Golgi reassembly stacking protein 55 interacts with
membrane-type (MT) 1-matrix metalloprotease (MMP) and
furin and plays a role in the activation of the MT1-MMP
zymogen
Christian Roghi
1,2
, Louise Jones
2
*, Matthew Gratian
2
, William R. English
1,2
and Gillian Murphy
1,2
1 Cancer Research UK Cambridge Research Institute, The Li Ka Shing Centre, UK
2 Cambridge Institute for Medical Research, UK
Keywords
furin; GRASP55; intracellular traffic;
MT1-MMP; protease
Correspondence
C. Roghi, Cancer Research UK Cambridge
Research Institute, The Li Ka Shing Centre,
Robinson Way, Cambridge CB2 0RE, UK
Fax: +44 (0)1223 404573
Tel: +44 (0)1223 404472
E-mail: [email protected]
*Present address
KuDOS Pharmaceuticals Ltd, Cambridge
Science Park, UK
(Received 25 March 2010, revised 14 May
2010, accepted 28 May 2010)

l
MINT-7897821: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with MT3-
MMP (uniprotkb:
P51512)bytwo hybrid (MI:0018)
l
MINT-7897577: GRASP55 (uniprotkb:Q9R064) and MT1-MMP (uniprotkb:P50281) coloca-
lize (
MI:0403)byfluorescence microscopy (MI:0416)
l
MINT-7897366: MT1-MMP (uniprotkb:P50281) physically interacts (MI:0915) with
GRASP55 (uniprotkb:
Q9H8Y8)byanti bait coimmunoprecipitation (MI:0006)
Abbreviations
ECM, extracellular matrix; EGFP, enhanced green fluorescent protein; EYFP, enhanced yellow fluorescent protein; FACS, fluorescence-
activated cell sorting; GFP, green fluorescent protein; GRASP, Golgi reassembly stacking protein; GRASP55F, FLAG-tagged GRASP55;
IB, immunoblotting; ICD, intracellular domain; M2H, mammalian two-hybrid; MMP, matrix metalloprotease; MT1/EYFP, EYFP-tagged
MT1-MMP; MT1/MYC, Myc-tagged MT1-MMP; MT-MMP, membrane-type MMP; PDZ, PSD-95/SAP90 Drosophila septate junction protein
discs-large and epithelial tight junction ZO-1; TGF, transforming growth factor; TGN, trans-Golgi network.
3158 FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS
Introduction
Extracellular matrix (ECM) remodelling is a crucial
process occurring during cell migration and invasion
in various physiological (i.e. embryonic development,
ovulation, angiogenesis, wound healing) and patho-
logical processes, including rheumatoid arthritis,
tumour growth, invasion and metastasis [1]. Of all
the different proteolytic systems involved in ECM
turnover, the matrix metalloproteinases (MMPs) have
been reported to exert a dominant effect [2]. MMPs
are a large family of structurally and functionally

ated cellular events [15]. MT1-MMP ICD has been
involved in cell migration [16] and invasion into recon-
stituted basement membrane [17,18]. The MT1-MMP
ICD is also critical for the intracellular trafficking of
the enzyme [19–23] and its targeting to invadopodia in
invasive cells [24]. The ICD of MT1-MMP has been
found to modulate multiple signal transduction path-
ways [16,25–27] and participates in the homophilic
interaction between MT1-MMP monomers [28].
Recently, the LL
572
di-leucine motif has been reported
l
MINT-7897617, MINT-7897659, MINT-7897681, MINT-7897702, MINT-7897725, MINT-
7898032, MINT-7898011, MINT-7897907, MINT-7897884: GRASP55 (uniprotkb:Q9R064)
physically interacts (
MI:0915) with MT1-MMP (uniprotkb:P50281)bytwo hybrid (MI:0018)
l
MINT-7898002: MT1-MMP (uniprotkb:P50281) physically interacts (MI:0914) with Furin
(uniprotkb:
P09958)byanti bait coimmunoprecipitation (MI:0006)
l
MINT-7897500: MT1-MMP (uniprotkb:P50281) and Giantin (uniprotkb:Q14789) colocalize
(
MI:0403)byfluorescence microscopy (MI:0416)
l
MINT-7897750, MINT-7897394: GRASP55 (uniprotkb:Q9R064) physically interacts
(
MI:0915) with MT1-MMP (uniprotkb:P50281)byanti tag coimmunoprecipitation (MI:0007)
l

MI:0403)byfluorescence microscopy (MI:0416)
l
MINT-7897938: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with PC5/6B
(uniprotkb:
Q04592)bytwo hybrid (MI:0018)
C. Roghi et al. Role of GRASP55 in MT1-MMP activation
FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS 3159
to influence the O-glycosylation pattern of MT1-MMP
[29]. Post-translational modifications of the MT1-
MMP ICD have also been reported with the palmitoy-
lation of the cysteine 574 (C
574
) residue [30] and the
phosphorylation of the tyrosine 573 (Y
573
) [31] and
threonine 567 (T
567
) [32] residues. The MT1-MMP
ICD has been reported to interact with the multifunc-
tional protein p32/gC1qR [21], a protein with homol-
ogy to members of the Cupin superfamily (MTCBP-1)
[33], as well as with the l2 subunit of the clathrin-
coated pits adapter protein 2 (AP-2) [18] and phospho-
caveolin-1 in src overexpressing cells [34].
Golgi reassembly stacking protein 55 (GRASP55) is
a peripheral Golgi matrix protein that has been impli-
cated, in vitro, in the post-mitotic stacking of Golgi cis-
ternae [35]. Cryo-electron microscopy has shown that
GRASP55 is found predominantly in the medial-cister-

