(Received for publication, April 22, 1997)
From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037
Integrins mediate signal transduction through
interaction with multiple cellular or extracellular matrix ligands.
Integrin v
3 recognizes fibrinogen, von Willebrand factor, and
vitronectin, while
v
1 does not. We studied the mechanisms for
defining ligand specificity of these integrins by swapping the highly
diverse sequences in the I domain-like structure of the
1 and
3
subunits. When the sequence CTSEQNC (residues 187-193) of
1 is
replaced with the corresponding CYDMKTTC sequence of
3, the ligand
specificity of
v
1 is altered. The mutant (
v
1-3-1), like
v
3, recognizes fibrinogen, von Willebrand factor, and vitronectin
(a gain-of-function effect). The
v
1-3-1 mutant is recruited to
focal contacts on fibrinogen and vitronectin, suggesting that the
mutant transduces intracellular signals on adhesion. The reciprocal
3-1-3 mutation blocks binding of
v
3 to these multiple ligands
and to LM609, a function-blocking anti-
v
3 antibody. These results
suggest that the highly divergent sequence is a key determinant of
integrin ligand specificity. Also, the data support a recent
hypothetical model of the I domain of
, in which the sequence is
located in the ligand binding site.
Integrins are a family of /
heterodimers of cell adhesion
receptors that mediate cell-extracellular matrix and cell-cell interactions (1-5). Integrin-ligand interactions are critically involved in the pathogenesis of many diseases in human and animal models. Although integrin-ligand interaction is a therapeutic target,
we poorly understand at the molecular level how integrins recognize
multiple ligands. Evidence suggests that the I or A domain, a set of
inserted sequences consisting of about 200 amino acid residues, of
several integrin
subunits (
M,
L,
1,
2) is important in
ligand binding and receptor activation (reviewed in Ref. 6 and
references therein). The presence of an I domain-like structure within
the
subunit has been suggested based on the similarity in
hydropathy profiles between the I domain and part of the
subunit
(7). Interestingly, this region of
has been reported to be critical
for ligand binding and its regulation (reviewed in Ref. 8) (Fig. 1).
The Asp-119 (
3) (9) and Asp-130 (
1) (10, 11) and the
corresponding residues in
2 and
6 are critical for ligand binding
(12, 13). A synthetic peptide of
3 (MDLSYSMKDDLWSI, residues
118-131) has been shown to produce a ternary complex with cations and
ligand (14). Also, the sequence DDLW (residues 126-129 of
3) was
shown to be critical for interaction with the RGD sequence using a
phage display system (15). A synthetic peptide of
3, DAPEGGFDAIMQATV
(residues 217-231 of
3), has been shown to bind to immobilized
fibrinogen (Fg),1 von
Willebrand's factor (vWf), and fibronectin (Fn) (16, 17). A synthetic
peptide of
3, SVSRNRDAPEG (residues 211-221 of
3), has been
reported to block binding of Fg to
IIb
3 (18, 19). We identified a
small region of
1 (residues 207-218, a regulatory epitope) that is
recognized by both activating and inhibiting anti-
1 antibodies (20).
These antibodies probably induce high or low affinity states,
respectively, by changing the conformation of the
1 subunit through
binding to the non-ligand binding site (20).
We and researchers at other laboratories have recently identified
residues critical for ligand binding in the putative I domain-like structure of 1 (6),
2 (21), and
3 (22). In
1, eight critical oxygenated residues are located in several separate predicted loop structures, which probably constitute multiple ligand/cation binding sites within the I domain-like structure of the
subunit. These critical oxygenic residues are conserved among integrin
subunits, indicating that these residues are ubiquitously involved in
ligand binding regardless of ligand and integrin species. We observed
that a large predicted loop region (residues 176-199 of
1) is
diverse among the
subunits (Fig. 1).
