|
|
|
|
|
|
|
||
© The Rockefeller University Press,
0021-9525/2001/12/1345 $5.00
The Journal of Cell Biology, Volume 155, Number 7, December 24, 2001 1345-1356
Article |
Cytoplasmic tail–dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity
Address correspondence to Motoharu Seiki, Ph.D., Professor, Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan. Tel.: 81-3-5449-5255. Fax: 81-3-5449-5414. E-mail: mseiki{at}ims.u-tokyo.ac.jp
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionMaterials and methodsReferences |
|---|
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 µ2 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.
Key Words: MT-MMP; metalloproteinase; internalization; invasion; migration
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Of all the MT-MMPs, MT1-MMP has been studied the most. It degrades various ECM components including collagens type I, II, and III, laminins 1 and 5, fibronectin, fibrin, and proteoglycans (Pei and Weiss, 1996; Ohuchi et al., 1997; Koshikawa et al., 2000). It also activates other MMPs on the cell surface including proMMP-2 (Sato et al., 1994) and proMMP-13 (Knauper et al., 1996). MT1-MMP is expressed mainly in the mesenchymal tissues of embryos such as bone, muscle, and fibroblastic tissues (Kinoh et al., 1996; Apte et al., 1997). In the MT1-MMP–null mouse, formation of skeletal tissues is disturbed, and extensive fibrosis occurs after birth (Holmbeck et al., 1999). Activation of proMMP-2 was severely suppressed in this mouse (Zhou et al., 2000) and isolated fibroblasts failed to digest type I collagen (Holmbeck et al., 1999).
Overexpression of MT1-MMP is associated with malignant tumors (Seiki, 1999). Because MT1-MMP expression has been shown to augment the invasive and metastatic potential of cancer cells in experimental systems (Sato et al., 1994; Tsunezuka et al., 1996), the enzyme is believed to play a key role in the invasion of malignant tumors. Invasion requires the dynamic and coordinated action of many cell surface molecules including proteinases and adhesion molecules (Horwitz and Parsons, 1999; Murphy and Gavrilovic, 1999). During this process, MT1-MMP is important not only for ECM degradation but also for stimulating cell locomotion (Koshikawa et al., 2000; Kajita et al., 2001). During cell migration, MT1-MMP localizes at the migration front (Sato et al., 1997), particularly near the layer to which the cells adhere (Kajita et al., 2001). However, it is still unclear how MT1-MMP activity is regulated at the migration front during cell migration.
In general, MT1-MMP activity on the cell surface is regulated by several different mechanisms. ProMT1-MMP has a recognition site for proprotein convertases at the end of the propeptide (Sato et al., 1994). Therefore, MT1-MMP appears on the cell surface in its active form after proprotein convertases cleave off the propeptide intracellularly (Yana and Weiss, 2000). The enzyme activity on the cell surface can be inhibited by the tissue inhibitors of metalloproteinases (TIMPs)-2, -3, and -4 but not by TIMP-1. Of the TIMPs, TIMP-2 is the major inhibitor of MT1-MMP (Strongin et al., 1995). TIMP-2 also participates in the activation of proMMP-2 by MT1-MMP mediating the binding of proMMP-2 to MT1-MMP through the formation of a tri-molecular complex (e.g., proMMP-2–TIMP-2–MT1-MMP) (Strongin et al., 1995; Butler et al., 1998; Kinoshita et al., 1998). ProMMP-2 in the complex can then be activated by the adjacent TIMP-2–free MT1-MMP. Homophilic oligomer formation through the PEX domain facilitates such bi-molecular events and stimulates the activation of proMMP-2 (Itoh et al., 2001). MT1-MMP is then downregulated through the processing by MMP-2 and MT1-MMP itself. The cleavage occurs within its catalytic domain and generates a 43-kD inactive fragment on the cell surface (Lehti et al., 1998; Stanton et al., 1998). Such an inactive fragment may compete with the formation of homo-oligomers of intact MT1-MMP molecules and eventually inhibit proMMP-2 activation (Itoh et al., 2001). Another proposed mechanism by which cell surface MT1-MMP activity can be regulated is through its removal from the cell surface by internalization and subsequent degradation. This is suggested by a recent experiment showing that MT1-MMP–bound TIMP-2 is internalized and degraded (Maquoi et al., 2000).