exogenous wild-type MT1-MMP cDNA (Fig. 1A),
aiming to detect the protease in the early secretory
pathway. Cells were then lysed and the extract was
A
60
50
40
IB: MT1-MMP
MT1-MMP
pCDNA3.1 Zeo+
kDa
+–
–+
+–
–+
60
50
40
IB: GRASP55
50
40
IB: β-actin
12
IgG
anti MT1-MMP
B
60
50
40
IB: GRASP55

with rabbit control IgGs (lane 1) or with the rabbit polyclonal anti-
body directed against MT1-MMP (lane 2) and analyzed by IB using
a monoclonal antibody to GRASP55. The arrow identifies immuno-
precipitated MT1-MMP. (C) Protein extracts prepared from HT1080
cells transiently transfected with pCDNA3.1 Zeo+ and MT1/MYC
(lane 2), pCDNA3.1 Zeo+ and GRASP55F (lane 3) and MT1/MYC
and GRASP55F (lane 4) were immunoprecipitated with the FLAG
M2 monoclonal antibody and the associated MT1-MMP was
detected by IB using the MYC tag monoclonal antibody. Expression
of the transfected construct was monitored in the input lysates
using specific antibodies. The black arrowhead indicates IgG (immu-
noglobulin heavy chain).
Role of GRASP55 in MT1-MMP activation C. Roghi et al.
3160 FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS
ABC
DEF
GHI
JKL
Fig. 2. Subcellular localization of GRASP55 and MT1-MMP in bbHT1080. Fixed and permeabilized bbHT1080 cells were incubated with anti-
bodies directed against MT1-MMP (A, D, J), GRASP55 (G, K), TGN46 (E) and giantin (B, H). The co-localization can be observed in yellow in
the merged panels (C, F, I, L). Arrowheads depict membranous structures where the proteins co-localize. Scale bar = 5 lm.
C. Roghi et al. Role of GRASP55 in MT1-MMP activation
FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS 3161
immunoprecipitated with nonspecific rabbit IgGs or
with the rabbit polyclonal antibody directed against
MT1-MMP. As shown in Fig. 1B, immunoprecipita-
tion of MT1-MMP led to the co-precipitation of a
small amount of endogenous GRASP55, as detected
by immunoblotting (IB). No GRASP55 was detected
when the pre-immune IgGs were used. Co-precipita-

with MT1/EYFP and the GRASP55-green fluorescent
protein (GFP) fusion protein as previously described
[35] and the localization of both proteins was studied in
live cells. Separation of the GFP and EYFP signals was
achieved using a Zeiss META confocal microscope (see
Fig. 3. Co-localization of MT1-MMP and GRASP55 in live cells. Four consecutive frames (4 s apart) of time lapse sequence collected from
HT1080 co-transfected cells with EYFP/MT1 and GRASP55-GFP. The arrowheads are examples of dynamic vesicles containing both fluore-
scent proteins. MT1/EYFP was pseudo-coloured in red during post-acquisition processing. The co-localization between GRASP55-GFP and
MT1/EYFP can be observed in yellow in the merged panels. Scale bar = 16 lm.
Role of GRASP55 in MT1-MMP activation C. Roghi et al.
3162 FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS
PGGGFFFRRHGTPRRLLYCQRSLLDKV
VP16-MT1
VP16
PGG
G
FFAAAHGTPRRLLYCQRSLLDKV
VP16VP16-MT1 FRR
A
PGGGFFFRRAAAPRRLLYCQRSLLDKV
VP16VP16-MT1 HGT
PGGGFFFRRHGTAAALLYCQRSLLDKV
VP16
VP16-MT1 PRR
PGGGFFFRRHGTPRRAAACQRSLLDKV
VP16
VP16-MT1 LLY
PGGGFFFRRHGTPRRLLYAAASLLDKV
VP16
VP16-MT1 CQR

2
3
4
GAL4 VP16GRASP55
MT1
VP16
GAL4
GAL4 GRASP55
MT1VP16
GAL4 VP16
+
+
+
+
C
VP16
+
GAL4 GRASP55
+
TGF-α
VP16GAL4
+
GAL4 VP16
1
2
3
Luminescence (arbitrary units)
GAL4 GRASP55
TGF-α
VP16

GAL4 GRASP55
+
MT1 PRR
VP16
GAL4 GRASP55
+
MT1
VP16
GAL4 GRASP55
MT1 LLY
VP16
+
2
3
4
5
6
*
*
***
+
GAL4 GRASP55
+
MT1 CQR
VP16
GAL4 GRASP55
+
MT1 SLL
VP16
+

Fig. 5. The LLY motif in the MT1-MMP ICD
is crucial for the interaction with GRASP55.
(A) Interaction between GAL4-GRASP55 and
VP16-MT1 or MT1-MMP ICD triple mutants.
(B) Cell lysates prepared from HT1080 cells
transfected with pCDNA3.1 Zeo+ and MT1/
MYC (lane 1), pCDNA3.1 Zeo+ and
GRASP55F (lane 2), pCDNA3.1 Zeo+
and MT1 LLY/MYC (lane 3), GRASP55F and
MT1 LLY/MYC (lane 4) and GRASP55F
and MT1/MYC (lane 5) were immunoprecipi-
tated with the FLAG M2 antibody.
MT1-MMP present in the immunoprecipi-
tate was detected by IB using the MYC tag
antibody. Levels of transfected proteins
were monitored in input lysates using
specific antibodies.
C. Roghi et al. Role of GRASP55 in MT1-MMP activation
FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS 3163
Materials and methods). As previously described in
fixed bbHT1080 (Fig. 2), we clearly observed, in live
HT1080 cells, the presence of both tagged proteins in
the same membrane compartment (Fig. 3, merged), thus
confirming the results that were observed previously in
fixed bbHT1080 cells (Fig. 2). Interestingly, we also
noted that MT1/EYFP and GRASP55-GFP also
co-localized in very dynamic unidentified cytoplasmic
membranous structures (Fig. 3, arrowheads).
MT1-MMP intracellular domain is involved in the
interaction with GRASP55

