The tetraspanin network modulates MT1-MMP cell surface trafficking

The International Journal of Biochemistry & Cell Biology 45 (2013) 1133–1144

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The tetraspanin network modulates MT1-MMP cell surface trafficking H.M. Schröder a,∗ , S.C. Hoffmann a,b , M. Hecker a , T. Korff a , T. Ludwig b a b

Institute of Physiology and Pathophysiology, Division of Cardiovascular Physiology, Heidelberg University, 69120 Heidelberg, Germany German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

a r t i c l e

i n f o

Article history: Received 10 August 2012 Received in revised form 11 February 2013 Accepted 21 February 2013 Available online 14 March 2013 Keywords: Cancer Tetraspanin MT1-MMP Cell surface trafficking Protein–protein interaction

a b s t r a c t The membrane-type 1 matrix metalloproteinase (MT1-MMP) drives fundamental physiological and pathophysiological processes. Among other substrates, MT1-MMP cleaves components of the extracellular matrix and activates other matrix-cleaving proteases such as MMP-2. Trafficking is a highly effective means to modulate MT1-MMP cell surface expression, and hence regulate its function. Here, we describe the complex interaction of MT1-MMP with tetraspanins, their effects on MT1-MMP intracellular trafficking and proteolytic function. Tetraspanins are credited as membrane organizers that form a network within the membrane to regulate the trafficking of associated proteins. In short, we found MT1-MMP to interact with the tetraspanin-associated EWI-2 protein by a yeast two-hybrid screen. Immunoprecipitation analysis confirmed this interaction and further revealed that MT1-MMP also stably interacts with distinct tetraspanins (CD9, CD37, CD53, CD63, CD81, and CD82) and the tetraspanin-like MAL protein. By using different MT1-MMP truncation constructs and mutants, we observed that all tetraspanins and MAL associated with the hemopexin domain of MT1-MMP. Moreover, this interaction was independent of O-glycosylation of MT1-MMP and exclusively occurred in the endoplasmic reticulum. Here, the respective subcellular compartment was identified by fitting the MT1-MMP interaction pattern to a model for post-translational processing of MT1-MMP. In addition, tetraspanins differentially affected the cell surface localization of MT1-MMP, its capacity to activate pro-MMP-2, and the collagen invasion capacity. Interestingly, the degree of tetraspanin-MT1-MMP association did not correlate with its impact on MT1MMP function. Tetraspanins thus distinctly affect MT1-MMP subcellular localization and function, and may constitute an effective mechanism to control MT1-MMP-dependent proteolysis at the cell surface. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Matrix metalloproteinases (MMPs) are a family of zincdependent endopeptidases that influence fundamental biological processes through the hydrolysis of miscellaneous substrates such as cell adhesion molecules and extracellular matrix proteins. Amongst the various MMPs, membrane-type 1 MMP (MT1-MMP) is pivotal to tissue remodeling. Its deficiency negatively impacts collagenolytic activity in mice and for example impairs connective tissue function during skeletal growth (Holmbeck et al., 1999). MT1-MMP is tightly regulated by multiple transcriptional and posttranscriptional mechanisms that control its expression and activity as well as sub-cellular localization during maturation of the enzyme (Itoh and Seiki, 2004; Ludwig et al., 2008b; Osenkowski et al., 2004; Zucker et al., 2002). MT1-MMP is synthesized as a pre-pro-enzyme that is directed to the endoplasmic reticulum. After removal of

∗ Corresponding author at: Institute of Physiology and Pathophysiology, Division of Cardiovascular Physiology, Heidelberg University, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany. Tel.: +49 6221 54 4067; fax: +49 6221 54 4038. E-mail address: [email protected] (H.M. Schröder). 1357-2725/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2013.02.020

its signal peptide, pro-MT1-MMP is transported to the Golgi network for O-glycosylation of its hinge domain (Ludwig et al., 2008b; Remacle et al., 2006). The protease is then activated in a post-Golgi compartment by proteolytic cleavage of the pro-peptide (Murphy et al., 1999). At the cell surface, the active 57 kDa MT1-MMP protein usually undergoes autocatalytic processing (Lehti et al., 2000; Sato and Takino, 2010; Toth et al., 2002). This yields the soluble catalytic domain and a membrane-tethered but catalytically inactive 44 kDa species (Lehti et al., 2000). Various factors and mechanisms regulate the cell surface processing of MT1-MMP including its Oglycosylation status and localization to membrane microdomains, the amount of bound tissue inhibitors of metalloproteinases type 2 (TIMP-2), or the indirect regulation by extracellular matrix components (Ellerbroek et al., 2001; Hernandez-Barrantes et al., 2000; Kirmse et al., 2011; Messaritou et al., 2009; Remacle et al., 2006; Yanez-Mo et al., 2008). In this context, cell surface trafficking has been identified as a pivotal posttranslational mechanism to regulate the biological functions of MT1-MMP. It is the most effective and rapid mechanism to regulate its proteolytic activity and it enables its fast recycling to the cell surface. In contrast, de novo synthesis and maturation of the enzyme is time and energy-consuming (Gingras and Beliveau, 2010; Poincloux et al., 2009). Interestingly,

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both clathrin-coated pits and caveolae have been proposed as alternate routes of trafficking in MT1-MMP internalization, but many aspects of MT1-MMP regulation still remain elusive (Annabi et al., 2001; Jiang et al., 2001). In order to gain more insight into how the protease is in fact controlled, we have conducted an extensive yeast two-hybrid screen that identified several new potential protein–protein interactions of MT1-MMP, amongst them the tetraspanin-associated protein IgSF8. Tetraspanins are integral membrane proteins that span the cell membrane four times. They constitute the main building blocks of specialized membrane microdomains, also referred to as tetraspanin-enriched microdomains (Berditchevski and Odintsova, 2007; Hemler, 2003). These domains are most likely composed of a core of several tetraspanins, which coordinate the interaction of other membrane proteins. In addition to this scaffolding function, tetraspanins have been reported to regulate various aspects of trafficking and biosynthetic processing of integral membrane proteins (Berditchevski and Odintsova, 2007; Charrin et al., 2002; Maecker et al., 1997). As such, overexpression of the tetraspanin CD63 has been shown to induce redistribution of H+ /K+ -ATPase from the plasma membrane to intracellular compartments in COS cells (Duffield et al., 2003). The tetraspanin-like myelin and lymphocyte protein (MAL, also known as VIP17) has been associated with the apical transport of lipids and proteins during myelogenesis in oligodendrocytes (Kim et al., 1995). Moreover, MAL can interfere with the internalization of aquaporin-2 by renal cells and therefore has been suggested to play a regulatory role in apical membrane protein stability (Kamsteeg et al., 2007). Furthermore, interaction of the EWI motif-containing protein 2 (EWI-2, also known as IgSF8) with tetraspanins CD9 and CD81 seems to contribute to the motility of T-cells as well as cancer cells (Clark et al., 2001; Stipp et al., 2001, 2003a). The interaction of EWI-2 with CD82, on the other hand, has been shown to yield the opposite effect on cell migration (Zhang et al., 2003). The aims of this study therefore were to (a) investigate the interaction of MT1-MMP with tetraspanins and (b) unravel their effects on MT1-MMP function.

