From the Department of Medical Biochemistry and
Microbiology, Biomedical Center, Box 582, Uppsala University, Uppsala
S-751 23, Sweden, the § Department of Biological
and Environmental Science, University of Jyväskylä,
Jyväskylä FIN-40351, Finland, the ¶ Department of
Biochemistry and Pharmacy, Åbo Akademi University, Turku FIN-20520,
Finland, and the
Department of Biochemistry, University of
Cambridge, Bldg. O, Downing Site, Tennis Court Rd., Cambridge
CB2 1QW, United Kingdom
Received for publication, October 8, 2002, and in revised form, December 20, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The integrins
The collagen family currently includes at least 24 members (1, 2),
and four different collagen-binding integrins
Studies of collagen-binding integrins in various in vitro
assays show that they take part in cell adhesion, cell migration, control of collagen synthesis, matrix metalloproteinase
synthesis, remodeling of collagen matrices, and influence such
complex processes as cell proliferation, cell differentiation,
angiogenesis, platelet adhesion/aggregation, and epithelial
tubulogenesis (10, 11).
Using transfected cells and recombinant I domain,
Integrin The integrin subunit Cyanogen bromide cleavage of collagen chains identified the
non-RGD-containing helical CB3 fragment of collagen Examination of the crystal structure of an Arginine interacts with negatively charged Asp-219 on the surface of
the Other modes of collagen binding also exist, and
The interaction between integrins and their physiological ligands
displays several requirements for divalent cations. First, the GER
glutamate residue binds directly to a Mg2+ ion coordinated
within the I domain MIDAS. Other divalent cations will serve this
purpose, notably Mn2+ and Co2+, but in nature
such ions are unlikely to be sufficiently abundant to contribute
significantly to the adhesion process. Integrins possess several other
divalent cation binding sites, three or four within the blades of the
In the present study we set out to study further the mechanism whereby
Production of Human Recombinant Integrin cDNAs encoding Synthesis of Peptides
Peptides were synthesized as carboxyl-terminal amides on
TentaGel R RAM resin in a PerSeptive Biosystems 9050 Plus
PepSynthesizer exactly as described in our earlier studies (30, 31).
Peptides were purified by reverse phase high performance liquid
chromatography (HPLC) on a column of Vydac 219TP101522 using a linear
gradient of 5-45% acetonitrile in water containing 0.1%
trifluoroacetic acid. Fractions containing homogeneous product were
identified by analytical HPLC on a column of Vydac 219TP54, pooled, and
freeze dried. All peptides were found to be of the correct theoretical mass by mass spectrometry. The triple-helical stability of each peptide
was assessed by polarimetry as described previously.
Solid Phase Binding Assay for The coating of a 96-well high binding microtiter plate (Nunc)
was done by exposure to 0.1 ml of PBS containing 5 µg/cm2
(15 µg/ml) collagens or 20 µg/ml synthetic triple-helical collagen peptides overnight at 4 °C. Type I rat (rat tail) collagen, type IV
mouse (basement membrane of Engelbreth-Holm-Swarm mouse sarcoma) collagen, and type IV human "cut" (human placenta) collagen were purchased from Sigma. Type IV human collagen and type II bovine collagen were purchased from Biodesign International and Chemicon, respectively. Type I bovine (bovine dermal) collagen was from Cellon
S. A. Blank wells were coated with a 1:1 solution of 0.1 ml Delfia®
Diluent II (Wallac) and PBS. Residual protein absorption sites on all
wells were blocked with a 1:1 solution of 0.1 ml of Delfia® Diluent
II and PBS. Recombinant proteins Cells
Murine C2C12 myoblast cells from the American Type Culture
Collection were provided by A. Starzinski-Powitz. The generation of
C2C12 cells stably transfected with integrin Antibodies
Rabbit antibodies to the cytoplasmic tail of Immunoprecipitation and Electrophoresis
Cell cultures were washed three times in Dulbecco's modified
Eagle's medium devoid of cysteine and methionine and metabolically labeled overnight in the presence of 25 µCi/ml
[35S]methionine/cysteine (pro-Mix 35S cell
labeling mix; Amersham Biosciences). Proteins were extracted from the
tissue culture dishes by the addition of 1 ml of solubilization buffer
(1% Triton X-100, 0.15 M NaCl, 20 mM Tris-HCl
pH 7.4, 1 mM MgCl2, 1 mM
CaCl2) containing protease inhibitors (1 mM
Pefabloc SC (Roche Molecular Diagnostics), 1% aprotinin, 1 µg/ml
pepstatin, 1 µg/ml leupeptin). Solubilized proteins were centrifuged
for 10 min at 15,000 × g. The centrifuged supernatant
was precleared by incubating with 100 µg/ml preimmune IgG and protein
A-Sepharose CL4B (Amersham Biosciences) for 2 h. After
centrifugation, immune IgG was incubated with the extract for 2 h.
Specifically bound proteins were recovered with protein A-Sepharose.
The precipitate was washed three times with buffer A (1% Triton X-100,
0.5 M NaCl, 20 mM Tris-HCl pH 7.4, 1 mM MgCl2, 1 mM CaCl2)
and three times with buffer B (0.1% Triton X-100, 0.15 M
NaCl, 20 mM Tris-HCl pH 7.4, 1 mM
MgCl2, 1 mM CaCl2) prior to
solubilization in electrophoresis sample buffer. Proteins were
separated on 6% SDS-polyacrylamide gels and processed for fluorography.
Cell Attachment Assay
General Setup--
Ligands and metal ions are described
separately for the two experiments. 24-well cell culture plates (Nunc)
were coated with ligands (500 µl to a 2-cm2 well) diluted
in PBS overnight at 4 °C, followed by blocking with 2% BSA in PBS
for 2 h at room temperature and then washed in Puck's saline (137 mM NaCl, 5 mM KCl, 4 mM
Na2CO3, 5.5 mM D-glucose, pH 7.0).
Transfected cells were trypsinized, washed four times in Puck's
saline, deeded into the wells at a concentration of 250,000 cells/well,
and were allowed to attach for 45 min at 37 °C and 5%
CO2. Wells were washed three times in Puck's saline, and
plates were rapidly frozen at Ca2+ Inhibition Setup--
Wells were coated with 10 µg/ml bovine collagen type I (Vitrogen®100, Cohesion) or 10 µg/ml
human plasma fibronectin. Wells were filled with Puck's saline, and
MgCl2 + EGTA was added to obtain a final concentration
(after addition of cells) of 2 mM MgCl2 and
0.01 mM EGTA. CaCl2 was added according to Fig.
1.