Furthermore, a recent structural model (23) and our preliminary model
(not shown) of the I domain-like structure of
suggest that the
sequence is also on the same side of the domain as residues critical
for ligand binding. We hypothesized that the predicted loop (especially the disulfide-linked short sequences, e.g. residues 187-193
of
1) is involved in ligand specificity of integrins.
v
3 has
been shown to recognize a wide variety of ligands, including Fn, Fg, vWf, and vitronectin (Vn);
v
1 is specific to Fn. We designed experiments, using
v
1 and
v
3 integrins, to determine
whether a diverse sequence in the predicted loop (e.g.
residues 176-199 in
1, residues 166-190 in
3) is involved in
ligand specificity of integrins.
mAb 4B4 (to human 1) (24) was kindly provided
by C. Morimoto (Dana-Farber Cancer Institute, Boston, MA); 8A2 (to
human
1) (25) by N. Kovach and J. Harlan (University of Washington, Seattle, WA); A1A5 (to human
1) (26) by M. Hemler (Dana-Farber Cancer Institute, Boston, MA); LM142 (to human
v), LM609 (to
v
3) (27), and P3G2 (to
v
5) (28) by D. Cheresh (Scripps); 15 (to human
3) (29) by M. H. Ginsberg (Scripps); and 15/7 (to
human
1) (30) by T. Yednock (Athena Neurosciences, San Francisco,
CA). P5D2 (to human
1) and polyclonal anti-
v cytoplasmic peptide
antibody were purchased from Chemicon (Temecula, CA).
A Fn 110-kDa fragment was prepared from bovine plasma Fn (Life Technologies, Inc.) as described (31). Bovine Fg was purchased from Daiichi Chemical (Tokyo, Japan). Purified vWf was provided by Z. Ruggeri (Scripps). Bovine Vn was purified according to Yatohgo et al. (32). Fg, Vn, and Fn 110-kDa fragment were coupled to CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer's instructions. The ligand concentration was 4.0, 1.2, and 2.3 mg/ml gel for Fg-, Vn-, and Fn 110-kDa fragment-Sepharose, respectively. The GRGDS peptide (6 mg/ml gel, Peptide Institute, Osaka, Japan) was coupled at the 6-carbon spacing arm of CH-Sepharose (Sigma) according to the manufacturer's instructions.
Transfection of Mammalian CellsHuman v and
3
cDNAs were provided by J. Loftus (Scripps). Ten µg of wild-type
human
v cDNA in pBJ-1 vector (33, 34) was transfected into
parental CHO-K1 cells (8 × 106 cells) together with 1 µg of pFneo plasmid containing a neomycin-resistant gene by
electroporation as described (35). After they were selected for G418
resistance, cells expressing
v were cloned by cell sorting in
FACStar cell sorter (Becton-Dickinson) with mAb LM142 (the cloned cells
are designated
v-CHO cells). Human
1 or
3 (WT/mutant) cDNA
in pBJ-1 vector was transfected into
v-CHO cells together with 1 µg of pCD-hygro plasmid with a hygromycin-resistant gene or into
parent CHO cells together with 1 µg of pFneo; cells were then
selected with hygromycin (500 µg/ml; Calbiochem) or G418 essentially
as described above. Cells expressing human
1 or
3 were cloned by
sorting with mAb A1A5 or 15 as described above. The flow cytometric
analysis was carried out using FACScan (Becton-Dickinson).
Wells of 96-well Immulon-2 microtiter plates (Dynatech Laboratories, Chantilly, VA) were coated with 100 µl of PBS (10 mM phosphate buffer, 0.15 M NaCl, pH 7.4) containing Fg, vWf, Fn, and Vn at a concentration of 10 µg/ml overnight at 4 °C. The remaining protein binding sites were blocked by incubating with 1% bovine serum albumin (Calbiochem) for 1 h at room temperature. Cells (105 cells/well) in 100 µl of Dulbecco's modified Eagle's medium containing 0.5 mg/ml bovine serum albumin were added to the wells and incubated at 37 °C for 1 h. After gently rinsing the wells three times with PBS to remove unbound cells, bound cells were quantified using endogenous phosphatase activity (36).