In this paper, we report that MT1-MMP is indeed internalized at the cell surface and this process depends on tyrosine and di-leucine motifs in its cytoplasmic tail. In particular, the LLY573 motif was found to be the binding site of a component of clathrin-coated pits, adaptor protein 2 (AP-2). Although internalization is expected to regulate the net amount of MT1-MMP present on the cell surface, we found that the level of MT1-MMP activity in cells expressing internalization-defective mutants did not change significantly in relation to those expressing the wild-type enzyme. Despite this, however, the MT1-MMP mutants, unlike wild-type MT1-MMP, failed to promote cell migration and invasion into reconstituted basement membrane. Because MT1-MMP localizes at the cell adherent edge and is actively internalized there, our results suggest that turnover of MT1-MMP at the adherent edge by internalization is an important step in regulating the enzyme during cell migration and invasion.
Biochemical methods have been used to study membrane proteinases. Although a biochemical approach is powerful, especially for soluble enzymes, it is not sufficient to characterize the cell surface events that are coupled with various cell functions, an equally important aspect of membrane protease regulation. From this point of view very little information about cell surface proteinases is available. Our study provides an interesting initial step to understanding the dynamic regulation of MT1-MMP coupled with cell locomotion.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
|
Almost 40% of the M2 antibody was released during the first 10-min incubation even in the cells fixed with 3% paraformaldehyde (unpublished data). Although we do not know how this weak binding occurs, a fraction of MT1-F may be processed partially from the NH2-terminal FLAG sequence. Thus, there is a possibility that the efficiency of MT1-F internalization is underestimated. We also analyzed the internalization using a monovalent Fab fragment derived from the M2 antibody and obtained similar results, indicating that internalization is not an artificial event caused by the cross-linking of MT1-MMP molecules by the divalent antibody (unpublished data).
To ensure that the acid-resistant radioactivity of 125I-M2 was a direct indication of the incorporation of molecules into the cells, the M2 antibody was visualized by immunostaining. The M2 antibody bound to cell surface MT1-F (Fig. 1 B, Surface fraction) was exclusively stained without permeation of the cells. The internalized antibody together with MT1-F was stained exclusively after removing the cell surface antibody by the acid wash and permeating the cell membrane. The M2 antibody bound to the cells expressing MT1-F, but not to the mock-transfected cells (Surface, 0 min). Internalized signals were not detected at time 0 (Intracellular, 0 min). The cell surface–bound M2 antibody decreased thereafter, suggesting that the M2 antibody was either being internalized together with the MT1-F molecules or was released from the surface. Intracellular signals of the M2 antibody increased according to the incubation time (Intracellular, 10 and 30 min) indicating that the M2 antibody bound to the cell surface MT1-F was incorporated into the cells. However both surface and intracellular signals became weak after a 60-min incubation (60 min). Thus, the internalization of the cell surface MT1-MMP molecules was visually confirmed again.
Internalization of MT1-MMP depends on sequences in its cytoplasmic tail
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 (
CAT) or the PEX domain (PEX) did not affect internalization (Fig. 2 D). However, deletion of the cytoplasmic tail (CP) or its substitution with the cytoplasmic tail derived from the interleukin 2 receptor 
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.
|
579) and eight (575) amino acids from the COOH terminus resulted in a slight decrease (20%) in internalization, whereas a significant reduction (60%) was observed with the 571 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 575 mutant constitute an important internalization signal. Consistent with this observation, internal deletion of the LLYC574 amino acids (571–574) reduced internalization by 50%.
|
CP; 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).
|
|
CP)–expressing cells and mock-transfected cells were examined, such internalized M2 antibody signals were not observed at all.
A defect in MT1-MMP internalization does not affect its ability to activate proMMP-2
To examine whether MT1-MMP internalization affects MT1-MMP activity on the cell surface, we analyzed the ability of the MT1-MMP mutants to activate proMMP-2, which is one of the important functions of MT1-MMP. MT1-MMP constructs with various mutations in the cytoplasmic domain were expressed in CHO-K1 cells, and their ability to activate the proMMP-2 in the culture medium was examined (Fig. 6 A). None of the mutations altered proMMP-2 activation significantly. Thus, reflecting the expression levels of MT1-MMP mutants on the cell surface (Figs. 3 B and 4 B), their net cell surface proteolytic activity did not change significantly compared with that of the wild-type enzyme.
|
CP) (Fig. 6 B). TIMP-2 was internalized if it was bound to MT1-F but not if it was bound to MT1-FCP (Fig. 6 C). Thus, the internalization of TIMP-2 was also dependent on the cytoplasmic sequence of MT1-MMP.