mutant (CHO) have been described previously (Ludwig et al., 2008b). The HA-tagged MT1-MMP constructs were cloned into the pcDNA3.1/Hygro vector (Invitrogen, Carlsbad, CA, USA) as described before (Kirmse et al., 2011). The following additional constructs were generated with regard to the domain composition of MT1-MMP: signal peptide (Met1 -Phe33 ), pro-domain (Ser34 Arg111 ), catalytic domain (Tyr112 -Gly284 ), hinge and hemopexin domain (Gly285 -Cys508 ), transmembrane domain (Ile525 -Val559 ), and the cytoplasmic tail (Phe560 -Val582 ) (see Fig. 4a). DNA constructs were first gel-purified as a polymerase chain reaction (PCR) product and cloned into the pCR2.1 Topo vector (Invitrogen) according to the manufacturer’s instructions. These constructs were then cloned into the pcDNA3.1/Hygro vector after digestion with HindIII and NotI. The cDNAs of the human tetraspanins CD9 (NM 001769), CD37 (NM 001774), CD53 (NM 001040033), CD63 (NM 001780), CD81 (NM 004356), CD82 (NM 002231), and the human tetraspanin-like MAL protein (NM 002371) were amplified via PCR from a human kidney cDNA library and sub-cloned with a FLAG-tag at the N-terminus into the pcDNA3.1/Hygro expression vector. The cDNAs of human MMP-1, MMP-2, MMP-9, MT3-MMP and EWI-2 were amplified via PCR from full-ORF clones that were kindly provided by the proteomics core facility of the German Cancer Research Center (DKFZ), Heidelberg, Germany. The corresponding gel-purified PCR products were sub-cloned into the pcDNA3.1/V5-His-TOPO expression vector (Invitrogen) encoding C-terminal V5-His-tagged proteins. The accuracy of all DNA constructs was confirmed by automated fluorescence sequencing. 2.3. Yeast two-hybrid screen

2. Materials and methods

The yeast two-hybrid screen was performed in collaboration with the proteomics core facility of the DKFZ. In short, four libraries were screened in total. These were HeLa cells (normalized), human brain, human fetal brain, and a full open reading frame mammalian gene collection. Two truncated MT1-MMP constructs consisting either of (i) the catalytic domain (Tyr112 -Gly284 ) or (ii) the catalytic, the hinge and the hemopexin domain (Tyr112 -Cys508 ) were used as bait. The experimental setup and selection of high-confidence interactions have been described elsewhere (Albers et al., 2005)

2.1. General reagents and antibodies

2.4. Procedures for cDNA transfection and sample preparation

Unless otherwise noted, all reagents were purchased from Sigma–Aldrich (Taufkirchen, Germany). All buffers and solutions were sterile filtered with Milliex-GS 0.22 mm syringe-driven filters (Millipore, Schwalbach, Germany). Anti ␤-actin mouse monoclonal antibody (mAb) (ab6276, Abcam, Cambridge, MA, USA), anti HA-epitope mAb (MMS-101R, Covance, Berkeley, CA, USA), anti V5-epitope rabbit polyclonal antibody (pAb) (GTX30564, GeneTex, Irvine, CA, USA), anti FLAG-epitope pAb (F7425, Sigma–Aldrich), and M2 anti-FLAG mAb (F1804, Sigma–Aldrich) were used as primary antibodies. Oregon green488-conjugated goat anti rabbit antibody (O6381, Invitrogen, Carlsbad, CA, USA), Alexa568conjugated goat anti mouse antibody (A11031, Invitrogen), and phycoerythrine-conjugated goat anti mouse antibody (115-116146, Jackson Dianova, Hamburg, Germany) were used as secondary antibodies.

COS cells were seeded in six-well plastic dishes (Greiner Bio-One, Frickenhausen, Germany) and transfected at 85–90% confluency 24 h after seeding. Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) was used for transfection according to the manufacturer’s instructions. For co-transfections, 200 ng DNA of MT1-MMP constructs and 300 ng DNA of tetraspanin or MAL constructs were used per cm2 of seeded cells. Unless otherwise noted, cells were lysed 24 h after transfection according to the following protocol: Cells were washed with ice-cold Hanks’ balanced salt solution (BSS; see Hoffmann et al., 2010) before the addition of 500 ␮l lysis buffer (50 mM Tris–HCl, 150 mM NaCl, pH 7.5; supplemented with 2× final concentration of complete protease inhibitor tablets with ethylenediaminetetraacetate (EDTA; Roche Diagnostics, Penzberg, Germany) and 1% Triton X-100 or 1% Brij96) per well. Cells were scraped off from the wells, incubated on ice for 45 min on a rocking platform, and the resulting whole cell lysates cleared by centrifugation (17,000 × g, 20 min, 4 ◦ C). Aliquots of 10 ␮l of cell lysates were mixed with reducing sample buffer (see Ludwig et al., 2008b) and heated (10 min, 56 ◦ C).

2.2. DNA constructs The cDNA of FLAG-tagged, full-length wild type (WT) human MT1-MMP was kindly provided by the laboratory of Motoharu Seiki (Institute of Medical Science, University of Tokyo, Japan) (Uekita et al., 2001). Insertion of the hemagglutinin (HA) tag between residues Gly511 and Gly512 in the stalk (linker-2) region of MT1-MMP and generation of the O-glycosylation-deficient

2.5. Tissue culture Cell culture media and reagents were purchased from Invitrogen (Carlsbad, CA, USA). COS cells were grown in flasks under standard

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conditions (37 ◦ C, 5% CO2 , and 100% humidity) in modified Eagle’s medium (MEM) supplemented with 2 mM l-glutamine (GlutaMAX1), 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany), and penicillin/streptomycin.