Affinity ChromatographyCells were harvested with 3.5 mM EDTA in PBS and washed with PBS. Cells (about 5 × 106) were then surface-labeled with 125I by using IODO-GEN (Pierce) (37), washed three times with PBS, and solubilized in 1 ml of 100 mM octyl glucoside in 10 mM Tris-HCl, 0.15 M NaCl, pH 7.4 (TBS), containing 2.5 mM MnCl2, 1 mM phenylmethylsulfonyl fluoride (Sigma) at 4 °C for 15 min. The insoluble materials were removed by centrifugation at 15,000 × g for 10 min. The supernatant was then incubated with a small amount of underivatized Sepharose 4B at 4 °C for 15 min to remove nonspecific binding material. The supernatant was incubated at 4 °C for 1 h with 200-500 µl of packed Fg-, Vn-, Fn 110-kDa fragment-, or GRGDS-Sepharose that had been equilibrated with TBS containing 2.5 mM MnCl2, 1 mM phenylmethylsulfonyl fluoride, 25 mM octyl glucoside (washing buffer). The unbound materials were washed with a 20 × column volume of washing buffer, and the bound materials were eluted with 20 mM EDTA instead of 1 mM MnCl2 in washing buffer; and then 0.5-ml fractions were collected. Twenty-µl aliquots from each fraction was analyzed by SDS-polyacrylamide gel electrophoresis using 7% polyacrylamide gel followed by autoradiography.
ImmunostainingGlass coverslips (Fisher) were treated with
10% KOH in methanol for 1 h at room temperature, washed three
times with distilled H2O, and stored in ethanol. Etched
coverslips were then coated with 50 µg/ml Fg, 50 µg/ml Fn, or 22 µg/ml Vn in PBS overnight at 4 °C and then blocked with 10 mg/ml
heat-denatured bovine serum albumin (Calbiochem) in PBS for 10 min at
room temperature. For plating experiments, cells were washed and then
detached with 2.5 mM EDTA/PBS. Detached cells were
isolated, washed, resuspended in Dulbecco's modified Eagle's medium,
and then replated on coated coverslips. Cells were allowed to attach
and spread for 2 h. Prior to fixation, cells were chilled on ice
for 5 min, washed with cold PBS, and then extracted with cold PIPES
buffer (0.1 M PIPES, pH 6.8, 1 mM
MgCl2, and 1 mM EGTA) containing 1% glycerol
and 0.5% Nonidet P-40 for 1-2 min. Extracted cells were washed with cold PIPES buffer and then fixed with 3.7% methanol-free formaldehyde (Polysciences) in PIPES buffer for 20 min at room temperature. Following fixation, cells were washed with PBS and then blocked with
10% normal goat serum (Life Technologies, Inc.)/PBS for 20 min at
37 °C. Human integrins were detected using either the anti-human 1 antibody P5D2 or the anti-human
3 antibody 15. Cells were immunostained for 1 h at 37 °C, washed, and then stained with a
fluorescein isothiocyanate-conjugated sheep anti-mouse IgG secondary antibody (Molecular Probes) for 30 min at 37 °C; cells were also labeled with rhodamine phalloidin (Molecular Probes) to detect actin
stress fibers. Stained cells were mounted in Fluoromount-G (Fisher) and
photographed using a Nikon Diaphot inverted microscope.
Site-directed mutagenesis of the 1 and
3 cDNA in a pBJ-1 vector was carried out using unique
restriction site elimination (38). The presence of mutations was
confirmed by DNA sequencing. Immunoprecipitation was carried out as
described previously (20).