the TGF-a ICD as previously observed using a yeast
two-hybrid assay [39].
The MT1-MMP LLY motif is important for the
interaction with GRASP55
We next sought to define the nature of the GRASP55
binding site in the MT1-MMP ICD. pACT plasmids
driving the expression of MT1-MMP ICDs containing
single-, double- and triple-point mutations were gener-
ated (Fig. 4A) and used in the M2H system. System-
atic analysis of the interaction between the triple
MT1-MMP ICD mutants and GAL4-GRASP55
revealed that, apart from the VP16-MT1 FFR
(Fig. 5A, lane 3) and VP16-MT1 DKV mutants
(Fig. 5A, lane 9), all the other triple mutants (Fig. 5A,
lanes 4–8) displayed a marked and significant reduc-
tion of luciferase activity compared to the wild-type
VP16-MT1 construct (Fig. 5A, lane 2). In particular,
the mutation of the LLY
573
motif (LLY571-573AAA)
(Fig. 5A, lane 6) resulted in a complete inhibition of
MT3
MT3 ILY
MT5
VP16-MT1
VP16-MT2
PGGGFFFRRHGTPRRLLYCQRSLLDKV
VP16
PGGGVQMQRKGAPRVLLYCKRSL QEW V
VP16

+
GAL4
VP16
GAL4 GRASP55
VP16
+
+
GAL4 GRASP55 VP16
1
2
3
**
GAL4
GRASP55

VP16
+
GAL4
VP16
+
GAL4 GRASP55 MT5VP16
+
GAL4 GRASP55
MT5 VTY
VP16
7
8
9
MT2
MT3

lane 4), although not to the level observed with the
LLY570-573AAA triple mutant (Fig. S2, lane 3), dem-
onstrating that the mutation of the whole LLY
573
motif is needed to abolish the interaction of MT1-
MMP ICD with GRASP55.
The importance of the LLY
573
motif in the MT1-
MMP ICD observed in the M2H assay was next con-
firmed by co-immunoprecipitation. HT1080 cells were
transiently co-transfected with GRASP55F together
with MT1/MYC or MT1 LLY/MYC (LLY570-573
AAA) triple mutant and total cell lysates were
subjected to immunoprecipitation using the FLAG tag
monoclonal antibody. As previously observed, we
detected a clear interaction between GRASP55F and
the wild-type MYC-tagged MT1-MMP (Fig. 5B, lane
5) when both proteins were expressed in HT1080 cells.
Mutation of the LLY
573
motif to AAA
573
in MT1-
MMP ICD led to a marked reduction in the amount
of MT1-MMP present in the immunoprecipitated
material (Fig. 5B, lane 4), thus confirming the impor-
tant role of the LLY
573
motif in the interaction

and VTY
636
sequences were found in MT3-MMP and
MT5-MMP ICDs, respectively (Fig. 6A). To test
whether MT2-, MT3- and MT5-MMP ICDs could also
interact with GRASP55, we generated VP16-MT2,
-MT3 and -MT5 chimeras (Fig. 6A). As shown in
Fig. 6B, all three ICDs (Fig. 6B, lanes 2, 5 and 8)
showed a clear interaction with GRASP55. We also
tested whether the LLY
660
motif in MT2-MMP, the
ILY
598
motif in MT3-MMP or the VTY
636
motif in
MT5-MMP could also be involved in the interaction
with GRASP55. Accordingly, VP16-MT2 LLY, VP16-
MT3 ILY and VP16-MT5 VTI triple mutants were
generated and used in the M2H assay. As previously
observed for MT1-MMP, mutation of the MT2-MMP
LLY
660
motif to AAA
660
significantly decreased
the interaction with GRASP55 (Fig. 6B, lane 3). By
Luminescence (arbitrary units)
500

+
TGF-α
VP16GAL4
+
GAL4
GRASP55
VP16
Luminescence (arbitrary units)
500
0 100 200 300 400
1
2
+
GAL4
GRASP55
VP16
+
P1GAL4
TGF-α
VP16
2
3
TGF-α
A
B
R3
Fig. 7. GRASP55 PDZ2 and region 3 are
important for the interaction with the
MT1-MMP ICD. (A) Interactions between
VP16-MT1 and GAL4-GRASP55, GRASP55

