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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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Internalization of MT1-MMP through clathrin-coated vesicles. (A) The interaction of the MT1-MMP cytoplasmic tail with the μ2 subunit of AP-2 was examined by yeast two-hybrid analysis. The cytoplasmic domain sequences derived from MT1-WT and MT1-LY/A were expressed as fusion proteins with the GAL4 DNA binding domain (bait), while the μ2 subunit was expressed as a protein fusion with the GAL4 transcription activation domain (prey). Protein–protein interaction was detected by the X-α-Gal assay. Three independent clones of transformed yeasts were analyzed for each plasmid combination. TGN38 was a positive control. (B) Colocalization of internalizing MT1-F and clathrin. Cells expressing MT1-F or its cytoplasmic mutants (L1Y/A and ΔCP) were incubated with Texas red–labeled anti-FLAG M2 antibody (red) on ice, washed, and then left at 37°C for 5 min to allow for internalization. Clathrin molecules were immunoreacted with goat anti-clathrin antibody and visualized with Alexa™488-conjugated anti–goat IgG (green). The colocalization of internalizing MT1-F and clathrin was observed by washing the cells with acid solution before their fixation and permeabilization. Signals were then examined by confocal laser microscopy. Bar, 5 μm.
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fig5: Internalization of MT1-MMP through clathrin-coated vesicles. (A) The interaction of the MT1-MMP cytoplasmic tail with the μ2 subunit of AP-2 was examined by yeast two-hybrid analysis. The cytoplasmic domain sequences derived from MT1-WT and MT1-LY/A were expressed as fusion proteins with the GAL4 DNA binding domain (bait), while the μ2 subunit was expressed as a protein fusion with the GAL4 transcription activation domain (prey). Protein–protein interaction was detected by the X-α-Gal assay. Three independent clones of transformed yeasts were analyzed for each plasmid combination. TGN38 was a positive control. (B) Colocalization of internalizing MT1-F and clathrin. Cells expressing MT1-F or its cytoplasmic mutants (L1Y/A and ΔCP) were incubated with Texas red–labeled anti-FLAG M2 antibody (red) on ice, washed, and then left at 37°C for 5 min to allow for internalization. Clathrin molecules were immunoreacted with goat anti-clathrin antibody and visualized with Alexa™488-conjugated anti–goat IgG (green). The colocalization of internalizing MT1-F and clathrin was observed by washing the cells with acid solution before their fixation and permeabilization. Signals were then examined by confocal laser microscopy. Bar, 5 μm.

Mentions: The binding of AP-2 to the cytoplasmic tail of membrane proteins is the first step in the process that causes internalization of the molecules via clathrin-coated pits. Tyrosine and di-leucine motifs are both potential binding sites for AP-2 (Hunziker and Fumey, 1994). Binding of AP-2 to the cytoplasmic tail of cell surface proteins is mediated by its subunit μ2, which recognizes tyrosine-containing motifs (Ohno et al., 1995). Such interactions can be detected by using the yeast two-hybrid system (Ohno et al., 1996). As we found that LLY573 serves as a cis-acting element in MT1-MMP internalization, we tested whether LLY573 is the binding site for the μ2 subunit. We used the 20–amino acid cytoplasmic tail of MT1-MMP as the “bait” by fusing it with the GAL4 DNA binding domain (GAL4DB), and the GAL4 activator domain fused with the μ2 fragment served as the “prey.” The trans-Golgi network–specific integral membrane protein (TGN38) was used as a positive control for these experiments as its cytoplasmic tail is known to bind to μ2 (Ohno et al., 1996). GAL4DB alone (Mock) did not interact with μ2, whereas TGN38 did (Fig. 5 A, TGN38). When the cytoplasmic tail of MT1-MMP was tested, it gave a strong positive signal similar to TGN38 (Fig. 5 A, MT1-WT), suggesting that it interacts with the μ2 subunit. To confirm the importance of the LLY motif in this interaction, it was mutated to alanines (MT1-L1Y/A) and examined. MT1-L1Y/A did not interact with μ2. Thus, the LLY573 motif appears to be the site that interacts with clathrin-coated pits.