Cell Attachment to Synthetic Peptides--
24-well plates were
coated with 10 µg/ml synthetic triple-helical collagen peptides at
4 °C overnight according to Fig. 4, or 10 µg/ml bovine collagen
type I (Vitrogen®100), or 10 µg/ml fibronectin. MgCl2
and CaCl2 were added to a final concentration of 2 mM MgCl2 and 0.01 mM
CaCl2.
Homology Modeling
Sequences of integrin The sequence alignment was made using the program MALIGN (43) in the
BODIL modeling environment5
(www.abo.fi/fak/mnf/bkf/research/johnson/bodil.html) using a structure-based sequence comparison matrix (44) with a gap penalty of 40.
Homology models were built with HOMODGE in BODIL. The amino acid side
chain rotamer library incorporated into BODIL was used to evaluate
alternative possibilities for side chain conformations for sequence
differences in the alignment of In the Influence of Ca2+ on Cell Attachment to
Collagen--
To compare the mechanism whereby
Collagen Preference of Binding to GER-containing Peptides--
Helical GFOGER and
GFOGER-like peptides have recently been shown to represent high
affinity integrin recognition motifs in collagens (32, 33). To
determine whether Modeling of Collagen Peptide Binding to the Glutamate in the Collagen Mimetic Tripeptide--
In the crystal
structure of the integrin
In the
In the Arginine in the Collagen Mimetic Tripeptide--
In the crystal
structure of the
In the
In the Phenylalanine in the Collagen Mimetic Tripeptide--
In the
When phenylalanine of the collagen mimetic tripeptide is mutated to
leucine, some favorable interactions would be lost, but this loss is
offset by the removal of unfavorable interactions with the main chain
oxygen atom of the residue at position 285 (tyrosine in
In the In recent years an increasing effort has been spent trying to
understand the mechanism whereby cells bind collagen. In vertebrates more than 24 different collagens exist, and the role of some of these
is yet unclear. Integrins are major receptors for collagens. A common
feature of the collagen-binding integrins is the presence of an The I domain is not found in integrin Gene knockout experiments and recombinant expression of the Both As a part of understanding the biological function of collagen-binding
integrins it is important to characterize all of the different
collagen-binding integrins with regard to collagen affinity, collagen
specificity, divalent ion requirements, and ligand recognition motifs.
Studies of Prior to this study no binding studies had been performed with the
The relatively low avidity for collagens estimated for both
The Despite the differences in collagen specificity, helical
GFOGER-like sequences are recognized by
1
1,
2
1,
10
1,
and
11
1 are referred to as a collagen
receptor subgroup of the integrin family. Recently, both
1
1 and
2
1
integrins have been shown to recognize triple-helical GFOGER (where single letter amino acid nomenclature is used,
O = hydroxyproline) or GFOGER-like motifs found in collagens,
despite their distinct binding specificity for various collagen
subtypes. In the present study we have investigated the mechanism
whereby the latest member in the integrin family,
11
1, recognizes collagens using C2C12
cells transfected with
11 cDNA and the bacterially expressed recombinant
11 I domain. The ligand binding
properties of
11
1 were compared with
those of
2
1.
Mg2+-dependent
11
1 binding to type I collagen required
micromolar Ca2+ but was inhibited by 1 mM
Ca2+, whereas
2
1-mediated
binding was refractory to millimolar concentrations of
Ca2+. The bacterially expressed recombinant
11 I domain preference for fibrillar collagens over
collagens IV and VI was the same as the
2 I domain.
Despite the difference in Ca2+ sensitivity,
11
1-expressing cells and the
11 I domain bound to helical GFOGER sequences in a
manner similar to
2
1-expressing cells and
the
2 I domain. Modeling of the
I domain-collagen peptide complexes could partially explain the observed preference of
different I domains for certain GFOGER sequence variations. In summary,
our data indicate that the GFOGER sequence in fibrillar collagens is a
common recognition motif used by
1
1,
2
1, and also
11
1 integrins. Although
10
and
11 chains show the highest sequence identity,
2 and
11 are more similar with regard to collagen specificity. Future studies will reveal whether
2
1 and
11
1
integrins also show overlapping biological functions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1
1,
2
1,
10
1 (3) and
11
1 (4) are known. The
3
1 integrin does not interact directly
with collagen, but it does act as a laminin receptor (5) that can
affect the activity of the collagen receptor
2
1 through receptor cross-talk (6).
1
1,
2
1,
10
1, and
11
1 possess an inserted, or I
domain1 closely related to
the von Willebrand factor A domain, which mediates binding to native
collagens. In different in vitro assays,
v
3 also appears to be able to interact
with type I collagen (7-9). However, this interaction most likely
involves RGD motifs in denatured or partially unfolded collagen chains.
1
1 has been shown to bind collagens, with
a preference for collagens IV and VI over collagen I and II. Collagen
XIII in vitro is also a ligand for
1
1 (12). Other identified ligands include
laminin-1/-2 (13, 14), the cartilage protein matrilin-1 (15), and the C-propeptide of collagen I (16). The affinity of
1
1 for laminin-1 has been reported to be
about 10-fold lower than for collagen IV (17). In accordance with this,
when the
1 integrin chain is expressed in K562 cells,
1
1 will bind type IV collagen, but it requires activation to bind laminin-1 (18). In Chinese hamster ovary cells,
1
1 integrin does not mediate
spreading on collagen II (12). It has been suggested that in these
cells a coreceptor is needed for
1
1-mediated spreading on collagen II.
2
1 and its
I domain have
been shown to bind a variety of collagens (19-21), the C-propeptide of
collagen I (16, 22), laminin-1 (23), laminin-2 (14), decorin (24), and the cartilage protein chondroadherin (25). Unlike the other ligands for
1
1 and
2
1,
chondroadherin does not support cell spreading. Early studies using
antibodies showed that
2
1 on some cells
(melanoma LOX cells), but not others (fibroblasts, platelets), mediated
the binding to laminin-1 (26).
10 was originally identified by
affinity purification of collagen type II-binding integrins from adult chondrocytes (3). The rather restricted expression of
10
1 to cartilage indicates that the
ligands are to be found in the cartilage extracellular matrix.
Intriguingly, using recombinant protein, the collagen binding
preference of the
10 I domain is most similar to that of
the
1 I domain, so that the
10 I domain prefers the basement membrane collagen IV and the beaded
filament-forming collagen VI over the interstitial collagens I and II
(27). In the same study, mutational analysis of the I domains showed
that the amino acid residues Arg-218 in
1 and
10 and Asp-219 in
2 are involved in
determining this collagen preference.
11 was initially detected in differentiating human fetal
muscle cells (28).