PDZ2 (P2; Fig. 7A, lane 5) and GRASP55 region 3
(R3; Fig. 7A, lane 6). However, no interaction was
found between VP16-MT1 and GAL4-GRASP55
PDZ1 (P1; Fig. 7A, lane 4), despite the expression of
the GAL4-GRASP55 PDZ1 chimera in HT1080 (data
not shown). TGF-a was previously reported to co-
immunoprecipitate with a very small amount of flagged
tagged GRASP55 PDZ1 domain [37]. In our hands, no
interaction between TGF-a ICD and GRASP55 PDZ1
could be observed in the M2H assay (Fig. 7B, lane 3).
The lack of interaction could result from a mis-folding
of GRASP55 PDZ1 subsequent to its fusion to the
GAL4 DNA binding domain. We therefore cannot rule
out an interaction between MT1-MMP ICD and the
GRASP55 PDZ1 domain.
GRASP55 binds to furin, PC5/6B and PC7
intracellular domains
The pro-convertase furin has previously been impli-
cated in the activation of pro-MT1-MMP [42].
Because MT1-MMP activation occurs during the
intracellular traffic of the protease, we tested whether
furin could interact, via its ICD, with GRASP55.
Accordingly, we generated a VP16-furin construct
500
0 100 200 300 400
1
*
+
FurinVP16GAL4
Luminescence (arbitrary units)

0 100 200 300 400
1
2
3
4
+
GAL4 P2 FurinVP16
+
R3GAL4 FurinVP16
5
6
A
B
C
Furin
GRASP55
Fig. 8. GRASP55 interacts with furin, PC5/
6B and PC7. (A) Interactions between
GAL4-GRASP55 and furin, PC5/6B or PC7
ICDs were tested using the M2H system.
(B) Interaction between VP16-furin and
GAL4-GRASP55, GRASP55 PDZ1 (GAL4-
P1), GRASP55 PDZ2 (GAL4-P2) or GRASP55
Region 3 (GAL4-R3). (C) Furin co-localized
with GRASP55. bbHT1080 cells, transfected
with a full-length furin cDNA, were
permeabilized and stained with polyclonal
antibodies against furin and GRASP55.
Arrows show examples of membrane
compartment containing GRASP55 and

64
*
C
IB: MT1-MMP
EGFP-furin ICD
64
50
kDa
pro-MT1-MMP
active-MT1-MMP
IB: FLAG
IB: Furin
Input
lysate
IB: MT1-MMP
50
64
64
98
IB: β-actin
12
50
36
GRASP55F
MT1/MYC
EGFP-furin ICD (μg)
B
1234
50
05 10

12 3 4
50
–
––++
+–+
–
–
––
++ +
++ +
–+
Fig. 9. GRASP55 is important for MT1-MMP–furin complex formation and activation of pro-MT1-MMP. (A) Lysates of bbHT1080 cells trans-
fected with pCDNA3.1 Zeo+ vector control (lane 1), pCDNA3.1 Zeo+ and furin (lane 2), pCDNA3.1 Zeo+ and GRASP55 (lane 3) or with furin
and GRASP55F (lane 4) were immunoprecipitated using the affinity-purified anti-MT1-MMP IgGs and the associated furin was detected by
IB. Levels of transfected proteins were monitored in input lysates using specific antibodies. Asterisks marks endogenous furin immunopre-
cipitated by MT1-MMP in bbHT1080. (B) Expression of EGFP-furin ICD disrupted the formation of the complex between MT1-MMP and
GRASP55. Lysates of HT1080 cells transfected with pCDNA3.1 Zeo+ vector control (lane 1), MT1/MYC and GRASP55F (lane 2), MT1/MYC
and GRASP55F and 0.5 lg EGFP-furin ICD (lane 3) or MT1/MYC and GRASP55F and 1.0 lg of EGFP-furin ICD (lane 4) were immunoprecipi-
tated with the FLAG antibody and the associated MT1-MMP was detected by IB using the MYC tag antibody. Top black arrowheads indicate
IgGs. The bottom black arrowhead indicates a crossreaction. (C) IB analysis of protein extracts prepared from the EGFP-negative (lane 1)
and -positive (lane 2) cell population sorted in Fig. S4. Equal amounts of total protein (6 lg) were loaded and the expression of MT1-MMP
(pro and active) was analyzed by IB. Protein loading was controlled using the b-actin polyclonal antibody. (D) Expression of furin decrease
MT1-MMP cell surface activity. HT1080 cells were transiently transfected with empty vector pCDNA3.1 Zeo+, furin, GRASP55, fu-
rin + GRASP55, MT1-MMP, MT1-MMP + furin, MT1-MMP + GRASP55 and MT1-MMP + GRASP55 + furin. 4b-Phorbol 12-myristate 13-ace-
tate was used at 50 ngÆlL
)1
. After 24 h, supernatants were collected and analyzed by zymography. Data represent the mean ± SEM of two
independent experiments.
C. Roghi et al. Role of GRASP55 in MT1-MMP activation
FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS 3167

cell lysates were subjected to immunoprecipitation
using the MT1-MMP antibody and immunocomplexes
were then probed with the furin polyclonal antibody
(Fig. 9A). No furin was immunoprecipitated with
MT1-MMP in bbHT1080 cells transfected with
pCDNA 3.1 Zeo+ alone (Fig. 9A, lane 1) or express-
ing only GRASP55 (Fig. 9A, lane 3). When furin was
expressed in bbHT1080 cells, a small amount of the
proprotein-convertase was found in the same complex
as MT1-MMP (Fig. 9A, lane 2, asterisk). Expression
of GRASP55F together with furin resulted in a signifi-
cant increase in the amount of furin co-immunoprecip-
itated with MT1-MMP (Fig. 9A, lane 4), therefore
suggesting that GRASP55 could enhance the forma-
tion of a complex between MT1-MMP and furin.
Disruption of the GRASP55–furin complex
reduces processing of pro-MT1-MMP
GRASP55 appears to play an important role in the
formation of the MT1-MMP–furin complex; therefore,
disruption of the interaction between GRASP55 and
furin should perturb the activation of pro-MT1-MMP
and reduce MT1-MMP activity at the cell surface. To
test this hypothesis, we generated an enhanced green
fluorescent protein (EGFP)-furin ICD construct in
which the extracellular domain of the proprotein-con-
vertase was replaced by EGFP. Expression of the
EGFP-furin ICD construct (1 lg) in HT1080 express-
ing MT1/MYC and GRASP55F led to a disruption of
the complex between these two proteins (Fig. 9B). We
next tested whether expression of the EGFP-furin chi-

