Identification of the Ligand Binding Site for the Integrin
9
1 in the Third Fibronectin Type III
Repeat of Tenascin-C*
Yasuyuki
Yokosaki
§¶,
Nariaki
Matsuura
,
Shigeki
Higashiyama**,
Isao
Murakami
§,
Masanobu
Obara
,
Michio
Yamakido§§,
Norikazu
Shigeto
,
John
Chen¶¶, and
Dean
Sheppard¶¶
From the Departments of
Internal Medicine and
§ Laboratory Medicine, National Hiroshima Hospital, 513 Jike, Saijoh, Higashi-Hiroshima 739-0041,
Department of
Pathology, School of Allied Health Sciences, ** Department of
Biochemistry, Faculty of Medicine, Osaka University, 2-2 Yamadaoka,
Suita, Osaka 565-0871, 
Department of
Developmental Science, Faculty of Science, Hiroshima University, 1-1 Kagamiyama, Saijoh, Higashi-Hiroshima 739-8526, §§ Department of Internal Medicine II, School of
Medicine, Hiroshima University, 1-2-3 Kasumi, Minamiku,
Hiroshima 734-8551, Japan, and the ¶¶ Lung Biology Center,
Department of Medicine, University of California,
San Francisco, California 94143
 |
ABSTRACT |
The integrin
9 subunit forms a
single heterodimer,
9
1 that mediates cell
adhesion to a site within the third fibronectin type III repeat of
tenascin-C (TNfn3). In contrast to at least 3 other integrins that bind
to this region of tenascin-C,
9
1 does not
recognize the common integrin recognition motif, Arg-Gly-Asp (RGD). In
this report, we have used substitution mutagenesis to identify a unique
ligand recognition sequence in TNfn3. We introduced mutations
substituting alanine for each of the acidic residues in or adjacent to
each of the exposed loops predicted from the solved crystal structure.
Most of these mutations had little or no effect on adhesion of
9-transfected SW480 colon carcinoma cells, but mutations
of either of two acidic residues in the B-C loop region markedly
reduced attachment of these cells. In contrast, cells expressing the
integrin
v
3, previously reported to bind to the RGD sequence in the adjacent F-G loop, attached to all mutant
fragments except one in which the RGD site was mutated to RAA. The
peptide, AEIDGIEL, based on the sequence of human tenascin-C in this
region blocked the binding of
9-transfected cells, but
not
3-transfected cells to wild type TNfn3. This
sequence contains a tripeptide, IDG, homologous to the sequences LDV,
IDA, and LDA in fibronectin and IDS in VCAM-1 recognized by the closely related integrin
4
1. These findings
support the idea that this tripeptide motif serves as a ligand binding
site for the
4/
9 subfamily of
integrins.
 |
INTRODUCTION |
Integrins are cell surface heterodimers that play roles in
essential biological processes including development and tissue remodeling (1-4). Ligand binding specificity depends in large part on
the specific
and
subunit present in each heterodimer. To date,
extracellular matrix proteins, cell surface immunoglobulin superfamily
molecules, and cadherins have been identified as ligands for integrins.
In most cases where the crystal structure of integrin ligands have been
solved, the ligand binding site includes at least one acidic residue,
generally displayed in an exposed peptide loop (5-7). The first short
peptide sequence identified as an integrin recognition sequence was the
tripeptide, Arg-Gly-Asp (RGD) (8), initially identified as a
cell-recognition sequence in the large extracellular matrix protein,
fibronectin (9, 10). Subsequently, this RGD sequence was found to be
present in several integrin ligands and to serve as a recognition
sequence for several different integrins (11). As the sequences of
multiple integrin
subunits were solved, it became clear that these
sequences could be divided into 3 subfamilies based on sequence
homology (1). One family includes
subunits with a characteristic
disulfide-linked cleavage site. A subset of these form heterodimers
that recognize RGD-containing ligands. Another includes
subunits
that contain an inserted domain close to the N terminus but no cleavage
site. These integrins generally do not recognize RGD-containing
ligands. Finally, a third family, including only two
subunits,
9 and
4, contains neither an inserted
domain nor a disulfide-linked cleavage site (12).