11 protein and mRNA expression
analysis in human embryos, however, revealed that expression is
localized to mesenchymal non-muscle cells in areas of highly organized
interstitial collagen networks. No expression was seen in muscle cells
in vivo (29). In the developing skeletal system,
10
1 and
11
1
thus show nonoverlapping, complementary expression patterns (11). In
accordance with the expression of
11
1 in
areas rich in interstitial collagens,
11
1
binds more efficiently to collagen I than to collagen IV (29).
1 I
as a cell-binding fragment that could be used to purify
1
1 (30). The
1 I and
2 I integrin binding site located within triple-helical
1 I CB3 has been identified as GFOGER (31, 32). Two
related sequences, GLOGER2
and GASGER, were identified elsewhere in collagen I (33), and other
GER-containing sequences in the collagen chains can also mediate cell
adhesion through
2
1.3
The GER motif thus appears to be a major cell adhesion motif used by
collagen-binding integrins.
2 I
domain-GFOGER complex suggested that other hydrophobic residues might
replace phenylalanine, which together with the glutamate and
arginine residues provided the main side chain interactions between the collagen-like peptide and the integrin (34). Interactions also occurred
with the main chain carbonyl group of the hydroxyproline residue,
suggesting that hydroxyproline itself may not be required specifically
for collagen-integrin interaction.
2 I domain, and although this appears a relatively nonspecific interaction, GEK will not substitute fully in human platelets. The ligand binding groove of the
2 I domain
is relatively deep compared with that of
1. Thus, the
coordination of Mg2+ in the metal ion dependent adhesion
site (MIDAS) may only be achieved by glutamate residues from the GER
motif, aspartate being too short for this purpose. This may not be the
case for other integrins, and the abundance of GDR triplets within the
collagens suggests the possibility that GDR motifs might serve as well. The present study was designed in part to test the possibility that
11, which lacks an acidic residue equivalent to Asp-219, and whose structure is not yet defined, might recognize GEK and GDR
triplets, both of which occur frequently in human collagens.
1
1 binds to collagen type IV using the
amino acids arginine and aspartate contributed from different collagen
chains (35, 36). The residues recognized by
1
1 in collagen XIII, lacking a GFOGER sequence, have not been identified. Studies with fragments of laminins
have shown that the I domain integrin binding sites are present in the
short arm of the
chain, but the exact region(s) has not yet been
mapped (13, 14).
subunit
propeller, and perhaps two within the
subunit
I-like domain. Some of these sites likely bind Ca2+ and
account for the biphasic role of Ca2+ in the competence of
the integrins. Adhesion of collagen to
2
1 in human platelets, in common with the binding of ligand by other integrins, has recently been shown to require micromolar
Ca2+ (37) and to be inhibited by millimolar
Ca2+. Conceivably the latter effect reflects competition
for Ser-123 lying between Mg2+ in the MIDAS and
Ca2+ in the ADMIDAS sites of the
subunit I-like domain,
such that high levels of Ca2+ render the integrin
subunit incapable of regulating
subunit function properly. The
requirement for Ca2+ appears to differ even for the same
integrin when expressed in different cells. Thus, although a biphasic
effect of Ca2+ on
2
1
competence in platelets can clearly be shown, the sensitivity of
2
1 to either high or low levels of
Ca2+ in HT1080 cells is much less
obvious.4
2
1 and
11
1
integrins bind collagens. Our data suggest that the approximated
Kd of the
11 I domain for type I
collagen is higher than that of the
2 I domain and that
in C2C12 cells the
11
1-mediated binding
to collagen I, but not that of
2
1, is
sensitive to the presence of Ca2+. However, both integrins
display a similar collagen specificity, and both recognize the helical
GFOGER sequence. Modeling of
2 I and
11 I
domain-ligand complexes could in part explain the observed differences
in
2 I and
11 I domain binding to
different collagen peptides. The results are potentially promising for
future attempts to generate reagents effective in blocking multiple
collagen-binding integrins simultaneously.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 I,
2 I, and
11 I Domains as Fusion
Proteins
1 I and
2 I
domains were generated by PCR as described earlier (27) using human
integrin
1 and
2 cDNAs as templates.
Vectors pGEX-4T-3 and pGEX-2T (Amersham Biosciences) were used to
generate recombinant glutathione S-transferase (GST) fusion
proteins of human
1 I and
2 I domains,
respectively. Human integrin
11 cDNA (4) was used as
a template when the
11 I domain was generated by PCR.
The PCR product having BamHI and EcoRI sites was
cloned to pGEX-KT, and the DNA sequence was checked by sequencing the
whole insert. The same vector was used for expression of recombinant
GST fusion proteins of the human
11 I domain. Competent
Escherichia coli BL21 cells were transformed with the
plasmids for protein production. 500 ml of LB medium (Biokar)
containing 100 µg/ml ampicillin was innoculated with a 50-ml
overnight culture of BL21/p
1 I, BL21/p
2
I, or BL21/p
11 I, and the cultures were grown at
37 °C until the A600 of the suspension
reached 0.6-1.0. Cells were induced with
isopropyl-1-thio-
-D-galactopyranoside and allowed to
grow for an additional 4-6 h before harvesting by centrifugation.
Pelleted cells were resuspended in PBS (pH 7.4) and then lysed by
sonication followed by the addition of Triton X-100 to a final
concentration of 2%. After incubation for 30 min on ice, suspensions
were centrifuged, and supernatants were pooled. Glutathione-Sepharose
(Amersham Biosciences) was added to the lysate, which was incubated at
room temperature for 30 min with gentle agitation. The lysate was then
centrifuged, the supernatant was removed, and glutathione-Sepharose
with bound fusion protein was transferred into disposable
chromatography columns (Bio-Rad). The columns were washed with PBS, and
fusion proteins were eluted using 30 mm glutathione. Purified
recombinant and glutathione-tagged
1 I,
2
I, and
11 I domains were analyzed by SDS and native
PAGE. The recombinant
1 I domain produced was 227 amino
acids in length, corresponding to amino acids 123-338 of the whole
1 integrin, whereas the
2 I domain was
223 amino acids long, which corresponded to amino acids 124-339 of the
whole
2 integrin. The carboxyl termini of the
1 I and
2 I domains contained 10 and 6 non-integrin amino acids, respectively. Recombinant
11 I
domain contains a total of 204 amino acids: at the amino terminus there
are 2 extra residues (GS) before the
11 I domain, which
starts from CQTY and ends with SLEG (residues 159-354); at the
carboxyl terminus there are 6 extra amino acids (EFIVTD). The
recombinant
11 I domain contains some GST as an impurity caused by endogenous protease activity during expression and
purification. Recombinant I domains were used as GST fusion proteins
for collagen binding experiments.