MT1-MMP led to a significant reduction in the levels
of MMP2 generated compared to cells expressing only
the protease. A significant decrease was also observed
when furin was co-expressed with MT1-MMP and
GRASP55. Taken together, our observations suggest
that furin-mediated disruption of the MT1-MMP
GRASP55 complex can lead to a reduction of the
MT1-MMP, and a consequent decrease of protease
activity at the cell surface. Our data also revealed that
intracellular furin levels are critical for the efficient acti-
vation of MT1-MMP. The results obtained suggest that
GRASP55 might act as a molecular bridge between
MT1-MMP and furin and is involved in the furin-medi-
ated activation of pro-MT1-MMP.
Role of GRASP55 in MT1-MMP activation C. Roghi et al.
3168 FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS
Discussion
In the present study, we have investigated the interac-
tion between GRASP55, a Golgi matrix protein, and
MT1-MMP. Using the M2H system and a series of
MT1-MMP ICD triple mutants, we discovered that
mutation of the LLY
573
motif to AAA
573
completely
inhibited the interaction between MT1-MMP ICD and
GRASP55, suggesting that this motif is important for
the interaction of MT1-MMP ICD with the PDZ2
domain of GRASP55. The interaction of PDZ

lated via tyrosine phosphorylation.
Surprisingly, MT1-MMP and furin ICDs were also
found to interact with the C-terminal region (region 3)
of GRASP55. Protein interactions with the region
located outside the PDZ domain have previously been
observed. Sox-4 and eIF5A [51,52] were reported to
interact only with the N-terminal region of syntenin-1,
thus allowing for the potential binding of different
proteins through the PDZ domain(s) and the N-termi-
nal region [53]. Our observation suggests that
GRASP55 could potentially interact with two MT1-
MMP molecules, therefore allowing for the homophilic
complex formation of the protease, as previously
reported [28,54]. Forcing the MT1-MMP ICD to
dimerize by fusing it to the coil-coiled region of
GM130 [39] was found to significantly enhance the
interaction between MT1-MMP and GRASP55
(Fig. S6). Furthermore, PDZ-containing proteins have
previously been reported to self-associate, generating
macromolecular complexes. This multimerization can
involve either of the PDZ domains, as for example in
glutamate receptor interacting protein 1 (GRIP1) [55]
or inactivation no afterpotential D (INAD) protein
[56]. However, it can also be a PDZ-independent
mechanism, as for example in the case of the PSD-95
protein, where dimer formation is mediated by the N-
terminal region of the protein [57]. GRASP65, which
is structurally related to GRASP55, has been found to
form dimers that can organize into higher-order oligo-
mers in interphase cells [58]. GRASP65 dimerization

sion of this mutated version of MT1-MMP. It is
important to note that perturbation of the GRASP55
interaction with TGF-a [37], as well as p24 proteins
[39], has been reported to affect the normal trafficking
of these proteins. The expression of the MT1-MMP
AAA
573
mutant at the cell surface [18] could result
from the intracellular traffic of MT1-MMP via an
alternative pathway, as previously described [60].
Alternatively, GRASP65, which is structurally related
to GRASP55, has previously been reported to interact
with transmembrane TGF- a, p24a, protein CD8a or
the frizzled receptor Fz4 [37–39] and therefore could
be involved in the intracellular traffic of MT1-MMP
AAA
573
to the cell surface. It would be interesting to
assess the role of GRASP65 with respect to MT1-
MMP trafficking to the cell surface.
MT1-MMP, similar to all the other MMPs, is syn-
thesized as a latent zymogen (pro-MT1-MMP) that is
activated by endoproteolytic cleavage of its N-terminal
inhibitory pro-domain peptide [6,61]. Furin has been
widely reported to be an activator of pro-MT1-MMP
and is considered to be physiologically relevant [42].
In the present study, we have reported that both
C. Roghi et al. Role of GRASP55 in MT1-MMP activation
FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS 3169
MT1-MMP and furin can interact with GRASP55.

motifs, respec-
tively, were found to be important for the interaction
of these MT-MMPs with GRASP55. By contrast,
mutation of the VTY
636
motif to AAA
636
in MT5-
MMP did not affect the binding of the protease with
GRASP55. Previous studies [66,67] have identified the
EWV
614
motif in MT5-MMP ICD as comprising an
important sequence for the interaction with the PDZ
proteins: Mint-3, AMPA binding protein and gluta-
mate receptor interacting protein (GRIP). It would
therefore be interesting to determine whether this motif
is also involved in the interaction between MT5-MMP
and GRASP55.
GRASP55 could potentially be considered as a
molecular bridge involved in connecting furin with var-
ious substrates. So far, it is unknown whether this
mechanism is specific for the type I transmembrane
MT-MMPs or whether it comprises a more general
mechanism for furin-mediated activation of transmem-
brane substrates. The activation of ADAM17 by furin
[68] and our observation of an interaction between
GRASP55 and the ICD of ADAM17 (C. Roghi and
L. Jones, unpublished results) would suggest a much
wider activation mechanism. A role of GRASP55 in