We have previously identified the third fibronectin type III repeat in
the extracellular matrix protein tenascin
(TNfn3)1 as a ligand for the
only known
9-containing integrin,
9
1 (13). TNfn3 is also recognized as a
ligand by at least three other integrins (14, 15),
8
1 (16),
v
3
(17), and
v
6 (18). The crystal structure
of TNfn3 has been solved and consists of a series of seven
strands
separated by six exposed loops (5).
8
1,
v
3, and
v
6
all bind to the RGD tripeptide contained within the F-G loop. However,
unlike each of these integrins,
9
1 could
mediate adhesion to a fragment in which this site was mutated to RAA, and adhesion was unaffected by RGD peptides, suggesting that the ligand
binding site was distinct from this RGD site (13). In the present
study, we have used substitution mutagenesis and synthetic peptides to
identify this site in an adjacent exposed loop on the same face of
tenascin-C as the RGD loop.
 |
EXPERIMENTAL PROCEDURES |
Cell Lines, Antibodies, and Reagents--
Human colon cancer
cells (SW480) were stably transfected with either the expression
plasmids pcDNAIneo
9, pcDNAIneo
3, or the empty vector
pcDNAIneo as described previously (19). Cells were maintained in
Dulbecco's modified Eagle's medium (BioWhittaker, Walkersville, MD)
supplemented with 1 mg/ml neomycin analog, G418 (Life Technologies,
Inc.). Anti-
9 monoclonal antibody Y9A2 was generated and
characterized in our laboratory as described previously (20).
Monoclonal antibody 15/7 that recognizes a ligand
binding-dependent epitope on the integrin
1
subunit (21) was obtained from Ted Yednock (Athena Neurosciences, South
San Francisco). The pGEX plasmids to express glutathione
S-transferase fusion proteins including the wild type third
fibronectin type III repeat of chicken tenascin-C (TNfn3) or a mutant
version in which the RGD site within the F-G loop was mutated to RAA
(18) were obtained from Kathryn Crossin (The Scripps Research
Institute, La Jolla, CA). Concentrations of recombinant proteins were
determined by the Bradford assay (Pierce) using bovine serum albumin as
a standard. Peptides were synthesized using Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry on a peptide
synthesizer (Model 432A, Perkin-Elmer) at Center Laboratory for
Research and Education, Osaka University, followed by purification with
C18-reversed phase column chromatography.
Cell Adhesion Assay--
As described previously (22), wells of
non-tissue culture-treated polystyrene 96-well flat-bottomed microtiter
plates (Nunc Inc., Naperville, IL) were coated by incubation with 100 µl of recombinant wild type or mutant tenascin fragment in
phosphate-buffered saline at 37 °C for 1 h. For blocking
experiments, cells were incubated in the presence or absence of soluble
peptide or Y9A2 on ice for 15 min before plating on tenascin fragments.
Wells were washed with phosphate-buffered saline, then blocked with 1%
bovine serum albumin in Dulbecco's modified Eagle's medium. 50,000 cells were added to each well in 200 µl of serum-free Dulbecco's modified Eagle's medium containing 0.5% bovine serum albumin. Plates
were centrifuged at 10 × g for 1 min, then incubated
for 1 h at 37 °C in a humidified atmosphere with 5%
CO2. Non-adherent cells were removed by centrifugation top
side down at 48 × g for 5 min. The attached cells were
fixed with 1% formaldehyde, stained with 0.5% crystal violet, and
excess dye was washed off with phosphate-buffered saline. The cells
were solubilized in 50 µl of 2% Triton X-100 and quantified by
measuring the absorbance at 595 nm in a Microplate Reader
(Bio-Rad).