1 I,
2 I, and
11 I Domains
1 I-GST,
2 I-GST, and
11 I-GST were added to the
coated wells at the desired concentration in Delfia® assay buffer and
incubated for 1 h at room temperature. Europium-labeled anti-GST
antibody (Wallac) was then added (typically 1:1,000), and the mixtures
were incubated for 1 h at room temperature. All incubations
mentioned above were done in the presence of 2 mM
MgCl2. Delfia® enhancement solution (Wallac) was added to
each well, and the europium signal was measured by time-resolved
fluorometry (Victor2 multilabel counter, Wallac). In every case, at
least three parallel wells were analyzed.
2 cDNA
or integrin
11 cDNA has been described previously
(29). Cells were cultured at 37 °C in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum and
antibiotics (Statens veterinärmedicinska anstalt, Uppsala). The
cells were grown to subconfluence and passaged every 2-3 days.
11
integrin have been described previously (4). To immunoprecipitate
1 integrins, a polyclonal antibody to rat integrin
1 chain was used (38).
20 °C for later assay using the
hexoseaminidase test as described previously (29). For each cell line
used, a cell number standard was made. Each experiment was performed in
triplicate. To minimize errors from unequal trypsinization stress
between cell lines and handling of plates, for example, data were
normalized as follows. For each plate the adhesion to 10 µg/ml
fibronectin (provided by S. Johansson, Uppsala University) was used as
the 100% reference level, and the background found on BSA-only coated
wells was used as the base-line (0%) reference level.
1 (accession code P56199
(39)) and
11 (Q9UKX5 (40)) were obtained from SWISS-PROT
(41). The crystal structure of the
2 I domain in complex
with the triple-helical collagen mimetic peptide (PDB code 1dzi (34))
was obtained from the Protein Data Bank (42).
1 and
11
I domain sequences with the template structure.
2 I domain-peptide complex structure, 1dzi, and
the three peptide chains (identical in sequence but having different interactions with the
2 I domain) of the collagen
mimetic tripeptide are labeled B, C, and D. The corresponding chain
labels are used for the tripeptides docked to the model structures
built for the
1 I and
11 I domains.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
11
1 and
2
1
recognize collagens, we used the satellite cell line C2C12 transfected
with
2 or
11 cDNAs (C2C12
2+ and C2C12
11+,
respectively). The parental cell line C2C12 expresses members of the
1 subfamily, such as
5
1
and
7
1 (45), but does not adhere to
collagen (29). C2C12 cells transfected with either
2 or
11 acquire the ability to interact with collagens I and IV, with a preference for collagen I (29). The
2
1-mediated binding of platelets to
collagen has been reported to require micromolar Ca2+ but
to be inhibited by millimolar Ca2+ in the presence of
Mg2+ (37). To test the effect of Ca2+ on cell
adhesion to collagen I, the transfected C2C12 cells were plated on
collagen I in the presence of Mg2+ and EGTA with increasing
concentrations of Ca2+ added. In the absence of
Ca2+, adhesion of cells expressing
11
1 was virtually absent, as reported for
human platelets (45), and a biphasic response to added Ca2+
was observed with peak adhesion occurring at a free Ca2+ of
around 30 µM and adhesion being substantially abolished
at 4 mM, with an IC50 of about 1 mM. In marked contrast, C2C12
2+
cell adhesion to collagen I was largely refractory to both the removal
of Ca2+ using EGTA or the subsequent addition of millimolar
Ca2+ (Fig. 1).
View larger version (17K):
[in a new window]
Fig. 1.
Effects of Ca2+ on
11
1-mediated
cell adhesion to collagen I. C2C12
2+
and C2C12
11+ cells were allowed to adhere
to collagen I in the presence of 1 mM Mg2+, 10 µM EGTA, and varying concentrations of Ca2+.
Adhesion was measured as described under "Materials and
Methods."
11 and
2 I Domains Differ in Their
Affinity for Collagen I--
To estimate the Kd
of
11 for collagen I, we produced an
11 I
domain in E. coli. Initial attempts to express the
11 I domain as a bacterial GST fusion protein yielded
low amounts of protein. We therefore expressed the
11 I
domain as a His-tagged fusion protein in Pichia pastoris.
After purification on nickel-Sepharose only low binding to collagen I
was noted, and background binding was high. Gel filtration revealed
that a majority of protein appeared in a high molecular weight
fraction, indicating aggregation (data not shown). Production of
11 I-GST in E. coli was subsequently optimized. Large scale expression of the
11 I domain
yielded enough protein to perform binding studies. Approximated
Kd values based on solid phase binding assays
(26) and the use of the Michaelis-Menten equation suggested a
relatively low
11 I domain avidity to collagen I
(750 ± 50 nM) when compared with the
2 I domain binding to collagen I (20 ± 5 nM (27)) (Fig.
2).
View larger version (14K):
[in a new window]
Fig. 2.
Binding of the
11 I domain to collagen as a function
of
11 I domain concentration.
Microtiter plates were coated with 15 µg/ml collagen types I
(rat col-I), II (human col-II), and IV
(mouse col-IV native) overnight. Diluent containing BSA was
used as a background control and to block the wells. The GST fusion
1 I domain (4-408 nM) was allowed to bind
for 1 h in the presence of 2 mM MgCl2. The
wells were washed three times. Bound
I domain was detected with
europium-labeled anti-GST antibody. Time-resolved fluorescence
measurements were used. The data are the means of three parallel
determinations (±S.D.).
11 I Domain--
Previous
studies from several laboratories have shown that
2
1 integrin prefers fibril-forming
collagens over network-forming type IV and beaded filament-forming type
VI collagen. The same pattern can be seen in the binding of the
2 I domain. Here
2- and
11-mediated binding to different collagens was compared. The
11 I domain was shown to prefer the fibril-forming
collagen types I and II (Fig. 3), whereas
its binding was weaker to type III (data not shown), a member of the
same collagen subgroup. Collagens IV and VI were poor ligands for the
11 I domain. Thus, in the terms of its binding pattern
the
11 I domain was closer to the
2 I
domain than to either the
1 or
10 I
domain.
View larger version (8K):
[in a new window]
Fig. 3.
Binding of the
11 I domain to ECM ligands.
Microtiter plates were coated with 15 µg/ml collagen types I
(rat col-I, bovine col-I), II (bovine
col-II), IV (human col-IV cut, mouse col-IV
native), and VI (human col-VI) overnight. Diluent
containing BSA was used as a background control and to block the wells.
GST fusion
11 I domain (408 nM) was allowed
to bind for 1 h in the presence of 2 mM
MgCl2. Wells were washed three times. Bound
I domain
was detected with europium-labeled anti-GST antibody. Time-resolved
fluorescence measurements were used. The data are the means of three
parallel determinations (±S.D.).