MT1-MMP is internalized at the adherent edge
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 (CP) (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(CP) was expressed, the signals at the cell adherent layer were not diminished even after a 30-min incubation (Adherent layer, CP). Internalization at this point was also not observed (Intracellular, CP). The number of cells that expressed MT1-F or MT1-F(CP) 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).
|
CP and L1Y/A) did not stimulate motility, whereas the S/A mutation, which has no effect on internalization, did enhance motility. Other cytoplasmic tail mutants that did not affect internalization also stimulated motility (unpublished data). Thus, the internalization of MT1-F correlates with its ability to promote cell migration. We also confirmed that the internalization defect of MT1-F does not affect the internalization of other cell surface molecules, using hTfnR as a representative molecule (unpublished data).
|
Correlation between internalization and invasion-promoting activity of MT1-MMP
It was reported previously that the MT1-MMP mutants with truncated cytoplasmic tails failed to promote invasion of the cells into Matrigel, a reconstituted basement membrane (Lehti et al., 2000). Invasion requires a combination of proteolytic and locomotive cell activities. As internalization-defective mutants also lost their ability to promote cell migration, there might be a possibility that the absence of invasion was caused by the impaired internalization of MT1-MMP. We expressed MT1-F and its mutants in CHO-K1 cells and cellular invasion of Matrigel was examined. The expression of MT1-F promoted a 6.5-fold increase in cell invasion (Fig. 9), whereas the expression of the catalytically inactive mutant (E/A) did not. Mutations that affect MT1-F internalization (571, 567, CP, and L1Y/A) all severely disturbed the invasion-promoting activity of the enzyme, whereas those that did not affect internalization (579, 575, and S/A) promoted invasion as efficiently as the wild-type enzyme. The inability of the internalization-deficient mutants to promote invasion was not due to the differences in their net proteolytic activity or the amounts of MT1-F on the cell surface, as demonstrated in Fig. 6, A and B. Thus, internalization of MT1-MMP seems to be important in regulating the enzyme during cell migration and invasion.
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Unexpectedly, mutations that abrogated the internalization did not cause drastic accumulation of MT1-MMP molecules on the cell surface. They also did not increase the extent by which MT1-MMP activated proMMP-2 (Fig. 6 A). This may indicate that there are yet unknown regulations that adjust the level of MT1-MMP on the cell surface. However, it is also possible that the transient overexpression used for the experiments was not appropriate for reflecting the effect of internalization on the cell surface amount of the enzyme. Nevertheless, it is clear that the internalization is dependent on a specific sequence in the cytoplasmic domain, and mutating this sequence abrogates internalization at the adherent edge.
Fine mapping of the cytoplasmic domain sequence with mutants revealed the critical residues to be the LLY573 motif, which is a binding site for the µ2 subunit of AP-2, a component of clathrin-coated vesicles. Confocal laser microscopic analysis also revealed that the internalized MT1-MMP colocalized with clathrin-coated vesicles, indicating strongly that the MT1-MMP molecules were in the vesicles (Fig. 5 B).
Of the six MT-MMP proteins, four are integral membrane proteinases with a cytoplasmic tail, and two are tethered to the membrane by GPI anchors (Itoh et al., 1999; Kojima et al., 2000). The reported consensus sequence for µ2 binding is YXXØ (X, any amino acid; Y, tyrosine; Ø, bulky hydrophobic amino acids; Ohno et al., 1996). The cytoplasmic sequence in MT1-MMP (YCQR) is close to the consensus but does not match completely because Ø is substituted by the bulky but hydrophilic arginine. The cytoplasmic tails of the other three transmembrane MT-MMPs all contain similar YXXR sequences. The di-leucine motif preceding the tyrosine in the MT1-MMP cytoplasmic sequence is also found in MT2- and MT3-MMP, but not in MT5-MMP. Consistent with this sequence conservation, we found that MT2- and MT3-MMP are internalized as effectively as MT1-MMP, whereas MT5-MMP is internalized less efficiently (unpublished data). GPI-anchored type MT4- and MT6-MMP that lack a cytoplasmic tail are not internalized efficiently (unpublished data).
Internalization-defective MT1-MMP cannot promote cell migration and invasion
Although a defect in MT1-MMP internalization did not affect its net amount on the cell surface nor its ability to activate proMMP-2, this defect severely disturbed its ability to promote cell invasion in Matrigel. This observation is consistent with a previous study showing that deletion of the MT1-MMP cytoplasmic tail reduced its invasion-promoting activity (Lehti et al., 2000). We found that all the mutations resulting in poor internalization also abrogated the invasion-promoting activity of MT1-MMP. Similarly, poor internalization of MT1-MMP also affected its ability to promote cell motility. Cell surface MT1-MMP has to be regulated precisely when it functions as a part of invasion machinery. In addition to its proteolytic activity, the enzyme has to be at the right site at the right time on the cell surface together with the right partners, and then it is inhibited by TIMP-2 or partially degraded. These "old molecules" have to be cleared off to assure substitution with the newly synthesized molecules. Thus, it is very plausible that the proteolytic activity of MT1-MMP is critical but not sufficient for a biological outcome such as invasion.