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recombinant human pro-MMP-2 (Biocol, Luckenwalde, Germany). After 24 h, cell culture supernatants were cleared by centrifugation and directly subjected to gelatin zymography. 2.11. Gelatin zymography assay

2.6. SDS-PAGE and Western blot analyses Standard protocols for Western blot analysis have been described elsewhere (Roush et al., 1998). Samples were loaded on 10% NuPage Novex Bis–Tris gels (Invitrogen, Carlsbad, CA, USA) and separated electrophoretically under constant current in a 3-(N-morpholino)propanesulfonic acid-buffered running buffer (Invitrogen). Proteins were transferred to nitrocellulose membranes using the iBlot dry blotting system (Invitrogen). Western blots were blocked with 5% dry milk in Tris-buffered saline containing 0.1% Tween-20 and incubated overnight with anti-ß actin mAb (1:10,000), anti HA-epitope mAb (1:1000), or anti V5-epitope rabbit pAb (1:1000).

The gelatinolytic activity of MMP-2 in conditioned medium was analyzed by zymography on 10% polyacrylamide gels containing 1 mg/ml gelatin. Equal volumes of sample were mixed with a non-reducing sample buffer and loaded in each lane. After electrophoresis, gels were washed twice with washing buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 2.7% Triton X-100 (w/v)) and once in washing buffer without Triton X-100, incubated in a developing buffer (50 mM Tris base, 40 mM HCl, 200 mM NaCl, 5 mM CaCl2 , 0.02% (w/v) Brij35) for 16 h at 37 ◦ C, and stained with SimplyBlue SafeStain (Invitrogen, Carlsbad, CA, USA). Proteins with gelatinolytic activity were visualized as clear zones in an otherwise blue gel.

2.7. Cell-surface biotinylation

2.12. Flow cytometry

Protocols and standard procedures for cell-surface protein biotinylation have been described previously (Gottardi et al., 1995). In brief, cell-surface biotinylation was carried out 24 h after co-transfection of COS cells in six-well plates with HA-tagged MT1-MMP and FLAG-tagged tetraspanin constructs. Cells were washed with Hanks’ BSS. 1 ml of biotinylation buffer (10 mM triethanolamine, 2 mM CaCl2 , 125 mM NaCl; pH 8.9) supplemented with 2.5 mM sulfo-NHS-SS-biotin (EZ-Link, Pierce) was added twice per well for incubations for 25 min each. Cells were washed with Hanks’ BSS supplemented with 100 mM glycine and once with Hanks’ BSS before they were lysed. Biotinylated proteins were captured by adding 40 ␮l of washed 50% slurry of streptavidinconjugated beads (Pierce Biotechnology, Rockford, IL, USA) to the cell lysates (see immunoprecipitation protocol).

Adherent cells were washed twice with ice-cold Hanks’ BSS and detached using the enzyme-free cell dissociation buffer (Gibco). Detached cells were washed twice and resuspended in phosphatebuffered saline (PBS) supplemented with 2% paraformaldehyde (PFA), 5% heat-inactivated FCS, 0.5% BSA, and 0.07% NaN3 . Fixed cells were incubated for 1 h with primary antibodies (mouse anti HA-epitope mAb, 1:50; rabbit anti FLAG-epitope pAb, 1:200) and 30 min with secondary antibodies (phycoerythrine-conjugated goat anti mouse and oregon green488-conjugated goat anti rabbit antibody). Fixed cells were extensively washed between and after incubations. Stained cells were analyzed on a Becton-Dickinson FACScan flow cytometer using the BD Cellquest and FlowJo software. 2.13. Fluorescence microscopy

2.8. Immunoprecipitation For co-immunoprecipitation, 4 ␮g M2 anti FLAG-epitope mAb were added to the cleared lysates each, and incubated at 4 ◦ C on a rotating platform. Immobilized protein A beads (Pierce) were washed, blocked with 1% bovine serum albumin (BSA) in lysis buffer for 1 h and washed again prior to addition of 40 ␮l of the 50% slurry of these beads to each pre-incubated lysate-antibody mix. After overnight incubation at 4 ◦ C on a rotating platform, beads were extensively washed in lysis buffer. Proteins were eluted by addition of 2× SDS-PAGE-sample buffer and heated to 65 ◦ C for 8 min followed by Western blot analyses to resolve immunoprecipitated protein complexes. 2.9. Enzymatic desialylation assay Enzymatic desialylation with sialidase A (GK80040, Prozyme Inc., San Leandro, CA) was performed according to the manufacturer’s instructions. In short, transfected cells were lysed, and 2 ␮l of lysate was mixed and incubated with 3 ␮l of 5× reaction buffer and 4 ␮l of sialidase A (activity > 5 units/ml) or solvent control in 15 ␮l total volume for 18 h at 37 ◦ C. The reaction was stopped by adding 6× reducing sample buffer and heating (8 min at 65 ◦ C). Samples were immunoblotted with anti HA-epitope mAb. 2.10. Pro-MMP-2 activation assay COS cells were co-transfected with MT1-MMP and tetraspanins or MAL and cultured for 24 h in OPTI-MEM (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 50 ng/ml final concentration

The immunofluorescence staining procedures applied here have been described previously (Duffield et al., 2003). In short, mouse anti HA-epitope mAb and rabbit anti FLAG-epitope served as primary antibodies. Alexa568-labeled goat anti mouse and Oregon green488-conjugated goat anti rabbit antibodies served as secondary antibodies. Confocal microscopy images were recorded with a Nikon Eclipse Ti inverted microscope (Nikon Instruments, Europe) equipped with an ERS-6FE spinning disc module (Perkin Elmer, Waltham MA, USA) and a 100× objective (100 × 1.4 NA; Nikon Instruments) in combination with the provided Nikon software (NIS-Elements AR2.3 build). For all combinations, the transfection efficiency was determined to be about 90% of total cell count. Therefore, the expression levels of MT1-MMP and tetraspanins or MAL were analyzed using wide-field fluorescence microscopy combined with differential interference contrast imaging at a 20-fold magnification (data not shown). 2.14. Collagen invasion assay Three-dimensional cell spheroids of a defined cell number were generated as described previously (Korff and Augustin, 1998; Korff et al., 2001, 2004) and adapted for COS cells. One day after co-transfection, COS cells were suspended in culture medium containing 0.25% (w/v) methylcellulose and seeded in nonadherent round-bottom 96-well plates (Greiner, Frickenhausen, Germany) for 24 h. Under these conditions, all suspended cells contribute to the formation of a single spheroid per well of defined size and cell number. Spheroids containing 750 cells each were embedded in neutralized collagen gels and covered with 200 ␮l growth medium