11
1 also differed from
2
1 with regard to its recognition
sequences, C2C12
2+ and C2C12
11+ cells were tested for their ability to
attach to different collagen-like peptides. C2C12
2+ cells adhered to GFOGER and GFOGEK
peptides as reported previously. C2C12
11+
cells also bound these peptides in a similar pattern, whereas untransfected cells failed to do so (Fig.
4).To confirm that the observed binding
occurred via the
I domain, I domains from
1,
2, and
11 were compared with regard to
binding to the different collagen-derived peptides (Fig.
5). In these studies we also included the
GLOGER peptide (33) which bound all I domains. The
11 I domain preferred the GFOGER peptide followed by the GFOGEK and GLOGER
peptides, much like the
2 I domain. The
1
I domain bound the GLOGER peptide as efficiently as the GFOGER peptide.
The peptide containing the sequence GASGER, reported as a weak binding
site for
2
1 and
1
1 showed only low capacity to bind any
of the I domains, and substitution of aspartate for glutamate within the GER triplet similarly abolished I domain binding. The sequence GPOGES, from the collagen I
2 chain where it corresponds
to GFOGER in the
1 I chain, was similarly without
significant binding activity. Comparing the overall peptide binding
pattern,
2 I and
11 I domains appear most
similar in their peptide binding preferences.
View larger version (20K):
[in a new window]
Fig. 4.
11
1
binds the helical GFOGER sequence. C2C12, C2C12
2+, and C2C12
11+
cells were allowed to adhere to synthetic collagen peptides in the
presence of 1 mM Mg2+ and 10 µM
Ca2+, and cell adhesion (triplicate wells) was evaluated
(±S.D.).
View larger version (16K):
[in a new window]
Fig. 5.
Binding of
1 I,
2 I, and
11 I domains to synthetic
triple-helical collagen peptides. Microtiter plates were coated
with 20 µg/ml collagen peptides. Diluent II containing BSA was used
as a background control and to block the wells. GST-fusion
1 I (A),
2 I (B),
and
11 I domains (C) were allowed to bind for
1 h in the presence of 2 mM MgCl2. The
wells were washed three times. Bound
I domains were detected with
europium-labeled anti-GST antibody. Time-resolved fluorescence
measurements were used. The data are the means of three parallel
determinations (±S.D.).
11 I
Domain--
The basic assumption in modeling was that the collagen
mimetic tripeptide GFOGER and its mutants bind to all of the integrin
I domains in a way similar to that seen in the crystal structure of
the complex between
2 I domain and the GFOGER
triple-helical peptide (34). The sequence identities of the
1 and
11 I domains to the
2 I domain are 51 and 45% respectively, thus experience dictates that high quality models will be produced. Only one region of
the
11 I domain model is uncertain, where Pro-310
(threonine in the
1 and
2 I domains) is
located within a region that corresponds to helix 6 of the open fold of
the
1 and
2 I domains. Proline generally
does not promote helix stability, so it is very likely that the helix
begins at or after position 310 in the
11 I domain. In
addition, the local alignment of residues 179 and 180 seems peculiar
because the charged residue Glu-180 would be buried, and the
hydrophobic residue Val-179 would be exposed toward the solvent. If
Glu-180 is buried, then the conserved residue Tyr-157 may change its
conformation in the
11 I domain and affect the binding
of the collagen mimetic tripeptide. Thus, it is possible that the
binding conformation seen in
2 I domain-tripeptide
complex structure may be different in the case of
11.
2 I domain in complex with the
collagen mimetic peptide (3 × GFOGER), the side chain of only one
of the glutamate residues in the collagen mimetic tripeptide, that of
the middle strand, chain C, interacts with the I domain. This glutamate
is coordinated to the metal ion of the MIDAS motif and thus, represents
a key interaction in tripeptide binding (Fig.
6). Moreover, even a conservative change,
mutation to aspartate, lowers the binding dramatically for the
1 I,
2 I, and
11 I domains
(GFOGDR; Fig. 4). Aspartate is one methylene group shorter than
glutamate and would not reach the metal ion of the MIDAS motif as
easily as glutamate can.
View larger version (55K):
[in a new window]
Fig. 6.
Stereo view of the interactions
between the arginine from chain C of the collagen mimetic tripeptide
and integrin 2 (1dzi)
(A),
1
(B), and
11
(C) I domains. Detailed interactions of Arg
Lys mutation of the collagen mimetic tripeptide with the integrin
2 I domain in the same region are shown in D.
The backbone of the collagen mimetic tripeptide is shown as
yellow cords, and the I domain backbone is shown in
blue. Strong electrostatic interactions are shown as
black dotted lines, and weak electrostatic interactions are
shown as yellow dotted lines.
2 I domain there is a leucine residue at position
286, which forms an interaction with phenylalanine of the collagen mimetic tripeptide from chain B, whereas both the
1 and
11 I domains have tyrosine at that position. Tyrosine
could either form a hydrophobic interaction with a planar face of the
Arg-148 side chain, or tyrosine could form a hydrogen bond with
glutamate from the collagen mimetic tripeptide chain B. If the latter
case is true then the hydrogen bond would not be possible in the
collagen mimetic tripeptide containing the Glu
Asp mutation.
1 I domain model, Arg-218 (aspartate in
2 and threonine in
11) may form an
additional interaction with the glutamate of chain D of the collagen
mimetic tripeptide. This interaction would still be possible in the
tripeptide containing the aspartate mutation.
2 I domain (1dzi), arginine in chain C
of the collagen mimetic tripeptide is bound to the area where Asp-219,
Asn-189, and Leu-220 are located. The N
and N
2 atoms of arginine
would interact with side chain carboxylate of Asp-219 but only weakly
because the angle is not optimal for forming a strong
hydrogen-bond/salt bridge. In addition, the side chain of Leu-220 forms
an optimal site for hydrophobic interactions with a planar face of the
arginine side chain (Fig. 6A). In both
1 and
11, the arginine of the tripeptide could form a salt
bridge with glutamate at the position equivalent to Asn-189 in
2 (Fig. 6, B and C). Furthermore,
in
11 there is a threonine equivalent to Asp-219 in
2 whose side chain hydroxyl group can accept a hydrogen
bond from the N
of arginine from the tripeptide (Fig. 6C). For
2, the Arg
Lys mutation in the
collagen mimetic tripeptide (GFOGEK) does not affect binding as
dramatically as seen for
1 and
11 (Fig.