During cell migration, MT1-MMP localizes predominantly to the cell adherent edge and the migration front. Because such localization is presumably important in regulating cell locomotion, we speculated first that the mutations in the cytoplasmic sequence affect such localization and eventually abrogate cell migration and invasion. However, this was not the case, as shown in Fig. 7. Consistent with the localization site of MT1-MMP, internalization of the enzyme was mostly observed there. Accordingly, whereas surface-labeled wild-type MT1-F disappeared over time at the adherent edge, the levels of the CP mutant decreased very slowly (Fig. 8, A and B). Thus, the mutations that prevent internalization of MT1-MMP molecules disturb the turnover of the enzyme at the migration front. The adherent edge and migration front are where the proteases and cell adhesion molecules that regulate cell locomotion and invasion congregate. However, our knowledge of the molecules involved and the way they operate in these processes is at present not sufficient to completely understand the role of MT1-MMP internalization. One possible idea is that internalization may be a mechanism to prevent accumulation of inactivated MT1-MMP, such as TIMP-2–inhibited or partially degraded molecules, which may disturb their substitution with freshly synthesized enzymes. If this is the case, internalization-defective mutants are expected to disturb the function of the wild-type enzyme. Indeed, expression of such mutant MT-MMP molecules in the cells expressing the wild-type enzyme inhibited the migration and invasion-stimulating activity of the wild-type MT1-MMP (unpublished data). This dominant-negative effect of the mutant against the wild-type enzyme indicates again that proteolytic activity of MT1-MMP is important but not sufficient to promote cell migration and invasion.
A recent study has described somewhat contrary results about the role of the MT1-MMP cytoplasmic tail on invasion-promoting activity. Namely, expression of the MT1-MMP mutant lacking a cytoplasmic tail still promoted invasion of COS-1 cells in a type I collagen gel, same as the wild-type enzyme did (Hotary et al., 2000). Because we used CHO-K1 cells and Matrigel for the assay, we tried to reproduce their experiments first. Expression of the MT1-F derivatives in COS-1 cells and analysis of the invasion in a type I collagen gel revealed that internalization-defective mutants also promoted invasion, confirming Hotary's results (unpublished data). However, when the same cells were analyzed in Matrigel, the results were similar to those obtained in this study using CHO-K1 cells. Thus, the requirement of the cytoplasmic tail for the invasion-promoting activity of MT1-MMP seems to differ depending on the matrix used for the assay. It should be noted that Matrigel contains complex ECM components, some of them cannot be degraded by MT1-MMP, whereas type I collagen gel contains a single component that is a good substrate for MT1-MMP (Ohuchi et al., 1997). Consequently, the collagen gel may be loosened more easily than Matrigel by MT1-MMP, and the cells may be able to move through the residual type I collagen matrix easily even when their locomotive force was reduced by the expression of internalization-defective mutants. Thus, the migration-promoting activity of MT1-MMP may play a more important role when the cells are moving through the residual Matrigel matrix rather than the loosened type I collagen gel.
Nakahara et al. (1997) have previously suggested that the cytoplasmic tail of MT1-MMP may play a role in localizing the enzyme to the protruding membrane structure known as the invadopodium, which is frequently associated with invasive cancer cells. In our experiments, CHO-K1 cells do not form obvious invadopodial structures and the localization of MT1-MMP at the cell adherent edge is not affected by mutations in the cytoplasmic tail. Similar observations were also made with COS-1 and HT1080 cells (unpublished data). Although the molecular characterization of invadopodia is not sufficient to understand how its formation is regulated (Chen and Wang, 1999), the cytoplasmic tail of MT1-MMP may interact with a certain molecule that exists in the invadopodia. It is also possible that the formation of the invadopodia itself might be disturbed by the internalization-defective MT1-MMP mutants because formation of these structures requires local invasion.
In conclusion, our study indicates that the cytoplasmic tail of MT1-MMP is critical for the internalization of MT1-MMP molecules at the cell surface, presumably after their incorporation into clathrin-coated vesicles. During cell migration, MT1-MMP localizes predominantly to the migration front and is internalized there. Mutations that disturb the internalization caused accumulation of the molecules at the adherent edge and abrogated cell migration and invasion. Thus, dynamic turnover of MT1-MMP molecules at the migration front by internalization appears critical to the migration-stimulating activity of MT1-MMP. This is a newly discovered mechanism that regulates the action of pericellular proteases during cell migration and sheds light on the dynamic events at the migration front that control cell locomotion, a field that is still unclear. Further study is needed to precisely delineate the sequential events that regulate proteases and cell adhesion molecules in relation to actin dynamics.
|
Materials and methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Construction of FLAG-tagged human MT1-MMP mutants and hTfnR
FLAG epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)–tagged MT1-MMP (denoted as MT1-F) was constructed as previously described (Itoh et al., 1999) and subcloned into the pSG5 expression vector (Stratagene). This plasmid was used as a template for PCR-based modification to generate mutant MT1-MMPs.