Mutagenesis--
Site-directed mutagenesis was performed with
the QuickChange site-directed mutagenesis kit (Stratagene, San Diego,
CA) as recommended by the manufacturer. Both strands of the expression plasmid were replicated by polymerase chain reaction using
Pfu DNA polymerase with two complementary primers designed
to introduce the desired mutation. The amplification product was
treated with DpnI endonuclease, specific for methylated DNA,
to digest the parental DNA template. Then XL-1 blue competent cells
were transformed with the polymerase chain reaction-generated nicked
plasmid. Plasmids from several isolated colonies were prepared by
Wizard mini-prep (Promega, Madison, WI), and inserts were sequenced by
ALFred autosequencer (Amersham Pharmacia Biotech, Uppsala, Sweden) with
fluorescent-tagged primers flanking the polylinker of the pGEX vector.
The verified mutated inserts were subcloned into pGEX vector that had
not been amplified by polymerase chain reaction.
Expression of Recombinant TNfn3 Fragments--
Glutathione
S-transferase fusion proteins containing either wild type or
variant TNfn3 were prepared by bacterial expression with the pGEX
plasmids as recommended by the manufacturer. Briefly, competent XL-1
blue cells were transformed by heat shock and grown on
ampicillin-containing plates. Individual colonies were picked and
propagated in 2 ml of 2× YT medium (16 g/liter tryptone, 10 g/liter
yeast extract, and 5 g/liter NaCl) with 100 µg/ml ampicillin at
37 °C overnight. 100 ml of 2× YT medium was inoculated with 1 ml of
the bacteria and incubated until A600 reached
0.5-2 at 30 °C, at which time
isopropyl-1-thio-
-galactopyranoside was added to a final
concentration of 0.1 mM. Cultures were grown for several
more hours, cells were collected and sonicated, and fusion proteins
were affinity-purified with glutathione-Sepharose 4B beads (Amersham
Pharmacia Biotech). Purity of the product was confirmed by 12.5%
SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue
staining.
Flow Cytometric Assessment of Peptide Ligation of
9
1--
Binding of authentic or
scrambled peptides to the ligand binding site of
9
1 was assessed by flow cytometry using
monoclonal antibody 15/7, which recognizes a ligand
binding-dependent epitope on the integrin
1
subunit. Mock- or
9-transfected SW480 cells were
incubated with the peptides VTDTTAL, AEIDGIEL, or with the scrambled
peptide GDLAIEEI in Dulbecco's modified Eagle's medium for 10 min at
4 °C. Cells were then incubated with antibody 15/7 at 15 µg/ml for
20 min at 4 °C at final peptide concentration of 100, 300, or 1,000 µM followed by incubation with secondary phycoerythrin-conjugated goat anti-mouse IgG. Induction of the epitope
recognized by 15/7 was then quantified on 5,000 cells with a Becton
Dickinson FACSort.
 |
RESULTS |
Attachment of
9 Transfectants to Mutant
TNfn3--
According to the published crystal structure of TNfn3 (5),
there are six extended loops separating seven
strands (Fig. 1). The previously characterized RGD
sequence is in the F-G loop. Since most integrin binding sites include
at least one acidic residue in an extended loop structure, we made a
series of alanine substitution mutations encompassing each acidic
residue present in or near a predicted loop, including the potential
loop at the N terminus of this repeat (Asp-775, Fig.
2). Fig. 2 shows the locations of each of
the substitution mutations described in this study. We could identify
acidic residues in or adjacent to five loops (all except the loop
between the E and F strands). For efficiency, we sometimes made
simultaneous mutations in more than one acidic residue.

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Fig. 1.
The crystal structure of the third
fibronectin type III repeat of tenascin-C (adapted from Ref. 5)
consisting of six extended loops separating seven strands. F-G
loop contains the RGD sequence. The recognition sequence AEIDGIEL for
9 1 identified in this study includes
portions of the B-C loop and the adjacent C strand.
