutant proteins after a 30-min incubation was analyzed as in Fig. 1. Values 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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Related In: Results  -  Collection

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

fig4: Effect of site-directed mutations on MT1-MMP internalization. (A) Schematic representation of the MT1-MMP cytoplasmic sequence and mutations. (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. Values indicate the mean ± SD of three experiments. (C) Internalization of MT1-F and its mutant proteins after a 30-min incubation was analyzed as in Fig. 1. Values are the mean ± SD of three experiments. The asterisks (*) indicate statistically significant differences (P < 0.001) between MT1-F and the mutant.
Mentions: The tyrosine (Y573) and di-leucine (LL572, referred to as L1) residues in the deleted sequence are found in the internalization motifs that have been reported previously (Heilker et al., 1999). Another di-leucine sequence (LL579, referred to as L2) was also found in the tail sequence. To determine which of these motifs participates in the MT1-MMP internalization, targeted mutations were introduced into one or more of the three motifs (Fig. 4 A). Mutation of either of the two di-leucine sequences to alanines (L1/A or L2/A) reduced internalization by 53 and 33%, respectively (Fig. 4 C). Mutation of both di-leucines showed a slightly additive effect (55% decrease). Mutation of tyrosine573 to alanine (Y/A) also reduced internalization by 40% and combining this mutation with L1/A (L1Y/A) inhibited it further (53%). Combined mutations in all three sites (L1YL2/A) reduced internalization to levels comparable with the cytoplasmic deletion mutant (ΔCP; 63%). In contrast, mutating either of the other two amino acids, i.e., serine577 (S/A) and valine582 (V/A), to alanine had no effect on internalization. The expression of the mutant MT1-MMPs and their cell surface amounts were confirmed and did not differ from the wild-type protein (Fig. 4 B).

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