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Blood, 1 January 2001, Vol. 97, No. 1, pp. 175-182
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Ligand binding to integrin v 3
requires tyrosine 178 in the v subunit
Shigenori Honda,
Yoshiaki Tomiyama,
Nisar Pampori,
Hirokazu Kashiwagi,
Teruo Kiyoi,
Satoru Kosugi,
Seiji Tadokoro,
Yoshiyuki Kurata,
Sanford J. Shattil, and
Yuji Matsuzawa
From the Department of Internal Medicine and Molecular
Science, Graduate School of Medicine, Osaka University, and the
Department of Blood Transfusion, Osaka University Hospital, Osaka,
Japan, and the Departments of Vascular Biology and Molecular and
Experimental Medicine, The Scripps Research Institute, La Jolla, CA.
 |
Abstract |
Integrin v 3 has been implicated
in angiogenesis and other biological processes. However, the
ligand-binding sites in v, a non-I-domain subunit,
remain to be identified. Recently in IIb, the other
partner of the 3 subunit, several discontinuous residues
important for ligand binding were identified in the predicted loops
between repeats 2 and 3 (W3 4-1 loop) and within repeat 3 (W3 2-3 loop). Based on these findings, alanine-scanning mutagenesis in
293 cells was used to investigate the role of these loops (cysteine [C]142-C155 and glycine [G]172-G181) of v in ligand
binding. Wild-type v 3 was able to bind
soluble fibrinogen following integrin activation either by 0.5 mM
manganese dichloride (MnCl2) or a mutation of
3 threonine (T)562 to asparagine. However, mutation of
tyrosine (Y)178 to alanine in the predicted G172-G181 loop of
v abolished fibrinogen binding, and alanine (A)
substitutions at adjacent residues phenylalanine (F)177 and tryptophan
(W)179 had a similar effect. Cells expressing Y178A v
also failed to bind to immobilized fibrinogen. Moreover, the Y178A
mutation abolished the binding of WOW-1 Fab, a monovalent
ligand-mimetic anti- v 3 antibody, and the
expression of 3 ligand-induced binding sites (LIBS)
induced by arginine-glycine-aspartic acid-tryptophan (RGDW). In sharp
contrast to the data obtained with IIb, none of the mutations in the predicted W3 4-1 loop in v impaired
ligand binding. These results implicate v Y178 in ligand
binding to v 3, and they suggest that
there are key structural differences in the adhesive ligand-binding
sites of v 3 and
IIb 3.
(Blood. 2001;97:175-182)
© 2001 by The American Society of Hematology.
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Introduction |
Integrins are a large family of 
heterodimeric adhesion receptors that is often subdivided into groups
based on 8 known integrin subunits.1 The
3-integrin subfamily is composed of
v 3, originally identified as the
vitronectin receptor, and IIb 3, a
platelet-specific receptor for fibrinogen and von Willebrand factor.
IIb 3 plays a crucial role in platelet
aggregation, normal hemostasis, and pathological thrombus
formation.2 On the other hand,
v 3 is expressed in a number of tissues,
including platelets, endothelial cells, vascular smooth muscle cells,
and osteoclasts, and it plays a key role in angiogenesis and bone
resorption.3,4
The IIb and v subunits are homologous and
36% identical in primary amino acid sequence,5 and most
ligands that bind to IIb 3, including
fibrinogen, von Willebrand factor, and vitronectin, also bind to
v 3. However, there are some distinctive
features between these 2 integrins.3 First, the
IIb subunit has been found only in combination with
3, whereas v can associate with at least
5 subunits ( 1, 3, 5,
6, and 8).6 Second, some ligands, such as osteopontin, matrix metalloproteinase-2, and adenovirus penton base, bind to v 3 but
not to IIb 3. Third, treatment of
IIb 3 with ethylenediamine tetraacetic
acid (EDTA) at 37°C dissociates the complex into its individual
subunits; v 3 remains a heterodimer.