MT1-F(EA) is the catalytically inactive mutant (Glu240 to Ala). MT1-F(PEX) is the hemopexin-like domain–deleted mutant (Ile318–Gly535), and MT1-F(CAT) is the catalytic domain–deleted mutant (Tyr112–Pro312). MT1-F(IL2R) is the chimera of the ectodomain of MT1-F and the transmembrane/cytoplasmic domain of IL2R. MT1-F(GPI) is the chimeric mutant of MT1-F (Met1–Cys508) and the GPI-anchoring signal derived from MT4-MMP (Gly526–Leu607). Cytoplasmic deletion mutants of MT1-MMP (579, 575, 571, 567, and CP) were generated by introducing a stop codon in the plasmid, and site-directed mutations (571–574, L1/A, Y/A, S/A, L2/A, V/A, L1Y/A, L1L2/A, and L1YL2/A) were generated by PCR-based mutation methods. All the PCR products were confirmed by DNA sequencing and the cDNAs subsequently generated were subcloned into a pSG5 mammalian expression vector (Stratagene).
Full-length hTfnR cDNA was amplified by reverse transcript PCR using total RNA obtained from HT1080 cells and subcloned into the pcDNA3.1(-) expression vector (Invitrogen).
Western blot analysis
Transfected cells were washed three times with PBS and lysed in SDS-PAGE loading buffer containing 2-mercaptoethanol. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Hybond-ECL; Amersham Pharmacia Biotech). After blocking the membrane with 10% fat-free dry milk in Tris-buffered saline (TBS; 20 mM Tris-HCl, pH 7.5, 150 mM NaCl), the membrane was probed with anti-FLAG M2 monoclonal antibody (Sigma-Aldrich) (5 µg/ml) to detect FLAG-tagged MT1-MMP (MT1-F) and its mutants. An hTfnR-specific antibody was used to detect control cells transfected with hTfnR. The membrane was further probed with alkaline phosphatase–conjugated anti–mouse IgG (Sigma-Aldrich) to visualize the antibody.
Iodination of human holo-transferrin, anti-FLAG M2 antibody, and rTIMP-2
The proteins were labeled with 125I using IODO-GEN (Pierce Chemical Co.) in TBS containing 0.05% Brij35. After the reaction, free 125I was removed by gel filtration on SephadexTM G-25 (Amersham Pharmacia Biotech).
Internalization assay
Transfected CHO-K1 cells were seeded onto gelatin-coated 12-well plates (106 cells/well). After 24 h, cells were incubated for 1 h on ice with 2 µg/ml 125I-labeled anti-FLAG M2 IgG, holo transferrin, or TIMP-2 in internalization medium (Ham's F-12 medium containing 0.1% FBS, 1% kanamycin, and MEM supplemented with nonessential amino acids). 10 µM of peptidyl hydroxamate MMP inhibitor, BB94 (provided by Dr. Peter D. Brown, British Biotech Pharmaceuticals Ltd., Oxford, UK), was included during the incubation with the M2 antibody to avoid the autodegradation of MT1-MMP. Cells were washed with chilled medium three times and incubated further at 37°C for the indicated period of time in each experiment. At the end of the incubation, culture supernatants were collected to measure the 125I-labeled proteins that had been released by the cells. 125I-labeled proteins on the cell surface were collected by washing the cells with acid solution (500 mM CH3COOH, 150 mM NaCl) for 5 min at 4°C. This allows more than 98% of cell surface–bound ligand to be recovered. After the acid wash, cells were dissolved in lysis buffer (100 mM NaOH, 150 mM NaCl) to measure the proteins that had been internalized. Radioactivity in each fraction was measured by a gamma counter.