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per 1 ml gel (24-well plates) after gel polymerization. The gels were incubated at 37 ◦ C, 5% CO2 and 100% humidity. After three days, the invasion and growth in three-dimensional collagen gels was digitally quantified using the Cell∧ D software (Olympus) analyzing at least 10 randomly selected spheroids per experimental group and experiment. 2.15. Statistical analyses All results are expressed as means ± SD. Probability values of p < 0.05 were considered as statistically significant. Differences between two matched experimental groups were analyzed by unpaired Student’s t test, differences among three or more experimental groups were analyzed by ANOVA followed by a Bonferroni post hoc test for selected pairs of groups with the Prism version 4.0c statistics software package (Graph Pad Software, San Diego, CA, USA). 3. Results 3.1. EWI-2 links with MT1-MMP and tetraspanins or the tetraspanin-like MAL protein In order to identify new interaction partners of MT1-MMP, a yeast two-hybrid screen was performed. Two MT1-MMP constructs consisting either of (a) the catalytic domain or (b) the catalytic, hinge and hemopexin domains were screened against four libraries (HeLa cells (normalized), human brain, human fetal brain, and a full open reading frame mammalian gene collection). In total, 28 proteins were identified as interaction partners of MT1-MMP (Supplemental Fig. S1). One potential interacting protein was the immunoglobulin superfamily member (IgSF) 8. IgSF8 is a single-pass membrane protein, also known as EWI-2. To validate this yeast two-hybrid result, a V5-tagged EWI-2 construct was generated and co-expressed with HA-tagged MT1-MMP in COS cells. Corresponding Triton X-100 or Brij96 extracts were tested for co-immunoprecipitation and confirmed the interaction between the two proteins (Fig. 1a). In addition, EWI-2 was found to form Triton-stable complexes with different tetraspanins such as CD9, CD37, CD53, CD63, CD81, CD82, and the tetraspanin-like MAL protein (Fig. 1b). 3.2. Tetraspanins or MAL robustly interact with MT1-MMP Next, we sought to elucidate whether tetraspanins or MAL can also stably interact with MT1-MMP and thereby regulate its function. Here, COS cells served as a particularly useful cellular expression system since they do not express MT1-MMP endogenously (Cao et al., 1998; Ludwig et al., 2008b). Moreover, the HA tag that does not interfere with MT1-MMP function was employed again to detect most forms of MT1-MMP (Ludwig et al., 2008b). Since MT1-MMP is subject to extensive post-translational modification (Fig. 2a and c), expression of the HA-tagged MT1-MMP construct in COS cells yielded a complex MT1-MMP protein pattern as detected by Western blot (Fig. 2b). Subsequently, FLAG-tagged tetraspanins or FLAG-tagged MAL were co-expressed with the HA-tagged MT1-MMP, and COS cell lysates were tested for coimmunoprecipitation (Fig. 3a, Supplemental Fig. S2). The IgG band (arrow) that is also detected by the secondary antibody displayed equal amounts of the anti-FLAG antibody used for immunoprecipitation. The tetraspanins CD37, CD53, CD82 and the tetraspanin-like MAL protein formed Triton-stable complexes with the 63 kDa proMT1-MMP protein. Associations of CD9, CD63 and CD81 with this MT1-MMP variant were also detected, but to a lesser extent (Fig. 3a).

Fig. 1. MT1-MMP and tetraspanins interact with EWI-2. (A) HA-tagged MT1-MMP or (B) FLAG-tagged tetraspanins (CD9, CD37, CD53, CD63, CD81, CD82) or the FLAG-tagged myelin and lymphocyte-associated protein (MAL) as a tetraspanin-like protein were co-expressed with V5-tagged EWI-2 in COS cells which were subsequently lysed in different detergents (T, Triton X-100; B, Brij96). Expression of an empty vector (–) served as control. Protein complexes were co-immunoprecipitated (IP), resolved on SDS-PAGE, and identified by immunoblotting (␤-actin: loading control).

To assess differences in the relative strength of these protein–protein interactions, the COS cells were lysed with different detergents. Extraction with Triton X-100 and Brij96 has previously been proven to be versatile in the identification of interactions with high affinity that are very likely to be direct, first-level tetraspanin–partner interactions (Claas et al., 2001; Duffield et al., 2003; Hemler, 2005; Levy and Shoham, 2005b; Yauch et al., 2000). Extraction with Brij96 however also preserves less robust interactions that are considered to be secondary in nature and most likely formed by heterophilic tetraspanin–tetraspanin contacts (YanezMo et al., 2009). We found that MT1-MMP-tetraspanin associations were less intense in Brij96 extracts compared to those in Triton extracts, but only additional associations with the inactive 44 kDa MT1-MMP protein were apparent for CD37, CD53, CD63 and the tetraspanin-like MAL protein (Fig. 3b, Supplemental Fig. S2). 3.3. Tetraspanins and MAL interact with the hemopexin domain of MT1-MMP In order to identify the MT1-MMP domain that interacts with tetraspanins or MAL, distinct HA-tagged MT1-MMP truncation constructs and mutants (Fig. 4a) were co-expressed with FLAGtagged tetraspanins or MAL and tested for co-immunoprecipitation. In general, the tetraspanins or MAL interacted only with those MT1-MMP proteins that contain the hemopexin domain (Fig. 4). MT1-MMP proteins lacking the catalytic domain (Cat) or both the pro and the catalytic domain (Pro–Cat) were therefore still capable of forming complexes with the tetraspanins and MAL

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Fig. 2. Model for the post-translational processing of MT1-MMP (Ludwig et al., 2008b). (I) The 23-amino acid signal peptide is removed upon synthesis in the endoplasmic reticulum (ER) to yield the inactive, non-glycosylated pro-form of MT1-MMP (a). (II) O-glycosylation of the hinge domain in the Golgi apparatus. (III) MT1-MMP is activated by the removal of its pro-domain by the pro-protein convertase activity of a furin or furin-like enzyme activity. (IV) Autocleavage causes shedding of the catalytic domain and results in the generation of a membranetethered remnant with the hinge domain at its N terminus (d). (B) Western blot analyses of wild type MT1-MMP expressed in COS cells. Cellular localization (a–d), ER: endoplasmic reticulum, Golgi, post-Golgi, PM: plasma membrane; different MT1-MMP processing types have been determined previously (see Ludwig et al., 2008b). (C) Products of MT1-MMPs metabolic processing (a–d). Source: Addapted from Ludwig et al. (2008b).