Finally, the ligand-binding function of
v 3, but not
IIb 3, is suppressed by calcium
(Ca2+).7
Generally, all integrins require divalent cations for ligand
recognition, and multiple residues important for ligand binding have
been identified on both and subunits.8 The
N-terminal region of integrin subunits has 7 repeats of homologous
sequences of about 60 amino acid residues. Some integrin subunits
(eg, 2, L, and M) contain
an inserted domain of about 200 amino acids residues (the I-domain)
between the second and the third repeats in the subunit, which is
critically involved in ligand binding.9,10 The crystal
structure of the I-domain has been determined, and the metal
ion-dependent adhesion site (MIDAS) motif that contributes to cation
binding, as well as ligand binding, has been clarified.11
Interestingly, a MIDAS-like motif essential for the ligand-binding
function was also identified in integrin subunits.12,13 On the other hand, integrin subunits,
such as v, IIb, and 4, do
not have the I-domain.
The structural basis for the interaction between non-I-domain subunits and their ligands remains elusive. For the IIb
subunit, peptide cross-linking studies have shown that the
histamine-histamine-leucine-glycine-glycine-alanine-lysine-glutamine-alanine-glycine-aspartic acid-valine (HHLGGAKQAGDV) sequence derived from the COOH terminus of
the -chain of fibrinogen interacts with residues 294-314, encompassing the second putative calcium-binding domain of
IIb.14 However, recent characterization of
molecular defects in Glanzmann thrombasthenia and mutagenesis studies
demonstrated several discontinuous residues important for ligand
binding: proline (P)145, D163, L183, G184, tyrosine (Y)189, Y190,
phenylalanine (F)191, G193, and D224.15-19 Springer has
proposed that the 7 N-terminal sequence repeats of integrin subunits are folded into a -propeller domain.20 The
proposed domains contain seven 4-stranded -sheets (W1-W7) arranged
in a torus around a pseudosymmetry axis. Interestingly, the
discontinuous residues identified in IIb as important
for ligand binding were located in the regions predicted to adopt a
-turn structure on the upper face of the -propeller model: P145
within the W3 4-1 loop; L183, G184, Y189, Y190, F191, and G193 within
the predicted W3 2-3 loop; and D224 within the predicted W4 4-1 loop.
Furthermore, the analysis of a Japanese variant of Glanzmann
thrombasthenia, KO, and alanine-scanning mutagenesis have shown that
D163 in the W3 4-1 loop (cystein [C]146-C167) is essential for ligand
binding.16 In contrast, the single available peptide
cross-linking study of v 3 showed that 2 distinct linear regions in v, residues 139-167 and
312-349, cross-link to an arganine-glycine-aspartic acid (RGD)
peptide.21
In this study, we took advantage of the data regarding ligand-binding
sites in IIb and investigated the role in ligand binding of the predicted loops between repeats 2 and 3 (W3 4-1 loop) and within
repeat 3 (W3 2-3 loop) in v. By performing
alanine-scanning mutagenesis of recombinant
v 3 expressed in 293 cells, we demonstrate a critical role for Y178 within the predicted W3 2-3 loop of
v in ligand binding. In contrast to IIb,
however, no mutations in the W3 4-1 loop affect ligand binding to
v 3, thereby implying key structural
differences in the adhesive ligand-binding sites of the 2 3 integrins.