5). In
2, the repulsion resulting from the charged amino
group of lysine positioned near the Leu-220 side chain would be offset
by the formation of a somewhat more optimal hydrogen bond/salt bridge
between lysine and Asp-219 (Fig. 6D). The mutation of
arginine to lysine reduces the binding affinity of collagen mimetic
tripeptide to the
1 and
11 I domains
because the salt bridge to glutamate, at the position equivalent to
Asn-189 in
2, cannot be maintained. When arginine of the
collagen mimetic tripeptide is mutated to lysine, lysine cannot reach
glutamate because a lysine residue is shorter than an arginine residue. Moreover, in
11 the lysine residue can form a hydrogen
bond with the threonine equivalent to Asp-219 in
2, and
thus, the effect of the mutation is not as dramatic as for
1.
2 I domain, the arginine from chain B of the
collagen mimetic tripeptide interacts mainly with other parts of the tripeptide and not with the I domain. N
is hydrogen-bonded to the
main chain oxygen of arginine in chain C, and the planar end of the
arginine side chain has a hydrophobic interaction with proline in
peptide chain C. In addition, hydrophobic interactions with the
hydrophobic part of the Glu-256 side chain and weak electrostatic interactions with the main chain oxygen atom of Ser-257 can be seen.
These interactions should be present and identical in each of the I
domains in this study. Thus, the effect of the mutation of arginine to
lysine, caused by chain C, should be same for all I domains.
2 structure, the arginine from chain D of the
collagen mimetic tripeptide is exposed to the solvent, and thus, the mutation can only have an indirect influence on I domain binding.
2 I domain structure, the phenyl ring of phenylalanine
in chain B of the collagen mimetic tripeptide is stacked with the
phenol ring of the conserved tyrosine (position 157 in
2). This phenylalanine also has hydrophobic
interactions with Leu-286 in
2. In addition, the
phenylalanine of chain B forms an unfavorable interaction with the main
chain oxygen atom of Tyr-285 (Fig. 7A ). A corresponding view of phenylalanine
interactions with the
1 I domain is shown in Fig.
7B.
View larger version (61K):
[in a new window]
Fig. 7.
Stereo view of the interactions between the
phenylalanine from chain B of the collagen mimetic peptide and integrin
I domains 2 (1dzi)
(A) and
1
(B). The collagen mimetic peptide backbone is
shown in yellow, and the I domain backbone is shown in
blue.
2 and
11; serine in
1).
Thus, there is a small change in the binding affinity of
1 and
2 when the Phe
Leu mutant is
compared with the "wild type" tripeptide (Fig. 5). The effects seen
for
11 are difficult to predict because the model is
inaccurate in this region. The binding affinity is lowered dramatically
when phenylalanine is replaced with alanine, resulting in the loss of
all favorable interactions (Fig. 5).
2 I domain structure, the phenylalanine in chain
C of the collagen mimetic tripeptide leans against the side chain of
Asn-154, which is conserved in the
1,
2,
and
11 I domains. This interaction is not very critical,
and thus the mutation of phenylalanine to leucine or alanine would not
affect the binding affinity by much. The phenylalanine in chain D is
exposed to the solvent, and thus it has practically no role in binding.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
I
domain that is directly involved in ligand binding.
chains from the invertebrate
Drosophila melanogaster but it is present in 9 of the 18 currently known vertebrate integrin
chains (11) including
L,
M,
X,
D, and
E, which are all involved in different aspects of leukocyte functions and pair exclusively with the
2
subunit (10). The overall importance of integrin-mediated cell-collagen interactions involving the
1,
2,
10, and
11 integrin chains is largely
unknown because of the limited information available for
10
1 and
11
1. Based on the appearance of I domain
integrin
chains during vertebrate evolution it is possible that
these integrin chains play important roles in vertebrate-specific
structures of the musculoskeletal system.
I
domains have yielded considerable information about the characteristics and functions of collagen-binding integrins
1
1 and
2
1
(11, 46, 47). Phylogenetically,
1 and
2
form a subfamily distinct from
10 and
11,
which most likely have formed through two distinct gene duplication events.
1 and
2 integrin chains are fairly
widely expressed throughout the body. Gene knockout experiments of
1 and
2 chains have shown that
inactivation of the individual collagen-binding integrins does not seem
to impair embryonic development (46, 47). Rather, mild phenotypes are
observed where either fibroblast and leukocyte interactions with
collagens or platelet interactions with collagens are affected. Recent
analysis of
10 and
11 expression (3, 29)
reveals a restricted embryonic expression pattern, which is not
overlapping but complementary. In the near future it will be important
to determine to what extent the collagen-binding integrins show
overlapping functions and to what extent different collagen-binding
integrins can functionally compensate for each other's absence.
Crossing different mice strains lacking certain collagen receptors will
shed light on these issues.
1 I,
2 I, and
10 I domains have shown that they bind collagens with
different specificity (27). This specificity seems in part to be
determined by residues located outside the MIDAS motif in the
I
domain. Data from several groups have convincingly shown that
1 prefers collagens IV and VI over collagen I and that
the preferences of
2 are opposite. More recently the
10 I domain was shown to display a collagen binding
specificity similar to that of
1 (27).
11 I domain. It thus appears that although
1 is, in terms of evolution, more similar to
2, and
10 is more closely related to
11, another grouping can be made based on their collagen
specificity. The finding that
11 I prefers interstitial
collagens over nonfibrillar collagens supports our previous cell
binding data (29), but the difference is even more pronounced at the I
domain level. A candidate amino acid that might play a role in
determining this preference is Thr-238 found in a position
corresponding to Arg-218 in
1.
10 I and
11 I domains is intriguing. Our
experience is that as recombinant GST fusion proteins, these I domains
are less soluble than
1 I and
2 I
domains, and they might have a tendency to form aggregates. This may
affect the Kd estimates. Furthermore, we have
shown that in the length of the produced protein a difference of one
amino acid residue might lead to changes in the avidity of collagen
binding (48). Thus, the approximated Kd values
should be used for comparing the binding of a recombinant
I domain with different collagens rather than for comparing the
I domains with each other. Low avidity may indicate that the major function for
10 and
11 is not that of firm adhesion
but that these integrins engage in dynamic interactions with collagen
during events such as cell migration. It is also possible that the true
ligands have not yet been identified. For example, for
10, a cartilage ligand, possibly a collagen other than
collagen II, might be the preferred ligand. In the case of
11, a perichondrium ligand other than collagen I might
bind this integrin with higher affinity.
2
1-mediated binding of platelets to
collagen I is inhibited by mM concentrations of
Ca2+ (37). The finding that
2
1 is not inhibited by Ca2+
when expressed in C2C12 cells is intriguing. In the case of platelets and C2C12 cells this difference might be related to the activation status of the integrin. Whereas platelets and leukocytes have a more
elaborate system for regulating integrin activation status, integrins
in C2C12 cells are expected to be mainly in the activated state,
displaying a higher affinity.