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Fig. 2.
Alignment of tenascin amino acid sequences of
human, chicken, pig, and mouse tenascin-C. The large arrow ( )
indicates initially mutated series of acidic residues in each loop.
Aspartic acid 816, 821, 825 were simultaneously mutated. The remaining
3 mutants generated for further experiments are indicated by small
arrows ( ). Asterisks (*) indicate the RGD sequence. Two-headed
arrows under the sequence alignment indicate predicted positions
of strands.
|
|
We have previously reported that both
9- and
3-transfected SW480 cells adhere to wild type TNfn3,
whereas mock-transfected cells do not adhere (19). To determine the
effects of each mutation on
9
1-mediated
adhesion, we performed cell adhesion assays on plates coated with wild
type TNfn3 or with TNfn3 expressing mutations in one or more loop. To
determine the specificity of any decreases in adhesion of
9-transfected cells, we also performed adhesion assays
on the same fragments with
3-transfected SW480 cells. As
previously reported, both
9- and
3-transfected cells adhered to wild type TNfn3, but only
9-transfected cells adhered to TNfn3 in which the RGD
sequence in the F-G loop was mutated to RAA (Fig. 3). Attachment of
9-transfected cells to wild type TNfn3 was completely
blocked by anti-
9 monoclonal antibody Y9A2. Mutations in
acidic residues in the N terminus (D775A), the C-C' loop (D812A), or
the C-C' and C'-E loops (D816A/D821A/D825A) had no effect on adhesion
of either transfectant. However a mutation in the B-C loop (E802A)
specifically and markedly reduced attachment of
9-transfected SW480 cells (Fig. 3, panel A).
The only other mutation that decreased adhesion of
9-transfected cells was a mutation in the A-B loop (D787A), which reduced adhesion by approximately 40%. Mock-transfected SW480 cells did not adhere to any of the TNfn3 fragments used in this
study (data not shown).

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Fig. 3.
Attachment of 9- (panel
A) and 3- (panel B) transfected SW480
cells to wild type TNfn3 or to TNfn3 expressing specific substitution
mutations. Numbers of mutated residues are shown on the
right. Attachment is expressed as absorbance at 595 nm. Each
bar represents the mean (±S.D.) of triplicate wells from a
representative experiment.
|
|
To examine the role of specific acidic residues in or adjacent to
either the B-C or A-B loops, we generated fragments expressing each of
3 additional mutations. Mutations of the aspartic acid residue just
proximal to the A-B loop (D784A) or of the most proximal glutamic acid
residue in the B-C loop (E800A) had no effect on adhesion. However,
mutation of the next glutamic acid residue in the adjacent C
strand
(E805A) reduced adhesion of
9-transfected cells to the
same degree as E802A, either at concentrations of 0.3 µg or at 3 µg/ml, without effect on adhesion of
3-transfectants (Fig. 4).

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Fig. 4.
Attachment of 9- and
3- transfected SW480 cells to mutated TNfn3 (panel
A, 0.3 µg/ml; panel B, 3 µg/ml) in which an
acidic residue in or adjacent to the A-B or B-C loop is mutated to
alanine. Numbers of mutated residues are shown on the right.
Attachment is expressed as absorbance at 595 nm. Each bar
represents the mean (±S.D.) of triplicate wells from a representative
experiment.
|
|
Effects of Synthetic Peptides on Cell Adhesion to TNfn3--
In an
attempt to further determine whether sites in the A-B loop or the B-C
loop were critical for
9
1-mediated
adhesion to TNfn3, we synthesized linear peptides corresponding to the amino acid sequence of each of these loops of human TNfn3 and evaluated
the ability of these peptides to inhibit adhesion of either
9-transfected or
3-transfected SW480
cells to 3 µg/ml wild type TNfn3 (Fig.