Indirect immunofluorescence staining
Transfected CHO-K1 cells were seeded on gelatin-coated coverslips. After 24 h, the cells were incubated for 1 h at 4°C with mouse anti-FLAG M2 (5 µg/ml) in internalization medium supplemented with 10 µM BB94. The cells were then washed with chilled medium three times, incubated at 37°C, and fixed with 3% paraformaldehyde in PBS. After blocking with 5% goat serum and 3% BSA in PBS for 1 h at room temperature, mouse anti-FLAG M2 antibody–treated cells were incubated with AlexaTM488-conjugated goat anti–mouse IgG (Molecular Probes, Inc.) or Cy3-conjugated goat anti–mouse IgG (Jackson ImmunoResearch Laboratories). For clathrin staining, fixed cells were incubated with goat anti-clathrin HC (C-20) antibody and visualized with AlexaTM488-conjugated rabbit anti–goat IgG (Molecular Probes, Inc.). Actin was stained with AlexaTM488-conjugated phalloidin. Fluorescence signals were detected using a Bio-Rad MRC-1024 confocal laser microscope.
Yeast two-hybrid analysis
Yeast two-hybrid assays were performed as described in the instructions for the MATCHMAKER two-hybrid kit using yeast strain Y190 (CLONTECH Laboratories, Inc.) with a modification described previously (Ohno et al., 1996). In brief, plasmids encoding the GAL4 binding domain fused with the MT1-MMP cytoplasmic domain (R563–V582) or the cytoplasmic domain mutant (LLY573/AAA) were constructed using a PCR-based technique and subcloned into the pGBT9 vector. These constructs were tested for interaction with GAL4 fused to activation domain µ2 using X--Gal (CLONTECH Laboratories, Inc.).
Gelatin zymography
Gelatin zymography was performed as described previously (Sato et al., 1994). Samples were mixed with SDS sample buffer without a reducing agent and separated in 7.5% acrylamide gels containing 0.1% gelatin. The gels were incubated at 37°C after removing SDS by washing with 2.5% Triton X-100–containing buffer. The gels were stained with Coomassie brilliant blue R250, and the gelatinolytic activities were detected as clear bands against a blue background.
Matrigel invasion assay
The Matrigel invasion assay was performed as described previously (Itoh et al., 2001) according to manufacturer's instructions (Becton Dickinson). MT1-MMP (wild-type or mutant) expression plasmids were cotransfected into CHO-K1 cells with the green fluorescent protein (GFP) expression plasmid. The transfected cells were suspended in Ham's F-12 medium without serum and seeded onto Matrigel-coated transwell filters (8-µm pore) in invasion chambers. FBS (10%)-containing medium was added to the lower chambers and incubated for 48 h. Noninvading cells remaining on the upper surface of the filter were removed and fixed with 3% paraformaldehyde. The GFP-positive cells appearing on the lower surface of the filter were counted by fluorescence microscopy. Each assay was performed in triplicate.
Phagokinetic track motility assay
The phagokinetic track motility assay was performed as described previously (Kajita et al., 2001). Colloidal gold–coated coverslips were placed in a 12-well plate and transfected cells were seeded at 3 x 103 cells/well. After 12 h of incubation, the phagokinetic tracks were visualized under bright-field illumination using a CoolSNAP-fx monochrome CCD camera (Roper Scientific). The track area was measured using NIH Image software v1.62.
|
Footnotes |
|---|
* Abbreviations used in this paper: AP-2, adaptor protein 2; ECM, extracellular matrix; GFP, green fluorescent protein; hTfnR, human transferrin receptor; IL2R, interleukin 2 receptor
-chain; MMP, matrix metalloproteinase; MT-MMP, membrane-type MMP; TIMP, tissue inhibitor of metalloproteinases. 
|
Acknowledgments |
|---|
Submitted: 21 August 2001
Revised: 29 October 2001
Accepted: 30 October 2001
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Apte, S.S., N. Fukai, D.R. Beier, and B.R. Olsen. 1997. The matrix metalloproteinase-14 (MMP-14) gene is structurally distinct from other MMP genes and is co-expressed with the TIMP-2 gene during mouse embryogenesis. J. Biol. Chem. 272:25511–25517.
Butler, G.S., M.J. Butler, S.J. Atkinson, H. Will, T. Tamura, S.S. van Westrum, T. Crabbe, J. Clements, M.P. d'Ortho, and G. Murphy. 1998. The TIMP2 membrane type 1 metalloproteinase "receptor" regulates the concentration and efficient activation of progelatinase A. A kinetic study. J. Biol. Chem. 273:871–880.
Chen, W.T., and J.Y. Wang. 1999. Specialized surface protrusions of invasive cells, invadopodia and lamellipodia, have differential MT1-MMP, MMP-2, and TIMP-2 localization. Ann. NY Acad. Sci. 878:361–371.
Heilker, R., M. Spiess, and P. Crottet. 1999. Recognition of sorting signals by clathrin adaptors. Bioessays. 21:558–567.