that were stable in Triton and Brij96 (Fig. 4a and c, Supplemental Figs. S3 and S4). Also, MT1-MMP proteins lacking the transmembrane domain and/or the cytoplasmic tail still associated with CD53 (Fig. 4b). 3.4. Tetraspanins or MAL interact with MT1-MMP in the endoplasmic reticulum independent of O-glycosylation When full-length MT1-MMP was co-expressed with the investigated tetraspanins or the MAL protein, only the pro-MT1-MMP species revealed an interaction (Fig. 3). This interaction occurs before the O-glycosylation of MT1-MMP. The same could be observed for the Cat and Pro–Cat truncation variants (Fig. 4b and c, Supplemental Figs. S3 and S4). The glycosylation state of these MT1-MMP metabolites was revealed by an enzymatic deglycosylation approach using sialidase A (Fig. 4d). These findings are summarized for the MAL protein as a representative example in Fig. 4e. In essence, the aforementioned observations raised the question as to whether or not O-glycosylation could modulate the interaction between tetraspanins or MAL with MT1-MMP. Glycosylation-deficient (CHO), truncated MT1-MMP mutants were thus co-expressed with tetraspanins or MAL. This approach revealed that neither did additional MT1-MMP metabolites interact with tetraspanins or MAL, nor did the lack of MT1-MMP

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Fig. 3. Tetraspanins and MAL form Triton-stable complexes with MT1-MMP. (A) HA-tagged MT1-MMP metabolites were co-expressed as synthetic, truncated constructs with either FLAG-tagged tetraspanins (CD9, CD37, CD53, CD63, CD81, CD82), the FLAG-tagged MAL protein, or an empty vector (–) as a control in COS cells. Protein lysates were co-immunoprecipitated (IP, anti FLAG), resolved on SDS-PAGE and identified by immunoblotting (IB). Lysate Mix: lysates of cells that individually expressed MT1-MMP and MAL were combined after lysis (50:50, v/v), and incubated overnight prior to immunoprecipitation. IgG: The IgG band indicates equal loading of the pull-down antibody that is also detected by the secondary antibody; for equal amount of isolated protein see Supplemental Fig. S2 (␤-actin). (B) COS cells co-expressing MT1-MMP and tetraspanins or the tetraspanin-like MAL protein (here: CD53 and MAL as examples for clearly detectable MT1-MMP binding partners) were lysed in 1% Triton X-100 or Brij96 to asses protein complex stability of different MT1-MMP metabolites.

O-glycosylation hamper the protein–protein interactions with tetraspanins or MAL (Fig. 4b and c). This observation applied to CD9, CD37, CD63, CD81, CD82, and MAL upon co-expression with an untruncated MT1-MMP CHO variant (data not shown). 3.5. Tetraspanins and MAL interact with other MMP family members To assess the specificity of tetraspanins as binding partners for MT1-MMP, other MMP family members containing a hemopexin domain, i.e. MT3-MMP, MMP-1, MMP-2 and MMP-9, were tested for co-immunoprecipitation with the tetraspanins or MAL. Hemopexin-like domains constitute a characteristic structural feature of almost all MMPs even though this domain is more differentiated than the catalytically active site of MMPs, possibly indicating a role in determining substrate specificity (Andreini et al., 2004). CD53 and the MAL protein – the most clearly detectable MT1-MMP interaction partners among the tested tetraspanins or tetraspanin-like proteins – in fact interacted both with V5-tagged MT3-MMP, MMP-1, MMP-2, and MMP-9 (Fig. 5a and b). 3.6. Tetraspanins differentially reduce MT1-MMP cell surface abundance Next, the effects of tetraspanins or MAL on MT1-MMP subcellular localization were investigated. Co-expression of MT1-MMP with the different tetraspanins or MAL revealed that total MT1MMP abundance was not affected by CD53, CD63 and MAL (Fig. 6).

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Fig. 4. Tetraspanins and MAL interact with the hemopexin domain of MT1-MMP. (A) A hemagglutinin-tag (HA) was introduced for immunodetection of distinct MT1-MMP metabolites (sp: signal peptide; pro: pro-domain; st: stalk domain; tm: transmembrane domain; ct: cytoplasmic tail; WT: wild type; Cat: construct missing the catalytic domain; Pro–Cat: N-terminally truncated construct missing the catalytic and the pro-domain; CT: construct lacking the cytoplasmic tail; TM-CT: construct lacking the transmembrane and the cytoplasmic domain; CHO: O-glycosylation-deficient mutants). (B–E) Tetraspanin-MT1-MMP or MAL-MT1-MMP interactions can be allocated to the endoplasmic reticulum (ER) and are independent of MT1-MMP O-glycosylation. HA-tagged MT1-MMP constructs were co-expressed with FLAG-tagged tetraspanins or FLAG-tagged MAL (here: CD53 and MAL as examples for strongly detectable MT1-MMP binding partners) in COS cells. Protein lysates were co-immunoprecipitated (IP: anti FLAG), resolved on SDS-PAGE and detected by immunoblotting. The IgG band (IgG) indicates equal loading of the pull-down antibody that is also detected by the secondary antibody. (B) Co-expression of CD53 with different MT1-MMP truncation mutants. (C) Co-expression of MAL with selected MT1-MMP truncation mutants. (D) COS cells were transfected with distinct MT1-MMP mutants. Protein lysates were incubated with or without sialidase A (SiA), which cleaves the glycosidic linkages of sialic acids, and MT1-MMP metabolites were analyzed by immunoblotting. Encircled numbers indicate the sequential processing of MT1-MMP. Arrows indicate metabolites that are shifted due to SiA treatment and therefore are identified as glycosylated metabolites. Triangles indicate non-glycosylated metabolites that are not shifted upon SiA treatment. (E) Subcellular localization of MT1-MMP metabolites in correlation to their interaction with tetraspanins (ER: endoplasmic reticulum, Golgi, post-Golgi, PM: plasma membrane).