) mouse monoclonal
antibody were obtained from Millipore (UK) Ltd
(Watford, UK). Polyclonal anti-TGN46 sheep (IF:
5 lgÆmL
)1
) serum was obtained from Serotec Ltd (Oxford,
UK). b-actin (IB: 0.1 ngÆmL
)1
) and anti-furin (IF:
8.6 lgÆmL
)1
, IB: 8.6 l gÆmL
)1
) rabbit Polyclonal antibody
were from Abcam plc (Cambridge, UK). Giantin mouse
monoclonal antibody was obtained from H P. Hauri
(University of Basel, Switzerland) [70]. All secondary
antibodies were obtained from Jackson ImmunoResearch
Europe Ltd (Soham, UK) and used in accordance with
the manufacturer’s instructions.
Cell culture conditions and transfections
All cell culture reagents were obtained from Invitrogen Ltd
(Paisley, UK), unless otherwise indicated. HT1080 Human
fibrosarcoma cells (from Cancer Research UK Research
Services, London, UK) were maintained in DMEM con-
taining 10% (v/v) fetal bovine serum (Hyclone Laborato-
ries Inc., UT, USA), 2 mml-glutamine, 100 UÆmL
)1
penicillin and 100 lgÆmL
)1

annealing overlapping oligonucleotides and ‘filling up’ the
single strand regions with KOD DNA polymerase
(2.5 units; Merck Biosciences Ltd, Nottingham, UK). The
resulting double-stranded DNA was cloned in frame with
VP16. MT1/MYC has a MYC tag between P
312
and T
313
in the hinge domain of MT1-MMP. LLY570-573AAA
MT1/MYC and G2A GRASP55F mutants were generated
using the QuikChange II site-directed mutagenesis kit
(Stratagene, Amsterdam, the Netherlands). Full-length rat
GRASP55 cDNA and N-terminally EGFP-tagged rat
GRASP55 (GRASP55-GFP) were obtained from F. Barr
and colleagues [35]. GRASP55F in pCDNA3.1 Zeo+ con-
tained a C-terminal FLAG tag. Full-length GRASP55,
GRASP55 PDZ1 (amino acids 1–107), GRASP55 PDZ2
(amino acids 84–172) and GRASP55 region 3 (amino acids
173–454) were fused to GAL4 DNA binding domain in the
pBIND Checkmate Mammalian Two-Hybrid System vector
(Promega).
To generate the MT1/EYFP, oligonucleotides coding for
MT1-MMP signal sequence (amino acids 1–30) were
annealed and inserted upstream of EYFP in pEYFP-N1
(Clontech-Takara Bio Europe, Saint-Germain-en-Laye,
France). DNA coding for MT1-MMP hinge, hemopexin,
stalk, transmembrane and cytoplasmic domains (amino
acids 283–582) was amplified by PCR and cloned down-
stream of EYFP. VP16-PC5/6B, VP16-PC7 and VP16-furin
were generated by fusing VP16 to ICDs of mouse PC5/6B

4
, 2.7 mm KCl, 1.47 mm KH
2
PO
4
) and lysed in
passive lysis buffer (200 lL; Promega) for 15 min at room
temperature. After centrifugation (13 000 g for 5 min at
room temperature), Firefly and Renilla reniformis luciferase
activities were measured in 20 lL of lysate using the
Dual-Luciferase Reporter Assay System (Promega) and a
SpectraFluor Plus plate reader (Tecan UK, Reading, UK).
Firefly luciferase activity was normalized to Renilla lucifer-
ase activity and is presented as relative luminescence units.
Each transfection was performed in duplicate and two inde-
pendent measurements were read per sample. All numerical
values are shown as the mean ± SEM. The graphs pre-
sented are representative of at least three experiments.
Immunoprecipitation
HT1080 cells (2 · 10
5
per well in a six-well plate) were
transfected (1–2 lg per construct) for 16 h. Cells were then
washed twice in ice-cold NaCl/P
i
and lysed for 5 min in
600 lL of lysis buffer per well [10 mm Tris–HCl, pH 7.4,
150 mm NaCl, 1% (v/v) Triton X-100, 0.5% (v/v) Nonidet
P-40, 1 mm EDTA, 1 mm EGTA, 1 mm sodium vanadate]
containing protease inhibitor (Roche Diagnostics Ltd). For

FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS 3171
Live cell time-lapse microscopy
Transfected HT1080 cells (500 ng of each construct) were
imaged with a confocal Zeiss LSM510 META microscope
(Zeiss Axiovert 200M platform, · 63/1.4 NA Oil Plan-Apo-
chromat lens; Carl Zeiss Ltd, Welwyn Garden City, UK).
The Argon Ion laser (Lasos Lasertechnik GmbH, Jena,
Germany) output was restricted to 3% of maximum
(< 0.3 mW at focal plane) to minimize phototoxicity and
bleaching. Coverslips (PeCon GmbH, Erbach, Germany)
were placed in a POC-R imaging chamber (PeCon GmbH)
containing phenol-red free Hepes-buffered medium and
kept at 37 ° C using a Heating Insert-P (PeCon GmbH).
Time series images (optical section thickness of 1.7 lm)
were collected in a lambda acquisition mode at one frame
every 4 s for 10–20 min. Reference EGFP and EYFP emis-
sion spectra were captured from singly transfected cells.
EGFP/EYFP signal unmixing was carried out using Zeiss
lsm510 software, version 3.2SP2. Confocal images were
post-processed with volocity software (Improvision Sys-
tems, Coventry, UK). Movies are presented at ten frames
per second, representing a speed increase of ·40.
FACS
bbHT1080, transfected for 24 h with the EGFP-furin ICD
construct, were washed with NaCl/P
i
and detached from the
plastic using NaCl/P
i
containing 5 mm EDTA (5 min at