Holmbeck, K., P. Bianco, J. Caterina, S. Yamada, M. Kromer, S.A. Kuznetsov, M. Mankani, P.G. Robey, A.R. Poole, I. Pidoux, et al. 1999. MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell. 99:81–92.
Horwitz, A.R., and J.T. Parsons. 1999. Cell migration—movin' on. Science. 286:1102–1103.
Hotary, K., E. Allen, A. Punturieri, I. Yana, and S.J. Weiss. 2000. Regulation of cell invasion and morphogenesis in a three-dimensional type I collagen matrix by membrane-type matrix metalloproteinases 1, 2, and 3. J. Cell Biol. 149:1309–1323.
Hunziker, W., and C. Fumey. 1994. A di-leucine motif mediates endocytosis and basolateral sorting of macrophage IgG Fc receptors in MDCK cells. EMBO J. 13:2963–2967.
Itoh, Y., M. Kajita, H. Kinoh, H. Mori, A. Okada, and M. Seiki. 1999. Membrane type 4 matrix metalloproteinase (MT4-MMP, MMP-17) is a glycosylphosphatidylinositol-anchored proteinase. J. Biol Chem. 274:34260–34266.
Itoh, Y., A. Takamura, N. Itoh, Y. Maru, H. Sato, N. Suenaga, T. Aoki, and M. Seiki. 2001. Homophilic complex formation of MT1-MMP facilitates proMMP-2 activation on the cell surface and promotes tumor cell invasion. EMBO J. 20:4782–4793.
Kajita, M., Y. Itoh, T. Chiba, H. Mori, A. Okada, H. Kinoh, and M. Seiki. 2001. Membrane-type 1 matrix metalloproteinase cleaves cd44 and promotes cell migration. J. Cell Biol. 153:893–904.
Kinoh, H., H. Sato, Y. Tsunezuka, T. Takino, A. Kawashima, Y. Okada, and M. Seiki. 1996. MT-MMP, the cell surface activator of proMMP-2 (pro-gelatinase A), is expressed with its substrate in mouse tissue during embryogenesis. J. Cell Sci. 109:953–959.
Kinoshita, T., H. Sato, A. Okada, E. Ohuchi, K. Imai, Y. Okada, and M. Seiki. 1998. TIMP-2 promotes activation of progelatinase A by membrane-type 1 matrix metalloproteinase immobilized on agarose beads. J. Biol. Chem. 273:16098–16103.
Knauper, V., H. Will, C. Lopez-Otin, B. Smith, S.J. Atkinson, H. Stanton, R.M. Hembry, and G. Murphy. 1996. Cellular mechanisms for human procollagenase-3 (MMP-13) activation. Evidence that MT1-MMP (MMP-14) and gelatinase a (MMP-2) are able to generate active enzyme. J. Biol. Chem. 271:17124–17131.
Kojima, S., Y. Itoh, S. Matsumoto, Y. Masuho, and M. Seiki. 2000. Membrane-type 6 matrix metalloproteinase (MT6-MMP, MMP-25) is the second glycosyl-phosphatidyl inositol (GPI)-anchored MMP. FEBS Lett. 480:142–146.
Koshikawa, N., G. Giannelli, V. Cirulli, K. Miyazaki, and V. Quaranta. 2000. Role of cell surface metalloprotease MT1-MMP in epithelial cell migration over laminin-5. J. Cell Biol. 148:615–624.
Lehti, K., J. Lohi, H. Valtanen, and J. Keski-Oja. 1998. Proteolytic processing of membrane-type-1 matrix metalloproteinase is associated with gelatinase A activation at the cell surface. Biochem. J. 334:345–353.
Lehti, K., H. Valtanen, S. Wickstrom, J. Lohi, and J. Keski-Oja. 2000. Regulation of membrane-type-1 matrix metalloproteinase activity by its cytoplasmic domain. J. Biol. Chem. 275:15006–15013.
Maquoi, E., F. Frankenne, E. Baramova, C. Munaut, N.E. Sounni, A. Remacle, A. Noel, G. Murphy, and J.M. Foidart. 2000. Membrane type 1 matrix metalloproteinase-associated degradation of tissue inhibitor of metalloproteinase 2 in human tumor cell lines. J. Biol. Chem. 275:11368–11378.
Murphy, G., and J. Gavrilovic. 1999. Proteolysis and cell migration: creating a path? Curr. Opin. Cell Biol. 11:614–621.
Nagase, H., and J.F. Woessner, Jr. 1999. Matrix metalloproteinases. J. Biol. Chem. 274:21491–21494.
Nakahara, H., L. Howard, E.W. Thompson, H. Sato, M. Seiki, Y. Yeh, and W.T. Chen. 1997. Transmembrane/cytoplasmic domain-mediated membrane type 1-matrix metalloprotease docking to invadopodia is required for cell invasion. Proc. Natl. Acad. Sci. USA. 94:7959–7964.