MT1-MMP levels, on the other hand, appeared to be reduced in the presence of CD9, CD37, CD81 or CD82. In terms of subcellular localization, immunofluorescence analyses revealed that MT1-MMP is predominantly present on the plasma membrane under control conditions (Supplemental Fig. S5) but accumulates in a perinuclear compartment when co-expressed with the tetraspanins or

MAL. In the presence of CD53, CD63 or the MAL protein, MT1-MMP was partially present at the plasma membrane, but virtually absent when co-expressed with CD9, CD37, CD81 or CD82. To differentiate between various forms of MT1-MMP present on the surface of the COS cells, cell surface proteins were biotinylated and immunoprecipitated with streptavidin. By this approach only the catalytically

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CD9 and CD37 have the strongest effect on the abundance of MT1MMP on the cell surface followed by CD81 and CD82. Only these two tetraspanins significantly reduced the amount of the mature protease on the cell surface. 3.7. Tetraspanins differentially reduce MT1-MMP-dependent pro-MMP2 activation and the invasion/growth capacity of COS cells within three-dimensional collagen gels Finally, we were interested in the effects of the tetraspanins or the MAL protein on the proteolytic activity of MT1-MMP expressed in COS cells. First, activation of pro-MMP-2 by MT1-MMP was used as a versatile measure for MT1-MMP activity that has also been proven to be biologically relevant for cleavage of extracellular matrix proteins (Sato et al., 1994). Thus, COS cells were co-transfected with MT1-MMP and the tetraspanins or MAL and cultured in a serum-reduced medium (OPTI-MEM) supplemented with a defined initial amount of recombinant pro-MMP-2 for 24 h followed by zymography analysis of MMP-2 activity. Remarkably, all tetraspanins, but not the MAL protein, decreased the activation of pro-MMP-2 by MT1-MMP (Fig. 7a), a finding that correlates well with the reduced level of catalytically active MT1-MMP on the cell surface under these conditions (compare with Fig. 6f). Moreover, the invasion and migration capacity of MT1-MMPtransfected COS cells when co-expressed with the tetraspanins or the MAL protein were analyzed within a three-dimensional (3D) collagen matrix. Therefore, a highly sensitive spheroid-based 3D collagen invasion assay was applied. Notably, the expression of all tested tetraspanins-except CD53 and the tetraspanin-like MAL protein-significantly reduced the invasion and migration capacity of MT1-MMP-transfected COS cells compared to control conditions (Fig. 7b).

Fig. 5. Tetraspanins or MAL interact with other MMP family members. (A) FLAGtagged CD53 and (B) FLAG-tagged MAL were co-expressed with different MMPs (HA-tagged: MT1-MMP; V5-tagged: MT3-MMP, MMP-1, MMP-2, MMP-9) and lysed in 1% Triton X-100. Protein lysates were co-immunoprecipitated (IP: anti FLAG), resolved on SDS-PAGE and detected by immunoblotting (anti HA and anti V5 at the same time). The IgG band (IgG) indicates equal loading of the pull-down antibody that is also detected by the secondary antibody.

active mature 57 kDa protease and in particular the inactive 44 kDa autocatalytic cleavage product were detected on the membrane of the COS cells (Fig. 6a). The abundance of both protein species was reduced in the presence of the tetraspanins but not the MAL protein. CD9 and CD37 clearly reduced the 57 kDa variant, whereas CD81 and CD82 had a more pronounced effect on the inactive 44 kDa MT1-MMP (Fig. 6a). To validate these findings, flow cytometry with COS cells expressing the HA-tagged MT1-MMP construct with an additional FLAG-tag in the catalytic domain (double-tagged MT1-MMP; Fig. 6b) and antibodies recognizing either tag was performed. By this approach, double-positive cells were analyzed toward their level of total MT1-MMP at the cell surface by detecting both the 57 and the 44 kDa MT1-MMP variants (HA-positive; Fig. 6c). Moreover, the FLAG-tag-derived staining intensity of those cells was analyzed that corresponded exclusively to the abundance of the catalytically active protease (57 kDa) at the cell surface. To this end, co-expression of the tetraspanins or the MAL protein with the double-tagged MT1-MMP essentially reproduced the results of the biotinylation approach. CD37 for example clearly reduced the total amount of the two MT1-MMP proteins on the cell surface, and in part that of the 57 kDa variant, while the MAL protein had no effect (Fig. 6d). Note that the tetraspanins or MAL with an intracellular FLAG-tag were not detected by cell surface staining. Combining the flow cytometry and biotinylation data (Fig. 6e and f) revealed that

4. Discussion Over the last decades, many studies have substantiated the pivotal role of MT1-MMP in physiological and pathological processes ranging from morphogenesis to cancer dissemination (Holmbeck et al., 2004; Itoh, 2006; Itoh and Seiki, 2004). Given the complexity and diversity of the processes driven by MT1-MMP, regulation of its activity is likely to be equally complex. In fact, many aspects of MT1MMP function and regulation still remain to be elucidated (Egeblad and Werb, 2002; Kirmse et al., 2011; Ludwig, 2005; Ludwig et al., 2008a). Here, we report on a relatively new aspect, the interaction of MT1-MMP with members of the tetraspanin family or transmembrane proteins. 4.1. MT1-MMP-tetraspanin associations Even though tetraspanin proteins show only little association with most other proteins under conditions of 1% Triton X-100 (Yanez-Mo et al., 2009), we observed robust Triton-stable interactions of MT1-MMP with tetraspanins CD37, CD53, CD82, the tetraspanin-like MAL protein, or the tetraspanin-associated EWI2 protein by using a co-expression/immunoprecipitation approach in COS cells. In line with this observation, interaction of the tetraspanin CD151 with integrin ␣3␤1 has been found to be Tritonstable as well, and most likely direct in nature as proven by chemical cross-linking (Berditchevski et al., 2001; Yanez-Mo et al., 2009; Yauch et al., 2000). Furthermore, associations of CD9 with the tetraspanin-associated protein CD9P-1 sustained conditions of 1% Triton X-100 (Charrin et al., 2001). We also observed weaker associations of MT1-MMP with CD9, CD63, and CD81 that were maintained in 1% Brij96. Complexes that are stable in Brij96 may involve a larger number of potential interaction partners (Wang et al., 2011; Yanez-Mo et al., 2009). However,

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Fig. 6. Tetraspanins regulate MT1-MMP cell surface localization. MT1-MMP was co-expressed with tetraspanins or MAL in COS cells for 24 h. (A) Pull-down of biotinylated cell surface proteins (IP: streptavidin) followed by immunoblotting (IB) of cell surface MT1-MMP metabolites. Only the catalytically active 57 kDa and the inactive 44 kDa metabolite are present at the cell surface. (B) For flow cytometry experiments, a double-tagged MT1-MMP construct (HA + FLAG) allowed to distinguish cell surface expression of the 57 kDa metabolite (FLAG+ ) from the total cell surface MT1-MMP (mix of 57 kDa and 44 kDa metabolite; HA+ ). (C) Analysis strategy of the flow cytometry experiments: double-positive cells were gated and analyzed for their cell surface fluorescence intensity. (D) Histograms show fluorescence intensities of COS cells co-expressed with the double-tagged MT1-MMP construct and either CD37, CD53, the MAL protein (dark gray), or an empty vector as a control (light gray). Note that tetraspanins with an intracellular FLAG-tag are not detected by cell surface staining. (E and F) Results from cell surface biotinylation (densitometric analyses) and the flow cytometry experiments (mean fluorescence intensities) were combined (total n = 5) and analyzed for cell surface localization for (E) the catalytically active 57 kDa MT1-MMP metabolite and (F) total MT1-MMP (*p < 0.05; **p < 0.01 vs. control).