5). We designed these peptides based on the human sequence to increase the likelihood that any effective peptide could serve as a starting point for drug design. The peptide Ala-Glu-Ile-Asp-Gly-Ile-Glu-Leu (AEIDGIEL) based on the B-C loop caused
concentration dependent inhibition of adhesion of
9
transfectants with no effect on adhesion of the
3-transfected control cells (Fig. 5). In contrast, the
peptide Val-Thr-Asp-Thr-Thr-Ala-Leu (VTDTTAL) corresponding to the A-B
loop was without effect. In addition, the scrambled peptide GDLAIEEI,
based on AEIDGIEL, had no effect on cell adhesion. These data further
suggest that the B-C loop is critical to ligand binding.

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Fig. 5.
Effects of synthetic peptides on the
attachment of 9- or 3-transfected SW480
cells to TNfn3. Cells were incubated with either the peptide
AEIDGIEL corresponding to the B-C loop region, the scrambled peptide
GDLAIEEI, the peptide VTDTTAL corresponding to A-B loop region, or
without peptide ( ) before plated onto wells coated with 3 µg/ml
TNfn3. Panel A shows attachment of
9-transfected cells in the presence of 3 different
concentrations of peptides (100, 300, or 1000 µM).
Panel B shows attachment of 3-transfected
cells in the presence of the highest concentration of peptides (1000 µM). Attachment is expressed as absorbance at 595 nm.
Each bar represents the mean (±S.D.) of triplicate wells
from a representative experiment.
|
|
Effects of Synthetic Peptides on Expression of a Ligand
Binding-dependent Epitope on the Integrin
1
Subunit--
As further confirmation of the role of the B-C loop
sequence in directly interacting with the
9
1 integrin, we examined the effects of
each peptide on expression of the epitope recognized by the monoclonal
antibody 15/7, an antibody that has previously been shown to be made
accessible following direct interaction of
1-integrins
with ligand (21). To determine whether any peptide-induced expression
of the 15/7 epitope was specifically due to interaction of peptides
with
9
1, and not to interaction with
other
1-integrins expressed on SW480 cells, we compared the effects
of each peptide on
9-transfected or mock-transfected
cells. The AEIDGIEL peptide, but not the VTDTTAL or scrambled peptides,
significantly induced the epitope recognized by antibody 15/7 on
9-transfected cells (Fig.
6). None of the peptides examined
affected 15/7 expression of mock-transfected cells, demonstrating that
induction of expression was due to specific interaction of the AEIDGIEL
peptide with
9
1.

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Fig. 6.
Effects of synthetic peptides on expression
of a ligand binding-dependent epitope on the integrin
1 subunit. Panel A, 9- or
mock-transfected SW480 cells were incubated with the synthetic peptides
shown at 1000 µM, and 15/7 expression was analyzed by
flow cytometry. The expression level of the ligand
binding-dependent epitope is compared in the absence of
peptides (open histogram) or in the presence of 1000 µM peptide (filled histogram). Panel
B, expression level of the ligand binding-dependent
epitope on 9-transfected SW480 cells (indicated by the
mean fluorescent intensity) is compared for peptide concentrations of
0, 100, 300, or 1000 µM. Panel C, expression
level of the ligand binding-dependent epitope on
mock-transfected SW480 cells.
|
|
 |
DISCUSSION |
Previous work identified tenascin-C as a ligand for the integrin
9
1 and localized the binding site to
TNfn3 (13). In the present study, we have used substitution mutagenesis
and synthetic peptides to map the ligand binding site within this
repeat in more detail. The dramatic reduction of adhesion of
9-transfected, but not
3-transfected
cells to mutant fragments in which either the second or third acidic
residue in the B-C loop region was replaced with alanine identify this
region as critical for ligand binding. The effect of a peptide based on
this region, AEIDGIEL, but not of a corresponding scrambled peptide in
inhibiting adhesion of
9-transfected cells to wild type
TNfn3, and specific induction of a ligand binding-dependent
epitope on the
1 subunit by the same peptide, provide
further evidence that this region includes the ligand binding site.