Statistical analysis was performed using the graphpad
prism, version 5 (GraphPad Software, Inc., San Diego, CA,
USA). Statistical significance was calculated using Student’s
t-test. Statistical significance was defined as P < 0.05 (*),
P < 0.001 (**) and P < 0.0001 (***).
Acknowledgements
We would like to thank Drs F. Barr, J. Creemers,
K. Shennan, D. Edwards and H P. Hauri for providing
reagents used in the present study. We thank Greg
Veltri, Therese Martin and Michele Bones (CRI Flow
Cytometry core unit) and Jane Gray (CRI equipment
park) for their technical assistance. We thank Neil
Taylor, Sue Atkinson, Patricia Eisenach and Helen
Gillingham for their helpful discussions and for criti-
cally reading the manuscript. We would like to
acknowledge the support of Cancer Research UK and
Hutchison Whampoa Limited (C.R. and G.M.; CRUK
grant C100/A8243), the Medical Research Council
(L.J.; grant 71393), the Wellcome Trust (M.G.; grant
079895/Z/06/Z), the British Heart Foundation
(W.R.E.; intermediate fellowship FS/03/055/15910)
and the University of Cambridge.
References
1 Zucker S, Pei D, Cao J & Lopez-Otin C (2003)
Membrane type-matrix metalloproteinases (MT-MMP).
Curr Top Dev Biol 54, 1–74.
2 Noe
¨
l A, Gilles C, Bajou K, Devy L, Kebers F, Lewalle
JM, Maquoi E, Munaut C, Remacle A & Foidart JM

1–11.
10 Vihinen P & Kahari VM (2002) Matrix metalloprotein-
ases in cancer: prognostic markers and therapeutic
targets. Int J Cancer 99, 157–166.
11 Genis L, Galvez BG, Gonzalo P & Arroyo AG (2006)
MT1-MMP: universal or particular player in angiogene-
sis? Cancer Metastasis Rev 25, 77–86.
12 Sabeh F, Ota I, Holmbeck K, Birkedal-Hansen H,
Soloway P, Balbin M, Lopez-Otin C, Shapiro S, Inada
M, Krane S et al. (2004) Tumor cell traffic through the
extracellular matrix is controlled by the membrane-
anchored collagenase MT1-MMP. J Cell Biol 167,
769–781.
13 Hotary KB, Allen ED, Brooks PC, Datta NS, Long
MW & Weiss SJ (2003) Membrane type I matrix
metalloproteinase usurps tumor growth control imposed
by the three-dimensional extracellular matrix. Cell 114,
33–45.
14 Ueda J, Kajita M, Suenaga N, Fujii K & Seiki M
(2003) Sequence-specific silencing of MT1-MMP
expression suppresses tumor cell migration and
invasion: importance of MT1-MMP as a therapeutic
target for invasive tumors. Oncogene 22, 8716–8722.
15 Gingras D & Beliveau R (2009) Emerging concepts in
the regulation of membrane-type 1 matrix metallopro-
teinase activity. Biochim Biophys Acta 1803, 142–150.
16 Gingras D, Bousquet-Gagnon N, Langlois S, Lacham-
bre MP, Annabi B & Beliveau R (2001) Activation of
the extracellular signal-regulated protein kinase (ERK)
cascade by membrane-type-1 matrix metalloproteinase

cling of MT1-MMP and MT3-MMP through the trans-
Golgi network. Identification of DKV582 as a recycling
signal. J Biol Chem 279
, 9331–9336.
24 Nakahara H, Howard L, Thompson EW, Sato H, Seiki
M, Yeh Y & Chen WT (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.
25 Sounni NE, Devy L, Hajitou A, Frankenne F, Munaut
C, Gilles C, Deroanne C, Thompson EW, Foidart JM
& Noel A (2002) MT1-MMP expression promotes
tumor growth and angiogenesis through an up-regula-
tion of vascular endothelial growth factor expression.
FASEB J 16, 555–564.
26 D’Alessio S, Ferrari G, Cinnante K, Scheerer W,
Galloway AC, Roses DF, Rozanov DV, Remacle AG,
Oh ES, Shiryaev SA et al. (2008) Tissue inhibitor of
metalloproteinases-2 binding to membrane-type 1
matrix metalloproteinase induces MAPK activation and
cell growth by a non-proteolytic mechanism. J Biol
Chem 283, 87–99.
27 Rozanov DV, Savinov AY, Williams R, Liu K, Golub-
kov VS, Krajewski S & Strongin AY (2008) Molecular
signature of MT1-MMP: transactivation of the
downstream universal gene network in cancer. Cancer
Res 68, 4086–4096.
28 Lehti K, Lohi J, Juntunen MM, Pei D & Keski-Oja J
(2002) Oligomerization through hemopexin and
cytoplasmic domains regulates the activity and turnover