Ohno, H., J. Stewart, M.C. Fournier, H. Bosshart, I. Rhee, S. Miyatake, T. Saito, A. Gallusser, T. Kirchhausen, and J.S. Bonifacino. 1995. Interaction of tyrosine-based sorting signals with clathrin-associated proteins. Science. 269:1872–1875.
Ohno, H., M.C. Fournier, G. Poy, and J.S. Bonifacino. 1996. Structural determinants of interaction of tyrosine-based sorting signals with the adaptor medium chains. J. Biol. Chem. 271:29009–29015.
Ohuchi, E., K. Imai, Y. Fujii, H. Sato, M. Seiki, and Y. Okada. 1997. Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. J. Biol. Chem. 272:2446–2451.
Pei, D. 1999. Identification and characterization of the fifth membrane-type matrix metalloproteinase MT5-MMP. J. Biol. Chem. 274:8925–8932.
Pei, D., and S.J. Weiss. 1996. Transmembrane-deletion mutants of the membrane-type matrix metalloproteinase-1 process progelatinase A and express intrinsic matrix-degrading activity. J. Biol. Chem. 271:9135–9140.
Sato, H., T. Takino, Y. Okada, J. Cao, A. Shinagawa, E. Yamamoto, and M. Seiki. 1994. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature. 370:61–65.
Sato, T., M. del Carmen Ovejero, P. Hou, A.M. Heegaard, M. Kumegawa, N.T. Foged, and J.M. Delaisse. 1997. Identification of the membrane-type matrix metalloproteinase MT1-MMP in osteoclasts. J. Cell Sci. 110:589–596.
Seiki, M. 1999. Membrane-type matrix metalloproteinases. APMIS. 107:137–143.
Stanton, H., J. Gavrilovic, S.J. Atkinson, M.P. d'Ortho, K.M. Yamada, L. Zardi, and G. Murphy. 1998. The activation of ProMMP-2 (gelatinase A) by HT1080 fibrosarcoma cells is promoted by culture on a fibronectin substrate and is concomitant with an increase in processing of MT1-MMP (MMP-14) to a 45 kDa form. J. Cell Sci. 111:2789–2798.
Strongin, A.Y., I. Collier, G. Bannikov, B.L. Marmer, G.A. Grant, and G.I. Goldberg. 1995. Mechanism of cell surface activation of 72-kDa type IV collagenase. Isolation of the activated form of the membrane metalloprotease. J. Biol. Chem. 270:5331–5338.
Tsunezuka, Y., H. Kinoh, T. Takino, Y. Watanabe, Y. Okada, A. Shinagawa, H. Sato, and M. Seiki. 1996. Expression of membrane-type matrix metalloproteinase 1 (MT1-MMP) in tumor cells enhances pulmonary metastasis in an experimental metastasis assay. Cancer Res. 56:5678–5683.
Werb, Z. 1997. ECM and cell surface proteolysis: regulating cellular ecology. Cell. 91:439–442.
Yana, I., and S.J. Weiss. 2000. Regulation of membrane type-1 matrix metalloproteinase activation by proprotein convertases. Mol. Biol. Cell. 11:2387–2401.
Zhou, Z., S.S. Apte, R. Soininen, R. Cao, G.Y. Baaklini, R.W. Rauser, J. Wang, Y. Cao, and K. Tryggvason. 2000. Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I. Proc. Natl. Acad. Sci. USA. 97:4052–4057.
Zuk, P.A., and L.A. Elferink. 1999. Rab15 mediates an early endocytic event in Chinese hamster ovary cells. J. Biol. Chem. 274:22303–22312.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
, radioactivity spontaneously released into the culture medium (Medium); 
, radioactivity released by acid wash (Cell surface); 
, acid wash–resistant radioactivity (Internalize). Mean values of three independent experiments are shown (mean ± SD). Transfected cells were also analyzed by Western blotting using anti-FLAG M2 or anti-hTfnR antibodies (inset). (B) CHO-K1 cells expressing MT1-F or mock-transfected cells were incubated with anti-FLAG M2 antibody, after which the cells were incubated at 37°C for the indicated periods of time. The cell surface and internalized MT1-F molecules were analyzed by immunolocalization using AlexaTM488-conjugated anti–mouse IgG. Internalized MT1-F molecules were observed by washing the cells with acid solution before fixation and permeabilization. Signals were observed by confocal laser microscopy. Bar, 10 µm.
) is plotted. Values in B and C are the mean ± SD of three experiments.