11
1 is not
expressed on platelets, so a direct comparison with
2
1 is not
possible.6 However, when
expressed in C2C12 cells,
11
1 binding to
collagen I requires µM Ca2+ and is sensitive
to mM concentrations of Ca2+, as
2
1 when expressed on the platelet
surface. The recent crystallization of soluble
v
3 supports a role of Ca2+
ions in allosteric modulation of integrin conformation (49). It is
possible that a high affinity interaction with collagen I, such as that
mediated by
2
1, is less affected by
Ca2+-induced allosteric conformational changes. Conversely,
a lower affinity interaction with collagen I, such as that mediated via
11
1 might be more sensitive to allosteric
changes in other regions of the receptor. This differential sensitivity
to Ca2+ might be physiologically important in the formation
and turnover of the musculoskeletal system, where the local
concentration of Ca2+ varies.
1
1,
2
1, and
as shown in this study, also by
11
1.
Careful analysis of the occurrence of GFOGER-like peptides has shown
that in addition to the CB3-derived sequences GFOGER and GFOGEK, which
are present in the central part of the collagen chain, an
amino-terminal
2 I and
1 I domain binding
site overlaps with the peptide GLOGER (33). As shown in this study, the
binding of the different I domains to different collagen peptides
varied somewhat. The glutamate in GFOGER was central for the binding of
all I domains, whereas the phenylalanine seemed to be more important
for
11 binding, and the arginine was especially
important for
1 binding. It is possible that in vivo collagen-binding integrins prefer certain sites on the
collagen molecules. In a particular cell expressing multiple collagen
receptors, a number of factors might determine which region in collagen
is bound by a particular integrin. Some of the factors that might affect ligand binding include local Ca2+ concentrations,
expression levels of the different receptors, and subcellular
localization within the cell. Collagen receptors have been shown to
affect collagen and matrix metalloproteinase synthesis. Already now it
is possible to envisage how GFOGER peptides have the potential to
become universal reagents blocking cell-collagen interactions. It will
be important to determine whether
10
1 also binds GFOGER peptides. Triple-helical collagen peptides might be
of use in conditions characterized by excessive collagen production such as various fibrotic conditions.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Swedish Research Council (to D. G.), the Medical Research Council (to R. W. F.), the British Heart Foundation (to R. W. F.), the Wenner-Gren Foundation (to W.-M. Z.), and the Kung Gustaf V:s 80-års Fond (to D. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed. Tel.: 46-18-471-4175; Fax: 46-18-471-4673; E-mail: donald.gullberg@imbim.uu.se.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M210313200
3 R. W. Farndale, P. R.-M. Siljander, and C. G. Knight, in preparation.
4 R. W. Farndale, P. R.-M. Siljander, and C. G. Knight, unpublished observation.
5 J. V. Lehtonen, V. V. Rantanen, D. J. Still, M. Gyllenberg, and M. S. Johnson, unpublished observation.
6 R. W. Farndale, unpublished observation.
2 Where single letter amino acid nomenclature is used, O = hydroxyproline.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: I domain, inserted domain; BSA, bovine serum albumin; GST, glutathione S-transferase; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Myllyharju, J., and Kivirikko, K. I. (2001) Ann. Med. 33, 7-21[Medline] [Order article via Infotrieve] |
2. |
Hashimoto, T.,
Wakabayashi, T.,
Watanabe, A.,
Kowa, H.,
Hosoda, R.,
Nakamura, A.,
Kanazawa, I.,
Arai, T.,
Takio, K.,
Mann, D. M.,
and Iwatsubo, T.
(2002)
EMBO J.
21,
1524-1534 |
3. |
Camper, L.,
Hellman, U.,
and Lundgren-Akerlund, E.
(1998)
J. Biol. Chem.
273,
20383-20389 |
4. |
Velling, T.,
Kusche-Gullberg, M.,
Sejersen, T.,
and Gullberg, D.
(1999)
J. Biol. Chem.
274,
25735-25742 |
5. | Carter, W. G., Ryan, M. C., and Gahr, P. J. (1991) Cell 65, 599-610[Medline] [Order article via Infotrieve] |
6. |
Hodivala-Dilke, K. M.,
DiPersio, C. M.,
Kreidberg, J. A.,
and Hynes, R. O.
(1998)
J. Cell Biol.
142,
1357-1369 |
7. | Agrez, M. V., Bates, R. C., Boyd, A. W., and Burns, G. F. (1991) Cell Regul. 2, 1035-1044[Medline] [Order article via Infotrieve] |
8. | Pfaff, M., Aumailley, M., Specks, U., Knolle, J., Zerwes, H. G., and Timpl, R. (1993) Exp. Cell Res. 206, 167-176[CrossRef][Medline] [Order article via Infotrieve] |
9. | Davis, G. E. (1992) Biochem. Biophys. Res. Commun. 182, 1025-1031[Medline] [Order article via Infotrieve] |
10. |
Bouvard, D.,
Brakebusch, C.,
Gustafsson, E.,
Aszodi, A.,
Bengtsson, T.,
Berna, A.,
and Fassler, R.
(2001)
Circ. Res.
89,
211-223 |
11. | Gullberg, D., and Lundgren-Åkerlund, E. (2002) Prog. Histochem. Cytochem. 37, 3-54[Medline] [Order article via Infotrieve] |
12. |
Nykvist, P., Tu, H.,
Ivaska, J.,
Käpylä, J.,
Pihlajaniemi, T.,
and Heino, J.
(2000)
J. Biol. Chem.
275,
8255-8261 |
13. |
Colognato-Pyke, H.,
O'Rear, J. J.,
Yamada, Y.,
Carbonetto, S.,
Cheng, Y. S.,
and Yurchenco, P. D.
(1995)
J. Biol. Chem.
270,
9398-9406 |
14. |
Colognato, H.,
MacCarrick, M.,
O'Rear, J. J.,
and Yurchenco, P. D.
(1997)
J. Biol. Chem.
272,
29330-29336 |
15. |
Makihira, S.,
Yan, W.,
Ohno, S.,
Kawamoto, T.,
Fujimoto, K.,
Okimura, A.,
Yoshida, E.,
Noshiro, M.,
Hamada, T.,
and Kato, Y.