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extracts that had been supplemented with bivalent cations (Charrin et al., 2003). Eventually, EWI-2 can be credited as a major component of the tetraspanin network and as a linker of MT1-MMP to tetraspanin-enriched microdomains (TEM), since we also detected protein–protein interactions between EWI-2 and MT1-MMP binding partners – namely CD37, CD53, CD63 and the tetraspanin-like MAL protein – that have not yet been described. 4.2. MT1-MMP interacting domain and subcelluar localization of tetraspanin-MT1-MMP complexes The deletion of MT1-MMP distinct domains revealed that the tetraspanins or MAL interact with the hemopexin domain of MT1-MMP. In fact, the N-terminal region of CD63 has previously been reported to interact with the hemopexin domain of MT1-MMP (Takino et al., 2003). Based on the interaction pattern of glycosylation-deficient MT1-MMP mutants and the recently established model for the spatiotemporal processing of MT1-MMP (Ludwig et al., 2008b), MT1-MMP-tetraspanin associations were narrowed down to the endoplasmic reticulum, where they seem to occur independently from O-glycosylation of MT1-MMP. MT1MMP appears to be mainly present at the plasma membrane upon control conditions. It accumulates, however, in a perinuclear zone resembling the endoplasmic reticulum when co-expressed with tetraspanins or the MAL protein. Other tetraspanins such as uroplakins in fact undergo homophilic interactions in the endoplasmic reticulum that seem to be a prerequisite to exit from this compartment (Tu et al., 2002). Other studies revealed that homodimers and heterodimers of CD9, CD81 and/or CD151 are also present in the trans-Golgi network (Kovalenko et al., 2004).

Fig. 7. Tetraspanins affect pro-MMP2 activation by MT1-MMP and the 3D collagen matrix invasion by COS cells. (A) COS cells were co-transfected with MT1-MMP and tetraspanins or the MAL protein and cultured in medium containing pro-MMP2 (50 ng/ml) for 24 h. Here, the capacity of MT1-MMP to activate pro-MMP-2 was used as a functional readout for MT1-MMP activity. MMP-2 gelatinase activity in supernatants was analyzed by gelatin SDS-gel zymography (control: empty vector; n = 3; *p < 0.05 vs. control). (B) COS cells co-transfected with MT1-MMP and tetraspanins or MAL were cultured as 3D spheroids and embedded in collagen gels. After 3 days, the invasion and migration distance of COS cells in the 3D collagen gels were quantified. Representative image sections (white dashed line: spheroid body) of each experimental group are shown; at least 12 randomly selected spheroids per experimental group and experiment were analyzed; the figure shows one out of two experiment with similar results (control: empty vector; *p < 0.05 vs. control **p < 0.01 vs. control).

conditions of 1% Brij96 did not alter the MT1-MMP-tetraspanin interaction pattern per se. Only the catalytically inactive 44 kDa MT1-MMP variant was found to be additionally associated with CD37, CD53, CD63, and MAL. This might be a different interaction taking place, e.g. at the plasma membrane rather than in the endoplasmic reticulum where Triton-stable interactions were present. In previous studies and in line with our findings, interactions of MT1-MMP with CD63 (Takino et al., 2003), CD81, as well as with EWI-2 have been described (Kolesnikova et al., 2009). Moreover, CD9, CD81 and TSPAN12 have been selectively coimmunoprecipitated and co-localized with MT1-MMP in different cancer cell lines (Lafleur et al., 2009), and the tetraspanin CD151 was found to associate with MT1-MMP and integrin a3ß1 forming a trimeric complex (Yanez-Mo et al., 2008). Also in agreement with our data, Triton-stable interactions of EWI-2 with CD9 (Glazar and Evans, 2009) or CD81 have been reported (Montpellier et al., 2011), and complexes of EWI-2 with CD82 were preserved in Brij97 extracts (Zhang et al., 2003). Moreover, CD9 and CD81 formed stable complexes with EWI-2 in 1% digitonin, whereas complexes of CD53, CD82, and CD151 with EWI-2 were only present in Brij97

4.3. Associations of other MMP family members with tetraspanins and MAL Other MMP family members harboring a hemopexin domain such as MT3-MMP, MMP-1, MMP-2, and MMP-9 also revealed an interaction with tetraspanins, here CD53. The association of CD53 with the secreted MMPs is particularly interesting since only three endogenous soluble ligands have been identified for tetraspanins so far: PSG17, an IgSF member for mouse CD9 (Waterhouse et al., 2002), the hepatitis C virus envelope protein E2 for CD81 (Little et al., 2004), and pro-MMP-7 for CD151 facilitating pro-MMP-7 maturation to MMP-7 (Shiomi et al., 2005). Thus, tetraspanins may not exclusively interact with MT1-MMP but also with other membrane-bound or secreted MMPs. This may imply a general function of tetraspanins for MMP regulation. 4.4. Effects of tetraspanins on the abundance of distinct MT1-MMP variants According to the overall level of expression analyzed by cell surface biotinylation or flow cytometry, CD9 and CD37 had the strongest effect on the localization of MT1-MMP on the cell surface, namely that of the mature protease, followed by CD81 and CD82, which rather displaced the inactive 44 kDa cleavage product that comprises up to 80% of total MT1-MMP on the cell surface. This might be a consequence of the overexpression of MT1-MMP which could facilitate its autocatalytic degradation (Remacle et al., 2006). Alternatively, the inactive cleavage product might interfere with other negatively charged surface molecules such as cadherins and thus adhesion to the cell surface due to freely accessible negatively charged sialic acids in its hinge region (Remacle et al., 2006). In addition, it may compete with homodimerization of the mature protease, which seems essential, e.g. for its activation of pro-MMP-2 (Ingvarsen et al., 2008; Itoh et al., 2006, 2008).

