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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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Identification of the domain involved in MT1-MMP internalization in CHO-K1 cells. (A) Schematic representation of MT1-MMP mutants used in this experiment. SP, signal peptide; Pro, propeptide; CAT, catalytic domain; H, hinge; PEX, hemopexin-like domain; TM, transmembrane domain; CP, cytoplasmic domain; FLAG, FLAG epitope; GPI, glycosylphosphatidylinositol anchor. (B) Western blot analysis of MT1-MMP mutants expressed in CHO-K1 cells. Molecules expressed in the transfected cells were detected by anti-FLAG M2 antibody. The asterisks (*) indicate the nonspecific band. (C) Amount of MT1-F and mutants expressed on the cell surface calculated from the bound 125I-labeled anti-FLAG M2 antibody. Values are the mean ± SD of three experiments. (D) Internalization of MT1-MMP mutants after a 30-min incubation. Experiments were performed as described in Fig. 1 and the Materials and methods. 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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fig2: Identification of the domain involved in MT1-MMP internalization in CHO-K1 cells. (A) Schematic representation of MT1-MMP mutants used in this experiment. SP, signal peptide; Pro, propeptide; CAT, catalytic domain; H, hinge; PEX, hemopexin-like domain; TM, transmembrane domain; CP, cytoplasmic domain; FLAG, FLAG epitope; GPI, glycosylphosphatidylinositol anchor. (B) Western blot analysis of MT1-MMP mutants expressed in CHO-K1 cells. Molecules expressed in the transfected cells were detected by anti-FLAG M2 antibody. The asterisks (*) indicate the nonspecific band. (C) Amount of MT1-F and mutants expressed on the cell surface calculated from the bound 125I-labeled anti-FLAG M2 antibody. Values are the mean ± SD of three experiments. (D) Internalization of MT1-MMP mutants after a 30-min incubation. Experiments were performed as described in Fig. 1 and the Materials and methods. 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 internalization of surface proteins depends on specific sequences in the cytoplasmic tail that direct incorporation of the molecules into clathrin-coated vesicles (Heilker et al., 1999). MT1-MMP itself may bear such signals on its cytoplasmic tail. Alternatively, it may associate with other proteins having such internalization motifs. We first located the MT1-MMP domain that is essential for internalization by constructing various deletion mutants as shown in Fig. 2 A. Comparable levels of expression for each construct were confirmed by Western blotting (Fig. 2 B). The amount of each mutant on the cell surface was measured by the amount of cell surface–bound 125I-M2 (Fig. 2 C). The internalization of each mutant was then analyzed after a 30-min incubation. Deletion of either the catalytic (ΔCAT) or the PEX domain (ΔPEX) did not affect internalization (Fig. 2 D). However, deletion of the cytoplasmic tail (ΔCP) or its substitution with the cytoplasmic tail derived from the interleukin 2 receptor α chain (IL2R) dramatically reduced internalization (Fig. 2 D). We also tested another chimeric MT1-F molecule, MT1-F(GPI), wherein the MT1-F transmembrane domain and the cytoplasmic tail had been substituted with the GPI-anchoring signal derived from MT4-MMP. The internalization of this mutant molecule was also reduced (Fig. 2 D). These observations indicate that the cytoplasmic tail of MT1-MMP contains the critical sequences required for internalization. These sequences are either internalization signals or the site through which MT1-MMP interacts with other molecules that contain internalization signal motifs.


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)

Identification of the domain involved in MT1-MMP internalization in CHO-K1 cells. (A) Schematic representation of MT1-MMP mutants used in this experiment. SP, signal peptide; Pro, propeptide; CAT, catalytic domain; H, hinge; PEX, hemopexin-like domain; TM, transmembrane domain; CP, cytoplasmic domain; FLAG, FLAG epitope; GPI, glycosylphosphatidylinositol anchor. (B) Western blot analysis of MT1-MMP mutants expressed in CHO-K1 cells. Molecules expressed in the transfected cells were detected by anti-FLAG M2 antibody. The asterisks (*) indicate the nonspecific band. (C) Amount of MT1-F and mutants expressed on the cell surface calculated from the bound 125I-labeled anti-FLAG M2 antibody. Values are the mean ± SD of three experiments. (D) Internalization of MT1-MMP mutants after a 30-min incubation. Experiments were performed as described in Fig. 1 and the Materials and methods. 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

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fig2: Identification of the domain involved in MT1-MMP internalization in CHO-K1 cells. (A) Schematic representation of MT1-MMP mutants used in this experiment. SP, signal peptide; Pro, propeptide; CAT, catalytic domain; H, hinge; PEX, hemopexin-like domain; TM, transmembrane domain; CP, cytoplasmic domain; FLAG, FLAG epitope; GPI, glycosylphosphatidylinositol anchor. (B) Western blot analysis of MT1-MMP mutants expressed in CHO-K1 cells. Molecules expressed in the transfected cells were detected by anti-FLAG M2 antibody. The asterisks (*) indicate the nonspecific band. (C) Amount of MT1-F and mutants expressed on the cell surface calculated from the bound 125I-labeled anti-FLAG M2 antibody. Values are the mean ± SD of three experiments. (D) Internalization of MT1-MMP mutants after a 30-min incubation. Experiments were performed as described in Fig. 1 and the Materials and methods. 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 internalization of surface proteins depends on specific sequences in the cytoplasmic tail that direct incorporation of the molecules into clathrin-coated vesicles (Heilker et al., 1999). MT1-MMP itself may bear such signals on its cytoplasmic tail. Alternatively, it may associate with other proteins having such internalization motifs. We first located the MT1-MMP domain that is essential for internalization by constructing various deletion mutants as shown in Fig. 2 A. Comparable levels of expression for each construct were confirmed by Western blotting (Fig. 2 B). The amount of each mutant on the cell surface was measured by the amount of cell surface–bound 125I-M2 (Fig. 2 C). The internalization of each mutant was then analyzed after a 30-min incubation. Deletion of either the catalytic (ΔCAT) or the PEX domain (ΔPEX) did not affect internalization (Fig. 2 D). However, deletion of the cytoplasmic tail (ΔCP) or its substitution with the cytoplasmic tail derived from the interleukin 2 receptor α chain (IL2R) dramatically reduced internalization (Fig. 2 D). We also tested another chimeric MT1-F molecule, MT1-F(GPI), wherein the MT1-F transmembrane domain and the cytoplasmic tail had been substituted with the GPI-anchoring signal derived from MT4-MMP. The internalization of this mutant molecule was also reduced (Fig. 2 D). These observations indicate that the cytoplasmic tail of MT1-MMP contains the critical sequences required for internalization. These sequences are either internalization signals or the site through which MT1-MMP interacts with other molecules that contain internalization signal motifs.

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