To determine whether a diverse sequence in the
predicted loop (residues 176-199 in 1 and residues 166-190 in
3) is involved in ligand specificity of integrins, we replaced the
CTSEQNC sequence of
1 with the corresponding CYDMKTTC sequence of
3 by site-directed mutagenesis. The CYDMKTTC sequence of
3 has
been reported to be disulfide-linked (39). The resulting mutant
1-3-1, wild-type
1, or wild-type
3 cDNA constructs were
transfected into either parental CHO cells or CHO cells expressing
wild-type human
v (
v-CHO). Parent CHO cells have been reported to
express endogenous hamster
v (40) but not
3 (41). Consistent with
these findings, we found that CHO cells express
v
5 using mAb P3G2
(data not shown). The cloned cells expressing WT or mutant
1 in
association with exogenous human
v are designated
v
1-,
v
1-3-1-,
v
3-CHO cells, and those with only endogenous
hamster
v are designated as
1-,
1-3-1-,
3-CHO
cells.
v
3 recognizes multiple ligands, including Fn, Fg, vWf, and Vn;
v
1 is specific to Fn on CHO cells. Therefore, we tested the
ligand specificity of the
1-3-1 mutant. As shown in Fig. 2A, we found that cells
expressing
v
3 or
v
1-3-1, but not
v
1, adhered to both
Fg and vWf. Adhesion of the
v
1-3-1- but not
v
3-CHO cells
was blocked by the inhibitory anti-human
1 mAb 4B4 (Fig.
2B), indicating that adhesion of the
v
1-3-1-expressing cells to Fg and vWf is mediated by human
1
sequences. Similar results were obtained with the
1-,
3-, and
1-3-1-CHO cells (Fig. 2, A and B). These
results suggest that the region spanning residues 187-193 of
1 or
177-184 of
3 is involved in the regulation of ligand
specificity.
Binding Specificity of
The specificity of the
interaction between v
1-3-1 and ligands was further analyzed by
affinity chromatography. Lysates from surface 125I-labeled
v
1-3-1-CHO cells (as well as control
v
1- and
v
3-CHO cells) were incubated with immobilized Fg or Fn 110-kDa fragments, and
bound materials were eluted with EDTA. As shown in Fig.
3A, bands corresponding to
v and
1 in size were eluted from Fg-Sepharose using a lysate of
v
1-3-1-CHO cells, while bands corresponding to human
v and
3 were eluted from Fg-Sepharose with a lysate of
v
3-CHO cells.
Immunoprecipitation of the eluate from
v
1-3-1 cells using
anti-
1 mAb A1A5 (Fig. 3C, lane 5) and
anti-
v mAb LM142 (Fig. 3D, lane 5) confirmed
that these two bands are human
v and
1 (
1-3-1). In contrast,
very low levels of
v and
1 were detected in the Fg-Sepharose
eluate with lysate of
v
1-CHO cells. These results suggest that
v
1-3-1 exhibits a much higher affinity for Fg than
v
1.
Similar results were obtained with Vn-Sepharose (data not shown),
suggesting that
v
1-3-1 shows a much higher affinity to Vn as
well. In experiments done in parallel, we have detected bands
corresponding to
v
1,
v
1-3-1, and
v
3 in the eluate
from Fn 110-kDa fragment-Sepharose with lysates from
v
1-,
v
1-3-1, and
v
3-CHO cells, respectively (Fig.
3B). Immunoprecipitation confirmed that the major
subunits in the eluates are
1,
1-3-1, and
3, respectively
(Fig. 3C). These results suggest that the
v
1-3-1
mutant, like
v
3, binds to Fg, Vn, and Fn 110-kDa fragments in a
solubilized form.
The
Next we determined if
the altered ligand specificity of the v
1-3-1 chimera affected
intracellular signaling. Cells were plated on Fn, Vn, or Fg, and
localization of the human integrin was determined by immunostaining
with anti-human
1 (
v
1 and
v
1-3-1) or anti-human
3
(
v
3). While all three receptors localized to focal adhesions in
cells plated on Fn (Fig. 4, A,
C, and F), only
1-3-1 and
3 localized to
focal adhesions in cells on Vn;
v
1-CHO cells did attach and
spread on Vn due to endogenous
v
5. However,
v
1 exhibited a
diffuse staining pattern. This result is consistent with the binding
data and indicates that the
v
1-3-1 chimera is able to generate
intracellular signals. In addition, we found that the
v
1-3-1
chimera, like
v
3, induced cell spreading and focal adhesion
formation in cells plated on Fg;
v
1 cells did not adhere to Fg.