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Materials and methods |
Monoclonal antibodies and peptides
The following monoclonal antibodies (mAbs) were used in the
study: LM609,22 a murine mAb specific for
v 3, and LM142,23 a mAb
specific for v (gift from Dr David Cheresh, The
Scripps Research Institute, La Jolla, CA); AP5,24 a mAb
specific for 3 (gift from Dr Thomas Kunicki, The Scripps
Research Institute); anti-LIBS1 (anti-ligand-induced binding site 1)
mAb (specific for 3)25 and anti-LIBS6 mAb
(specific for 3)26 (Dr Mark Ginsberg, The
Scripps Research Institute); and AP3 (specific for 3)27 (Dr Peter Newman, The Blood Center of
Southeastern Wisconsin, Milwaukee, WI). WOW-1 Fab,28 a
monovalent ligand-mimetic mAb specific for activated
v 3, was created by replacing the heavy chain hypervariable region 3 (H-CDR3) of PAC-1 Fab, a ligand-mimetic mAb specific for activated
IIb 3,29 with a single
integrin-binding domain of adenovirus penton base. We also used the
RGDW peptide (gift from Dr Jiro Seki, Fujisawa Pharmaceutical Co.,
Osaka, Japan).30
Construction of v expression vectors and cell
transfection
Wild-type (WT) v complementary DNA (cDNA)
(gift from Dr David Cheresh, The Scripps Research Institute) and WT
3 cDNA (gift from Dr Gilbert White, University of North
Carolina, Chapel Hill, NC) were cloned into mammalian expression vector
pcDNA3 (Invitrogen Corp, San Diego, CA). To introduce single alanine
substitutions into v, overlap extension polymerase chain
reaction (PCR) was carried out as previously described.16
For example, to generate the Y178 A (Y178A) v mutant,
we synthesized mismatched sense primer v178A-s,
5'-GGTCCTGGTAGCTTTGCATGGCAAGGTCAGC-3' (sense, nucleotides
[nt] 648-678; mismatched sequences underlined) and antisense primer
v178A-as,
5'-GCTGACCTTGCCATGCAAAGCTACCAGGACC-3' (antisense, nt
678-648; mismatched sequences underlined), which were constructed based
on the published sequence.31 PCR was performed by
using v cDNA as a template and primers
v258-s, 5'-GCAAACACCACCCAGCC-3' (sense, nt 258-274) and
v178A-as, or primers v178A-s and
v952-as, 5'-GCAGCCATCTGCTCGCCAG-3' (antisense, nt
952-934).
The 2 individually amplified PCR products were mixed and used as a
template for PCR using primers v258-s and
v952-as. The amplified PCR products were digested
with PpuMI and AflIII. The fragments digested with AflIII and XbaI
were isolated from the full-length v cDNA cloned into
pcDNA3. These 2 fragments were introduced together into the pcDNA3 that
had been digested with PpuMI and XbaI. The
nucleotide sequences of the inserts were confirmed by sequence
analysis. In a selected experiment, the C142-C155 loop in
v was swapped with the corresponding sequence of
IIb C146-C167. The WT or mutant v
construct was cotransfected into 293 cells with WT 3
construct by the calcium-phosphate method as previously
described.32 The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated fetal calf
serum (FCS) and analyzed 2 days after transfection.
Immunoprecipitation
Immunoprecipitation was performed as previously described with
slight modification.33 In brief, cells were
surface-labeled with sulfo-NHS-biotin (Pierce, Rockford, IL) and lysed
in a buffer containing 1% Triton X-100, 25 mM Tris-HCl
(tris[hydroxymethyl] aminomethane-hydrochloride), 100 mM sodium
chloride (NaCl) (pH 7.4), 0.1 mg/mL leupeptin, 4 µg/mL pepstatin A, 1 mM phenylmethylsulfonyl fluoride, and 10 mM benzamide. Then 200 µg
protein from each sample was immunoprecipitated with the mAb LM609, and
precipitated bands were identified on Western blots using
peroxidase-conjugated avidin.
Ligand-binding studies
Fibrinogen (Kabi, Stockholm, Sweden) was labeled with
fluorescein isothiocyanate (FITC) as previously
described34 and stored at 80°C until use.
FITC-fibrinogen binding to 293 cells was assessed by flow cytometry as
described.33 Briefly, 50-µL aliquots of 1.5 × 105 washed cells in
Ca2+-free-Tyrode-HEPES
(4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid) buffer containing
1 mM magnesium dichloride (MgCl2) were incubated with mAb
LM142, specific for v (5 µg/mL), for 30 minutes on
ice. After washing, 0.5 mM manganese dichloride (MnCl2) was added into the cell suspension to induce a high-affinity state of
v 3. Cells were then incubated with 150 µg/mL FITC-fibrinogen in the presence or absence of 1 mM RGDW peptide
and phycoerythrin (PE)-conjugated antimouse immunoglobulin G (IgG) (1:5
dilution) for 25 minutes at 22°C and then incubated with propidium
iodine (PI) (Sigma Chemical Co., St Louis, MO) for 5 minutes at 22°C. After washing, fibrinogen binding (FL1) was analyzed on the gated subset of single high v 3-expressing (FL2)
live cells (PI-negative, FL3). Specific fibrinogen binding was defined
as that inhibited by 1 mM RGDW peptide.