12734–12743.
34 Labrecque L, Nyalendo C, Langlois S, Durocher Y,
Roghi C, Murphy G, Gingras D & Beliveau R (2004)
Src-mediated tyrosine phosphorylation of caveolin-1
induces its association with membrane type 1 matrix
metalloproteinase. J Biol Chem 279, 52132–52140.
35 Shorter J, Watson R, Giannakou ME, Clarke M,
Warren G & Barr FA (1999) GRASP55, a second
mammalian GRASP protein involved in the stacking of
Golgi cisternae in a cell-free system. EMBO J 18, 4949–
4960.
36 Short B, Preisinger C, Korner R, Kopajtich R,
Byron O & Barr FA (2001) A GRASP55-rab2 effector
complex linking Golgi structure to membrane traffic.
J Cell Biol 155, 877–883.
37 Kuo A, Zhong C, Lane WS & Derynck R (2000)
Transmembrane transforming growth factor-alpha
tethers to the PDZ domain-containing, Golgi mem-
brane-associated protein p59/GRASP55. EMBO J 19,
6427–6439.
38 D’Angelo G, Prencipe L, Iodice L, Beznoussenko G,
Savarese M, Marra P, Di Tullio G, Martire G, De
Matteis MA & Bonatti S (2009) GRASP65 and
GRASP55 sequentially promote the transport of
C-terminal valine-bearing cargos to and through the
Golgi complex. J Biol Chem 284, 34849–34860.
39 Barr FA, Preisinger C, Kopajtich R & Korner R (2001)
Golgi matrix proteins interact with p24 cargo receptors
and aid their efficient retention in the Golgi apparatus.
J Cell Biol 155, 885–891.

(1996) Binding of the inward rectifier K
+
channel Kir
2.3 to PSD-95 is regulated by protein kinase A
phosphorylation. Neuron 17, 759–767.
48 Cao TT, Deacon HW, Reczek D, Bretscher A & von
Zastrow M (1999) A kinase-regulated PDZ-domain
interaction controls endocytic sorting of the beta2-
adrenergic receptor. Nature 401, 286–290.
49 Hegedus T, Sessler T, Scott R, Thelin W, Bakos E,
Varadi A, Szabo K, Homolya L, Milgram SL &
Sarkadi B (2003) C-terminal phosphorylation of MRP2
modulates its interaction with PDZ proteins. Biochem
Biophys Res Commun 302, 454–461.
50 Birrane G, Chung J & Ladias JA (2003) Novel mode of
ligand recognition by the Erbin PDZ domain. J Biol
Chem 278, 1399–1402.
51 Geijsen N, Uings IJ, Pals C, Armstrong J, McKinnon
M, Raaijmakers JA, Lammers JW, Koenderman L &
Coffer PJ (2001) Cytokine-specific transcriptional regu-
lation through an IL-5Ralpha interacting protein.
Science 293, 1136–1138.
52 Li AL, Li HY, Jin BF, Ye QN, Zhou T, Yu XD,
Pan X, Man JH, He K, Yu M et al. (2004) A novel
eIF5A complex functions as a regulator of p53 and
p53-dependent apoptosis. J Biol Chem 279, 49251–
49258.
53 Gimferrer I, Ibanez A, Farnos M, Sarrias MR, Fenutria
R, Rosello S, Zimmermann P, David G, Vives J, Serra-
Pages C et al. (2005) The lymphocyte receptor CD6

rapid trafficking and processing of MT1-MMP in furin-
negative colon carcinoma LoVo cells. Traffic 5, 627–641.
61 Pei D & Weiss SJ (1996) Transmembrane-deletion
mutants of the membrane-type matrix metalloprotein-
ase-1 process progelatinase A and express intrinsic
matrix-degrading activity. J Biol Chem 271, 9135–9140.
62 Stawowy P, Meyborg H, Stibenz D, Borges Pereira
Stawowy N, Roser M, Thanabalasingam U, Veinot JP,
Chretien M, Seidah NG, Fleck E et al. (2005) Furin-like
proprotein convertases are central regulators of the
membrane type matrix metalloproteinase-pro-matrix
metalloproteinase-2 proteolytic cascade in atherosclero-
sis. Circulation 111, 2820–2827.
63 Kang T, Nagase H & Pei D (2002) Activation of mem-
brane-type matrix metalloproteinase 3 zymogen by the
proprotein convertase furin in the trans-Golgi network.
Cancer Res 62, 675–681.
64 Wang X & Pei D (2001) Shedding of membrane type
matrix metalloproteinase 5 by a furin-type convertase:
a potential mechanism for down-regulation. J Biol
Chem 276, 35953–35960.
65 Will H & Hinzmann B (1995) cDNA sequence and
mRNA tissue distribution of a novel human matrix
metalloproteinase with a potential transmembrane
segment. Eur J Biochem 231, 602–608.
66 Wang P, Wang X & Pei D (2004) Mint-3 regulates the
retrieval of the internalized membrane-type matrix
metalloproteinase, MT5-MMP, to the plasma
membrane by binding to its carboxyl end motif EWV.
J Biol Chem 279, 20461–20470.

containing sodium dodecyl sulfate and copolymerized
substrates. Anal Biochem 102, 196–202.
Supporting information
The following supplementary material is available:
Fig. S1. MT1-MMP co-immunoprecipitates with
GRASP55F in HeLa cells.
Fig. S2. The LLY motif in the MT1-MMP ICD is
crucial for the interaction with GRASP55.
Fig. S3. MT1-MMP co-immunoprecipitates with
GRASP55F in HeLa cells.
Fig. S4. Selection of bbHT1080 cells expressing EGFP-
furin by FACS.
Fig. S5. Furin expression impairs activation of
pro-MMP2.
Fig. S6. Fusion of GM130 coiled-coil region to
MT1-MMP ICD increased its binding to GRASP55.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
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from supporting information (other than missing files)
should be addressed to the authors.
C. Roghi et al. Role of GRASP55 in MT1-MMP activation
FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS 3175