(1999)
J. Biol. Chem.
274,
11417-11423 |
16. | Davies, D., Tuckwell, D. S., Calderwood, D. A., Weston, S. A., Takigawa, M., and Humphries, M. J. (1997) Eur. J. Biochem. 246, 274-282[Abstract] |
17. | Pfaff, M., Gohring, W., Brown, J. C., and Timpl, R. (1994) Eur. J. Biochem. 225, 975-984[Abstract] |
18. | Wong, L. D., Sondheim, A. B., Zachow, K. R., Reichardt, L. F., and Ignatius, M. J. (1996) Cell Adhes. Commun. 4, 201-221[Medline] [Order article via Infotrieve] |
19. |
Dickeson, S. K.,
Mathis, N. L.,
Rahman, M.,
Bergelson, J. M.,
and Santoro, S. A.
(1999)
J. Biol. Chem.
274,
32182-32191 |
20. | Dickeson, S. K., and Santoro, S. A. (1998) Cell. Mol. Life Sci. 54, 556-566[CrossRef][Medline] [Order article via Infotrieve] |
21. | Tuckwell, D. S., Reid, K. B. M., Barnes, M. J., and Humphries, M. J. (1996) Eur. J. Biochem. 241, 732-739[Abstract] |
22. |
Weston, S. A.,
Hulmes, D. J.,
Mould, A. P.,
Watson, R. B.,
and Humphries, M. J.
(1994)
J. Biol. Chem.
269,
20982-20986 |
23. | Ettner, N., Gohring, W., Sasaki, T., Mann, K., and Timpl, R. (1998) FEBS Lett. 430, 217-221[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Guidetti, G.,
Bertoni, A.,
Viola, M.,
Tira, E.,
Balduini, C.,
and Torti, M.
(2002)
Blood
100,
1707-1714 |
25. |
Camper, L.,
Heinegard, D.,
and Lundgren-Akerlund, E.
(1997)
J. Cell Biol.
138,
1159-1167 |
26. | Elices, M. J., and Hemler, M. E. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9906-9910[Abstract] |
27. |
Tulla, M.,
Pentikäinen, O. T.,
Viitasalo, T.,
Käpylä, J.,
Impola, U.,
Nykvist, P.,
Nissinen, L.,
Johnson, M. S.,
and Heino, J.
(2001)
J. Biol. Chem.
276,
48206-48212 |
28. | Gullberg, D., Velling, T., Sjöberg, G., and Sejersen, T. (1995) Dev. Dyn. 204, 57-65[Medline] [Order article via Infotrieve] |
29. | Tiger, C.-F., Fougerousse, F., Grundström, G., Velling, T., and Gullberg, D. (2001) Dev. Biol. 237, 116-129[CrossRef][Medline] [Order article via Infotrieve] |
30. | Gullberg, D., Gehlsen, K. R., Turner, D. C., Ahlen, K., Zijenah, L. S., Barnes, M. J., and Rubin, K. (1992) EMBO J. 11, 3865-3873[Abstract] |
31. |
Knight, C. G.,
Morton, L. F.,
Onley, D. J.,
Peachey, A. R.,
Messent, A. J.,
Smethurst, P. A.,
Tuckwell, D. S.,
Farndale, R. W.,
and Barnes, M. J.
(1998)
J. Biol. Chem.
273,
33287-33294 |
32. |
Knight, C. G.,
Morton, L. F.,
Peachey, A. R.,
Tuckwell, D. S.,
Farndale, R. W.,
and Barnes, M. J.
(2000)
J. Biol. Chem.
275,
35-40 |
33. |
Xu, Y.,
Gurusiddappa, S.,
Rich, R. L.,
Owens, R. T.,
Keene, D. R.,
Mayne, R.,
Hook, A.,
and Hook, M.
(2000)
J. Biol. Chem.
275,
38981-38989 |
34. | Emsley, J., Knight, C. G., Farndale, R. W., Barnes, M. J., and Liddington, R. C. (2000) Cell 101, 47-56[Medline] [Order article via Infotrieve] |
35. | Eble, J. A., Golbik, R., Mann, K., and Kuhn, K. (1993) EMBO J. 12, 4795-4802[Abstract] |
36. | Golbik, R., Eble, J. A., Ries, A., and Kuhn, K. (2000) J. Mol. Biol. 297, 501-509[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Onley, D. J.,
Knight, C. G.,
Tuckwell, D. S.,
Barnes, M. J.,
and Farndale, R. W.
(2000)
J. Biol. Chem.
275,
24560-24564 |
38. |
Gullberg, D.,
Terracio, L.,
Borg, T. K.,
and Rubin, K.
(1989)
J. Biol. Chem.
264,
12686-12694 |
39. |
Briesewitz, R.,
Epstein, M. R.,
and Marcantonio, E. E.
(1993)
J. Biol. Chem.
268,
2989-2996 |
40. | Lehnert, K., Ni, J., Leung, E., Gough, S. M., Weaver, A., Yao, W. P., Liu, D., Wang, S. X., Morris, C. M., and Krissansen, G. W. (1999) Genomics 60, 179-187[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Bairoch, A.,
and Apweiler, R.
(2000)
Nucleic Acids Res.
28,
45-48 |
42. |
Berman, H. M.,
Westbrook, J.,
Feng, Z.,
Gilliland, G.,
Bhat, T. N.,
Weissig, H.,
Shindyalov, I. N.,
and Bourne, P. E.
(2000)
Nucleic Acids Res.
28,
235-242 |
43. | Johnson, M. S., and Overington, J. P. (1993) J. Mol. Biol. 233, 716-738[CrossRef][Medline] [Order article via Infotrieve] |
44. | Johnson, M. S., May, A. C., Rodionov, M. A., and Overington, J. P. (1996) Methods Enzymol. 266, 575-598[Medline] [Order article via Infotrieve] |
45. | Velling, T., Collo, G., Sorokin, L., Durbeej, M., Zhang, H. Y., and Gullberg, D. (1996) Dev. Dyn. 207, 355-371[CrossRef][Medline] [Order article via Infotrieve] |
46. | Gardner, H., Kreidberg, J., Koteliansky, V., and Jaenisch, R. (1996) Dev. Biol. 175, 301-313[CrossRef][Medline] [Order article via Infotrieve] |
47. |
Holtkotter, O.,
Nieswandt, B.,
Smyth, N.,
Muller, W.,
Hafner, M.,
Schulte, V.,
Krieg, T.,
and Eckes, B.
(2002)
J. Biol. Chem.
277,
10789-10794 |
48. |
Käpylä, J.,
Ivaska, J.,
Riikonen, R.,
Nykvist, P.,
Pentikäinen, O.,
Johnson, M.,
and Heino, J.
(2000)
J. Biol. Chem.
275,
3348-3354 |
49. |
Xiong, J. P.,
Stehle, T.,
Diefenbach, B.,
Zhang, R.,
Dunker, R.,
Scott, D. L.,
Joachimiak, A.,
Goodman, S. L.,
and Arnaout, M. A.
(2001)
Science
294,
339-345 |