For WOW-1 Fab binding to v 3, cells in
Ca2+-free-Tyrode-HEPES buffer containing 1 mM
MgCl2 were incubated with 5 µg/mL WOW-1 Fab in the
presence of 0.5 mM MnCl2 for 30 minutes at 22°C. After washing, cells were incubated with 5 µg/mL Alexa-conjugated goat antimouse IgG F(ab')2 (Molecular Probes, Eugene, OR) for 25 minutes on ice and then incubated with PI for 5 minutes at 22°C.
After washing, WOW-1 binding (FL1) was analyzed on the gated subset of
single, living cells. WOW-1 binding to untransfected 293 cells was
routinely taken as a measure of nonspecific binding because this value
was similar to that obtained for WOW-1 binding to
v 3 transfected cells in the presence of 1 mM RGDW.
For the induction of LIBS on v 3, washed
cells in Tyrode-HEPES buffer containing 1 mM MgCl2 and 1 mM
calcium dichloride (CaCl2) were incubated with 1 mM RGDW
for 30 minutes at 22°C. The cells were then incubated for 30 minutes
with AP5, anti-LIBS1, or anti-LIBS6 at a final concentration of 5 µg/mL. After washing, cells were incubated with FITC-conjugated goat
F(ab')2 antimouse IgG for 25 minutes. The mixtures were
incubated with PI for an additional 5 minutes and washed, and mAb
binding was analyzed on the gated subset of single, living cells.
Adhesion assays
Adhesion assays were performed as described by Faull et
al.35 Wells of 96-well microtiter plates were coated with
up to 1 µg fibrinogen per well in 100 µL phosphate-buffered saline
(PBS) and incubated at 4°C overnight. After washing with PBS, wells were blocked with PBS containing 1% bovine serum albumin (BSA) (Sigma)
for 90 minutes at 22°C. To determine background adhesion, control
wells were coated with 1% BSA. Cells were washed twice with PBS and
resuspended in DMEM containing 0.1% BSA at a concentration of
1 × 106 cells per mL. Then, 100-µL aliquots of cell
suspension were added to wells in triplicate. The plate was incubated
in a humidified 37°C incubator for 60 minutes. After washing with
PBS, the adherent cells were checked by visual inspection, and
adhesion was quantified by measuring endogenous cellular acid
phosphatase activity in an enzyme-linked immunosorbent assay.
 |
Results |
Expression of v 3 mutants in
293 cells
Recent studies have demonstrated that several residues within the
C146-C167 loop and the G184-G193 loop in the IIb
subunit, which correspond to the W3 4-1 loop and W3 2-3 loop in the
proposed -propeller model, respectively, play a critical role in
ligand binding. Figure 1 shows the
corresponding regions (C142-C155 and G172-G181) in the v
subunit and the residues we replaced with alanine. The WT or mutant
v construct was transiently cotransfected into 293 cells
with the WT 3 construct, and
v 3 surface expression was examined by
flow cytometry using the v 3
complex-specific mAb LM609. As shown in Figure
2A, the surface expression of mutant v 3 was 70% to approximately 123% of WT
v 3. Similar results were obtained when
mAb LM142 was used to quantify v and mAb AP3 was used to
quantify 3 (data not shown).

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| Figure 1.
Amino acid sequences of the predicted W3 4-1 loop
between the N-terminal repeats 2 and 3 and the predicted W3 2-3 loop
within repeat 3 in the subunits of 3 integrins.
Both loops are located on the upper face of the -propeller
model.20 The asterisks indicate that the residues were
substituted by alanine in this study. This figure is adapted from a
-propeller model proposed by Springer,20 and the arrows
indicate strands.