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 MT1-MMP through clathrin-coated vesicles. (A) The interaction of the MT1-MMP cytoplasmic tail with the μ2 subunit of AP-2 was examined by yeast two-hybrid analysis. The cytoplasmic domain sequences derived from MT1-WT and MT1-LY/A were expressed as fusion proteins with the GAL4 DNA binding domain (bait), while the μ2 subunit was expressed as a protein fusion with the GAL4 transcription activation domain (prey). Protein–protein interaction was detected by the X-α-Gal assay. Three independent clones of transformed yeasts were analyzed for each plasmid combination. TGN38 was a positive control. (B) Colocalization of internalizing MT1-F and clathrin. Cells expressing MT1-F or its cytoplasmic mutants (L1Y/A and ΔCP) were incubated with Texas red–labeled anti-FLAG M2 antibody (red) on ice, washed, and then left at 37°C for 5 min to allow for internalization. Clathrin molecules were immunoreacted with goat anti-clathrin antibody and visualized with Alexa™488-conjugated anti–goat IgG (green). The colocalization of internalizing MT1-F and clathrin was observed by washing the cells with acid solution before their fixation and permeabilization. Signals were then examined by confocal laser microscopy. Bar, 5 μm.
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Related In: Results  -  Collection

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fig5: Internalization of MT1-MMP through clathrin-coated vesicles. (A) The interaction of the MT1-MMP cytoplasmic tail with the μ2 subunit of AP-2 was examined by yeast two-hybrid analysis. The cytoplasmic domain sequences derived from MT1-WT and MT1-LY/A were expressed as fusion proteins with the GAL4 DNA binding domain (bait), while the μ2 subunit was expressed as a protein fusion with the GAL4 transcription activation domain (prey). Protein–protein interaction was detected by the X-α-Gal assay. Three independent clones of transformed yeasts were analyzed for each plasmid combination. TGN38 was a positive control. (B) Colocalization of internalizing MT1-F and clathrin. Cells expressing MT1-F or its cytoplasmic mutants (L1Y/A and ΔCP) were incubated with Texas red–labeled anti-FLAG M2 antibody (red) on ice, washed, and then left at 37°C for 5 min to allow for internalization. Clathrin molecules were immunoreacted with goat anti-clathrin antibody and visualized with Alexa™488-conjugated anti–goat IgG (green). The colocalization of internalizing MT1-F and clathrin was observed by washing the cells with acid solution before their fixation and permeabilization. Signals were then examined by confocal laser microscopy. Bar, 5 μm.
Mentions: The binding of AP-2 to the cytoplasmic tail of membrane proteins is the first step in the process that causes internalization of the molecules via clathrin-coated pits. Tyrosine and di-leucine motifs are both potential binding sites for AP-2 (Hunziker and Fumey, 1994). Binding of AP-2 to the cytoplasmic tail of cell surface proteins is mediated by its subunit μ2, which recognizes tyrosine-containing motifs (Ohno et al., 1995). Such interactions can be detected by using the yeast two-hybrid system (Ohno et al., 1996). As we found that LLY573 serves as a cis-acting element in MT1-MMP internalization, we tested whether LLY573 is the binding site for the μ2 subunit. We used the 20–amino acid cytoplasmic tail of MT1-MMP as the “bait” by fusing it with the GAL4 DNA binding domain (GAL4DB), and the GAL4 activator domain fused with the μ2 fragment served as the “prey.” The trans-Golgi network–specific integral membrane protein (TGN38) was used as a positive control for these experiments as its cytoplasmic tail is known to bind to μ2 (Ohno et al., 1996). GAL4DB alone (Mock) did not interact with μ2, whereas TGN38 did (Fig. 5 A, TGN38). When the cytoplasmic tail of MT1-MMP was tested, it gave a strong positive signal similar to TGN38 (Fig. 5 A, MT1-WT), suggesting that it interacts with the μ2 subunit. To confirm the importance of the LLY motif in this interaction, it was mutated to alanines (MT1-L1Y/A) and examined. MT1-L1Y/A did not interact with μ2. Thus, the LLY573 motif appears to be the site that interacts with clathrin-coated pits.

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