One other mutation, D787A, involving an acidic residue in the A-B loop
also decreased adhesion of
9-transfected SW480 cells. On
the basis of this result, we cannot exclude a role for this region in
binding to
9
1. However, several pieces of
evidence suggest that the A-B loop is unlikely to be the principal site of interaction with this integrin. First the degree of inhibition caused by this mutation was much less than that caused by mutations in
the B-C loop. This partial inhibition was not due to a contribution by
other adjacent acidic residues to
9
1
adhesion, since mutation of the only adjacent acidic residue had no
effect on adhesion. Furthermore, a peptide corresponding to this
region, VTDTTAL, had no effect on adhesion and did not induce the
ligand-dependent epitope on the
1 subunit.
It is thus likely that the D787A mutation inhibited adhesion by
altering the conformation of a critical region of the B-C loop on the
opposite face of TNfn3.
Several ligand binding sites for integrins have now been mapped within
fibronectin type III repeats and immunoglobulin domains (23-27). Most
of these sites have been mapped to short linear sequences involving
exposed peptide loops. In every case these sequences have included a
critical acidic residue. Many of these sites include the classic
integrin recognition motif, RGD, but binding to RGD sites is largely
restricted to integrins that contain the closely related
subunits
(
5,
8,
IIb, and
v) containing a disulfide-linked cleavage site. Based on
its amino acid sequence,
9 is most similar to
4 (28).
Recognition sites for the integrin
4
1 in
the ligands fibronectin and VCAM-1 have been extensively investigated.
In fibronectin, 3 sites have been identified: LDV (23) in the
alternatively spliced IIICS type III repeat, IDAP (24) in C-terminal
heparin binding domain, HepII, and KLDAPT (25) in the fifth type III repeat. In VCAM-1, the binding sequence for
4
1 is IDSP (26, 27). All of these
recognition sequences contain a homologous tripeptide composed of a
leucine or isoleucine followed by an acidic residue, followed by a
neutral amino acid with a small side chain. The sequence we identified
in this study, AEIDGIEL, contains a homologous tripeptide, IDG.
Recognition of the importance of this motif could facilitate the
identification of other integrin ligands.
The synthetic peptide used in this study, AEIDGIEL, specifically
inhibited
9
1-mediated cell adhesion. The
potency of this peptide was quite low, however, requiring a
concentration of 1000 µM to achieve approximately 50%
inhibition. This particular peptide is thus not likely to itself be
useful as a reagent to examine the functional significance of
9
1 in vivo. Nonetheless, since the crystal structure of TNfn3 has been solved, this peptide and the
known structure of the B-C loop could serve as starting points for the
development of drugs (8, 29) specifically targeting this integrin.
 |
ACKNOWLEDGEMENT |
We thank Dr. Kathryn L. Crossin (The Scripps
Research Institute) for the pGEX plasmid to express TNfn3 and TNfn3RAA,
and Dr. Ted Yednock (Athena Neuroscience) for supplying antibody
15/7.
 |
FOOTNOTES |
*
This work was supported in part by Research Grant for
Cardiovascular Diseases 8C-1, Grants-in-aid for Cancer Research 7-16 and 9-39 from the Ministry of Health and Welfare (to Y. Y.), by a
grant from the Tsuchiya Memorial Foundation for Medical Research (to
Y. Y.) and by National Institutes of Health Grant HL/AI33259 (to
D. S.).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: Dept. of Internal
Medicine, National Hiroshima Hospital, 513 Jike, Saijoh
HigashiHiroshima 739-0041, Japan. Tel.: 81-824-23-2176; Fax:
81-824-22-4675; E-mail: yokosaki{at}hirosima.hosp.go.jp.
1
The abbreviation used is: TNfn3, third
fibronectin type III repeat.
 |
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