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| Figure 2.
Surface expression of v 3
mutants in transiently transfected 293 cells.
(A) The surface expression of transfected
v 3 was analyzed 2 days after transfection
by flow cytometry. Cells expressing WT or mutant
v 3 were incubated with 5 µg/mL
v 3 complex-specific mAb LM609 for 30 minutes on ice and then washed once. Bound antibodies were detected by
FITC-conjugated goat F(ab')2 antimouse IgG. Relative
amounts of the binding were normalized to a 100% value for LM609
binding to cells expressing WT v 3. The
results are representative of 3 separate experiments. (B)
Immunoprecipitation showing the surface expression of transfected
v 3. Transiently transfected cells were
surface-labeled with sulfo-NHS-biotin and lysed in the lysing buffer
containing 1% Triton X-100. WT or mutant
v 3 was precipitated with LM609 (specific
for the v 3 complex) and separated on a
6% sodium dodecyl sulfate polyacrylamide gel under reducing
conditions. After transfer, a membrane was incubated with
peroxidase-conjugated avidin and developed with chemiluminescence. The
results are representative of 3 separate experiments.
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Because 293 cells normally express v 1 but
not v 3,36 we were concerned
that endogenous v might associate with transfected 3 and contribute to the total
v 3 expressed on these cells. However, by
comparing v 3 transfectants to
3 transfectants, we found that endogenous
v contributed no more than approximately 17% to the
total v 3 expressed. Immunoprecipitation
experiments employing LM609 further showed that the expression of
endogenous v associated with transfected
3 in 293 cells is low, and that the surface expression
levels of mutant F177A, Y178A, and
W179A v 3 are comparable to those of WT
v 3 (Figure 2B). These results indicate
that transfected v, not endogenous v, was
the major contributor toward v 3
expression in 293 cells. Moreover, none of the alanine-scanning mutants
of v adversely affected surface expression of
v 3.
The Y178A mutation in v abolishes soluble ligand
binding to v 3
To analyze the ligand-binding function of each mutant
v 3, we initially examined the binding of
FITC-conjugated soluble fibrinogen to v 3.
Because v 3 expressed on 293 cells is
present in a low-affinity state and does not bind soluble ligands,
cells were incubated with 0.5 mM MnCl2, which induces a
high-affinity state of integrins by a direct effect on the
extracellular domain (Figure
3A).37 To avoid even a
minimal contribution of endogenous v in 293 cells to
fibrinogen binding, we selectively analyzed the subset of transfectants expressing high levels of exogenous v 3
monitored by LM142. As shown in Figure 3B, a D119Y mutation within the
ligand-binding site of 3 abolished fibrinogen binding to
v 3 as expected,38 confirming
the specificity of the ligand-binding assay in this system. Moreover, a
Y178A mutation within the predicted G172-G181 loop of v
abolished fibrinogen binding, and both F177A and W179A mutations
adjacent to the Y178 also moderately impaired binding. However, none of
the mutations within the C142-C155 loop of v (S144A,
Q145A, D146A, D148A, D150A, Q152A, and G153A) disturbed fibrinogen
binding to the receptor.

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| Figure 3.
Ligand-binding function of
v 3 mutants.
(A) The binding of soluble fibrinogen and a ligand-mimetic mAb, WOW-1
Fab, to WT or mutant v 3 were examined in
the presence or absence of 0.5 mM MnCl2 by flow cytometry.
For fibrinogen binding, washed cells were first incubated with 5 µg/mL mAb LM142 (specific for v) for 30 minutes on
ice. After washing, cells with 0.5 mM MnCl2 were incubated
with 150 µg/mL FITC-conjugated fibrinogen and PE-conjugated antimouse
IgG (1:5 dilution) for 25 minutes at 22°C and then incubated with PI
for 5 minutes at 22°C. After washing, fibrinogen binding (FL1) was
analyzed on the gated subset of single, high
v 3 expression (FL2) and live cells
(PI-negative, FL3) as indicated. (B) Relative amounts of fibrinogen
binding are normalized to a 100% value for the binding to cells
expressing WT v 3 (% of WT). Fibrinogen
binding in the presence of 1 mM RGDW was used as a negative control.