Similar results were obtained with the
1-,
1-3-1-, and
3-CHO
cells that express lower levels of the transfected integrins (data not
shown). These results indicate that the
v
1-3-1 chimera is a
functional receptor and has the same signaling properties as
v
3.
The Reciprocal
To determine whether the reciprocal swapping
mutation has any effect on the ligand specificity of v
3, we
replaced the CYDMKTTC sequence of
3 (residues 177-184) with the
corresponding CTSEQNC sequence of
1 (the
3-1-3 mutation). The
resulting mutant
3-1-3 and WT
3 cDNA constructs were
transfected into
v-CHO cells, and cells stably expressing
v
3-1-3 or
v
3 were cloned by sorting (
v
3-1-3-CHO and
v
3-CHO cells, respectively). The levels of
v and
3
expression were comparable in clonal WT
v
3-CHO and
v
3-1-3-CHO cells used.
v
3-1-3-CHO cells showed
significantly lower adhesion activity than
v
3-CHO cells to both
Fg and vWf;
v
3-1-3-CHO cells required higher ligand
concentrations for adhesion than WT
v
3-CHO cells (Fig.
5, A and B). In
addition, solubilized
v
3-1-3 did not bind to either Fg or Vn
immobilized to Sepharose, although solubilized WT
v
3 did (Fig.
5C). These results suggest that the
3-1-3 mutation
significantly reduces binding of
v
3 to Fg, Vn, and vWf. Although
we observed that
v
3-1-3 mutant binds to Fn 110-kDa fragments and
to the GRGDS peptide on affinity chromatography (data not shown), we
could not determine whether the
3-1-3 mutation changes the binding
affinity of
v
3 to Fn 110-kDa fragment or the GRGDSP peptide
(because of the presence of other fibronectin receptors, endogenous
v
1 and
5
1).
The immunoprecipitation of whole lysate of v
3-1-3-CHO cells
using anti-
v and anti-
3 mAbs showed that anti-
v and anti-
3 co-precipitated
3-1-3 and
v subunits, respectively, suggesting that the
3-1-3 mutation does not affect the
-
association. However, the
v
3-1-3 mutant did not react with LM609, a
function-blocking anti-
v
3 mAb, upon immunoprecipitation (Fig.
6) and flow cytometric analysis (data not
shown), suggesting that the
3-1-3 mutation destroyed the LM609
epitope and that the CYDMKTTC sequence of
3 is closely located to
ligand binding sites of
v
3.
We established that swapping the CTSEQNC sequence of 1 with the
corresponding CYDMKTTC sequence of
3 induces significant changes in
ligand specificity of
v
1. The
1-3-1 mutation markedly increases affinity of
v
1 to Fg, vWf, and Vn (a gain-of-function effect). Since the
v
1-3-1 mutant is functional in cultured cells and transduces signals on adhesion to the ligands, the swapping did not
induce a detectable adverse effect on the other receptor functions
(e.g.
-
association and signal transduction). In
reciprocal experiments, swapping a disulfide-linked CYDMKTTC sequence
of
3 with the corresponding CTSEQNC sequence of
1 blocks the
binding function of
v
3 to Fg, vWf, and Vn. Taken together, the
present study suggests that a small disulfide-linked CYMKTTC sequence of
3 (and the CTSEQNC sequence of
1 as well) defines a novel site
of integrin
critical for ligand specificity. Sequence diversity among
subunits and localization within an I domain-like structure of
, close to putative ligand binding sites (see Introduction) is
consistent with the proposed function of the sequence. In a preliminary
study, we introduced mutations into the corresponding predicted loop of
the
2 subunit. We found that these mutations showed profound effects
on the ligand binding function of
L
2 integrin,2 indicating that
the diverse predicted loops of the
subunits are ubiquitously
involved in the regulation of ligand binding functions.