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4.5. Effects of tetraspanins and MAL on MT1-MMP activity and collagen invasion It was remarkable in this context that all tetraspanins except the tetraspanin-like MAL protein significantly reduced the ability of MT1-MMP to activate recombinant pro-MMP-2. Therefore, tetraspanins do not only affect the subcellular localization of MT1MMP but also its activity. Clearly, MT1-MMP does not associate exclusively with a single tetraspanin. In agreement with our data, interactions of MT1-MMP with CD81, a pool of tetraspanins (CD9, CD63, CD81, and CD151), and EWI-2 have been shown in lysates of U87 cells, an epithelial-like human glioblastoma–astrocytoma cell line (Kolesnikova et al., 2009). Notably, MT1-MMP-tetraspanin complexes were disturbed in the presence of EWI-2, which also affected MMP-2 activity. Furthermore, modulatory effects of tetraspanins on MT1-MMP function have been reported in cancer cells (Lafleur et al., 2009). More precisely, the simultaneous knockdown of two or three members of the tetraspanin family (CD9, CD81, TSPAN12) markedly decreased the proteolytic function of MT1-MMP, which is contrary to our data. However, it can be assumed that these tumor cells are different from COS cells and therefore tetraspanin functions might differ as well. The assumption that tetraspanin function might vary in different cell types is further substantiated by additional experiments that we performed in human umbilical arterial smooth muscle cells (HUASMC). Of note, the individual expression of the selected tetraspanins and the MAL protein in HUASMCs revealed a different pattern of 3D collagen invasion when compared to the situation in COS cells (compare Fig. 7b with Supplemental Fig. S6). CD9 and CD53 for instance rather promoted HUASMC collagen invasion compared to control, whereas CD82 significantly reversed this effect. Moreover, this spheroid-based 3D collagen invasion assay is suitable to assess MT1-MMP activity since the knockdown of MT1-MMP effectively blocked collagen invasion by the HUASMCs (Supplemental Fig. S7). In MT1-MMP-expressing COS cells, we observed a significantly reduced 3D collagen invasion when co-expressed with CD9, CD37, CD63, CD81, and CD82, but not with the MAL protein. This pattern assorts well with our previous finding where those tetraspanins clearly reduced the activation of pro-MMP-2 by MT1-MMP. CD53 however showed no such correlative effect: it significantly reduced MT1-MMP-dependend pro-MMP-2 activation but did not reduce the 3D collagen invasion capacity of COS cells. This might be due to the restricted expression of CD53 in lymphoid and myeloid cells whereas other tetraspanins such as CD9, CD63, CD81, CD82 are found in virtually all tissues (Maecker et al., 1997). A similar pleiotropic but functionally reversed situation was reported for the collagenolytic activity of MT1-MMP that was found to be regulated through association with tetraspanin CD151 in primary endothelial cells (Yanez-Mo et al., 2008). Here, MT1-MMP-mediated activation of pro-MMP-2 was enhanced by CD151 loss of function (siRNA-based knockdown) while paradoxically supporting MT1MMP-dependent collagen degradation. 4.6. Tetraspanin–partner associations during biosynthesis and functional consequences With regard to tetraspanin–partner associations during early biosynthesis and functional consequences, we observed that e.g. CD82 formed robust complexes with MT1-MMP and at the same time clearly reduced pro-MMP-2 activation by MT1-MMP as well as collagen invasion by the COS cells. However, MT1-MMP partners such as CD53 or the MAL protein with a likewise strong interaction only revealed weak or no such effects. This highlights the complexity and dynamics of the tetraspanin network. A similar discrepancy between the degree of interaction of tetraspanins with MT1-MMP

and alterations in its function was instructively reported for CD151 (Levy and Shoham, 2005a). This might be due to the participation of distinct tetraspanin domains in varied functions and cell types that may explain the pleiotropic effects of tetraspanins within the tetraspanin network (Shoham et al., 2006). It could be hypothesized therefore that functionally relevant tetraspanin–tetraspanin associations are more likely to occur in the Golgi compartment because they depend on palmitoylation (Berditchevski et al., 2002; Yang et al., 2002). Notably, all tetraspanins used in this study were found to be palmitoylated (Delandre et al., 2009; Flannery et al., 2010; Stipp et al., 2003b), and furthermore, MT1-MMP was reported to be palmitoylated as well – a posttranslational modification that determines MT1-MMP-dependent cell migration (Anilkumar et al., 2005). For MAL however, there is no report with regard to palmitoylation, and it therefore might integrate in different domains than tetraspanins do. This premise is supported by the finding that MAL preferably interacts with MT1-MMP in glycosphingolipid-rich microdomains that in turn are envisioned to be predominantly lipid raft-associated (Cheong et al., 1999; Le Naour et al., 2006). Moreover, MAL shows only about 15% protein sequence homology compared to the other tetraspanins used in this study (data not shown). Finally, MAL-MT1-MMP associations seemed not to be rate-limiting for MT1-MMP cell surface transport, internalization or activity in COS cells. In conclusion, localization to tetraspanin microdomains might represent a novel mechanism for regulating the enzymatic activity of MT1-MMP, whereby its subcellular localization and access to different substrates – such as components of the extracellular matrix, growth factors, signaling molecules and other proteases – are controlled through the regulation of its compartmentalization at the plasma membrane. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (LU 854/3-1; SFB-TR23 projects C5 and C6) and both the HBIGS and FRONTIER program within the Excellence Initiative at Heidelberg University. We thank Michael J. Caplan, Sarah Theissen, Gudrun Scheib, and the Nikon Imaging Center at Heidelberg University for their support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. biocel.2013.02.020. References Albers M, Kranz H, Kober I, Kaiser C, Klink M, Suckow J, et al. Automated yeast two-hybrid screening for nuclear receptor-interacting proteins. Molecular and Cellular Proteomics 2005;4:205–13. Andreini C, Banci L, Bertini I, Luchinat C, Rosato A. Bioinformatic comparison of structures and homology-models of matrix metalloproteinases. Journal of Proteome Research 2004;3:21–31. Anilkumar N, Uekita T, Couchman JR, Nagase H, Seiki M, Itoh Y. Palmitoylation at Cys574 is essential for MT1-MMP to promote cell migration. FASEB Journal 2005;19:1326–8. Annabi B, Lachambre M, Bousquet-Gagnon N, Page M, Gingras D, Beliveau R. Localization of membrane-type 1 matrix metalloproteinase in caveolae membrane domains. Biochemical Journal 2001;353:547–53. Berditchevski F, Gilbert E, Griffiths MR, Fitter S, Ashman L, Jenner SJ. Analysis of the CD151-alpha3beta1 integrin and CD151-tetraspanin interactions by mutagenesis. Journal of Biological Chemistry 2001;276:41165–74. Berditchevski F, Odintsova E. Tetraspanins as regulators of protein trafficking. Traffic 2007;8:89–96. Berditchevski F, Odintsova E, Sawada S, Gilbert E. Expression of the palmitoylationdeficient CD151 weakens the association of alpha 3 beta 1 integrin with the

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