Mechanisms by which the disulfide-linked sequences in a predicted loop
within the I domain-like structure of the subunits define ligand
specificity of integrins have yet to be studied. In preliminary
studies, we did not obtain evidence that the
1-3-1 mutation induces
constitutive activation of
1 integrins or induces drastic
conformational changes. We determined the reactivity of the
1-3-1
mutant to an activation-dependent anti-
1 mAb 15/7, which
recognizes the highly activated form of
1 integrin (30). The binding
profiles of 15/7 to the
1-3-1 mutant and wild-type
1 were
identical; binding of 15/7 was dependent on activation in both cases
(data not shown). The epitope for 15/7 has been localized within the
residues 354-425 of
1 (in the non-ligand binding region outside the
I domain-like region) (42). Therefore, there is a possibility that the
effect of the
1-3-1 mutation on conformation remains local
(e.g. within the I domain-like structure of
1) and 15/7
does not detect it. The amino acid residues surrounding the tripeptide
RGD of the ligands have been reported to be critical for receptor
specificity of snake venom disintegrins (43-45). One possible
mechanism is that the predicted loop structures of
3 or
1
interact with the residues surrounding the tripeptide RGD of ligands,
if we assume that the predicted loop structure of
is close to the
ligand binding site of
v
3 or
v
1. Another possibility is
that the predicted loops regulate the access of a group of ligands (in
the case of
v
3, Fg, Vn, and vWf) to the ligand binding site.
The CTSEQNC sequence of 1 (or the CYDMKTTC sequence of
3) is
located within a predicted
-turn in the putative I domain-like structure of the
subunit (6). A recent model of the
I
domain-like structure (23), the folding diagram, appears to be
consistent with our previous and present mutagenesis data (Fig.
7), and this model is similar to our
preliminary model (not shown). All of the residues critical for ligand
binding (e.g. Asp-130 and Glu-229 of
1) (6, 10) are
located in the upper face of the model (predicted as the ligand binding
site). Also, the regulatory epitope (residues 207-218 of
1) (20),
which is recognized by both activating and inhibiting anti-
1 mAbs,
is located in the non-ligand binding site (in the lower face) of the
domain. Interestingly, a diverse sequence in the predicted loop
(e.g. residues 176-199 in
1, residues 166-190 in
3),
which is involved in ligand specificity of integrins in the present
study, is located in the upper face of the domain in this model. The
finding that the
3-1-3 mutation blocked binding of the
function-blocking anti-
v
3 antibody LM609 supports the idea that
the predicted loop structure is close to the ligand binding site of
v
3. Taken together, the present and previous mutagenesis data
strongly support this model. Recently, Collins Tozer et al.
(22) published an interesting atomic model of the putative I domain of
3, which is based on the crystal structure of the
M I domain (7).
However, our mutagenesis data do not fit in very well with their model,
since 1) the sequence CYDMKTTC of
3, which is critically involved in
ligand binding to
v
3, is not close to the MIDAS site (apparently
in a non-ligand binding site) in their model, and 2) although Thr-197
of
3 is located in the MIDAS site of
3 in this model, the
corresponding residue of
1 (Thr-206) is very close to the regulatory
epitope. This epitope is probably located in a non-ligand binding site
of
1 because 1) binding of some mAbs actually activates, instead of inactivating, the
1 integrins, and 2) this epitope has recently been
shown to be an allosteric effector site of
1 (46), since the binding
of an inhibitory anti-
1 mAb 13 to the regulatory epitope is also
dramatically attenuated by ligands (Fn fragments or the GRGDS peptide).
Further biochemical and structural characterization of this region of
the
subunit may be required to substantiate these models.
v
3 has been shown to be involved in the progression of melanoma
and induction of neovascularization by tumor cells.
v
3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels (47, 48). We identified a critical region for
ligand binding and specificity of integrins using a gain of function
mutant of the
subunit. The predicted loop sequence of the
integrin
3 subunit is a new potential target for designing inhibitors of ligand binding functions of
v
3.
This is publication 10153-VB from The Scripps Research Institute.
We thank Drs. D. Cheresh, M. H. Ginsberg, M. E. Hemler, R. L. Juliano, J. Loftus, C. Morimoto, and Z. Ruggeri for valuable reagents.