Data represent the mean ± SE of 3 experiments. (C) WOW-1 Fab
binding. For WOW-1 binding, cells with 0.5 mM MnCl2 were
first incubated with 5 µg/ml WOW-1 Fab for 30 minutes at 22°C.
After washing, cells were incubated with 5 µg/mL Alexa-conjugated
antimouse IgG for 25 minutes on ice and then incubated with PI for 5 minutes at 22°C. After washing, bound antibodies were analyzed. WOW-1
binding to 293 cells was used as a negative control. Relative amounts
of WOW-1 Fab binding are expressed by the following formula: % binding
of WOW-1 Fab to WT v 3/% binding of LM609
to WT v 3. The
v IIb mutant represents a chimera in which
the C142-C155 loop in v was swapped with the
corresponding sequence of IIb C146-C167. Data represent
the mean ± SE of 3 experiments.
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To further examine the ligand-binding function of mutant
v 3, we next examined the binding of WOW-1
Fab, a monovalent ligand-mimetic anti- v 3
antibody. The Y178A mutation in v, as well as the D119Y mutation in 3, markedly inhibited WOW-1 binding to
MnCl2-treated cells. On the other hand, swapping of the
C142-C155 region of v with the corresponding region of
IIb (C146-C167) did not show a marked inhibition of
WOW-1 Fab binding. Because WOW-1 Fab is sensitive to changes in
v 3 affinity rather than
avidity,28 these results suggest that the Y178A mutation
disrupts the conformation of the ligand-binding pocket in
v 3.
Because the integrin activator MnCl2 may have additional
effects on cells, we cotransfected the WT or the mutant
v with the T562N 3 mutant, which
constitutively activates 3 integrins and eliminates the
need for MnCl2.33 As shown in Figure
4, T562N 3 augmented
fibrinogen binding when complexed with the WT v, but it
failed to induce fibrinogen binding when complexed with the Y178A v. These results indicate that Y178 in the
v subunit is critical for soluble ligand binding to
v 3.

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| Figure 4.
Effects of 3-activating mutant (T562N) on
fibrinogen binding.
WT ( ) or Y178A v ( ) construct was transiently
cotransfected with WT or T562N 3 construct into 293 cells. Fibrinogen binding to transfected
v 3 was determined by flow cytometry.
Cells were first incubated with 5 µg/mL LM142 (specific for
v) for 30 minutes on ice. After washing, cells were
incubated with 150 µg/mL FITC-conjugated fibrinogen and PE-conjugated
antimouse IgG for 25 minutes at 22°C in the presence or absence of 1 mM RGDW and then incubated with PI for 5 minutes at 22°C. After
washing, cells expressing high levels of transfected
v 3 were analyzed. In these experiments,
all were performed in the absence of MnCl2. The results are
representative of 3 separate experiments.
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The Y178A mutation in v prevents the induction
of LIBS epitopes by RGDW peptide
To further clarify the effects of the Y178A mutation on the
structure of v 3, the reactivities of
several mAbs (AP5, LIBS1, and LIBS6) specific for LIBS on
3 were examined in the absence of ligands. These
LIBS epitopes are believed to be outside the ligand-binding pocket of
the receptor. As shown in Figure 5A, there was no apparent difference in the reactivities of these mAbs
between WT v 3 and
Y178A v 3, suggesting that this mutation does not grossly alter the conformation of
v 3. The binding of activation-independent
ligands, such as RGD peptides to v 3, has
been shown to induce LIBS expression on the receptor.39 Indeed, 1 mM RGDW increased the binding of all 3 LIBS mAbs to WT
v 3. However, it failed to induce LIBS
expression on the Y178A v 3 mutant. These
data suggest that the Y178A mutation in v disturbs the
binding of small and macromolecular RGD ligands to
v 3.

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| Figure 5.
LIBS expression on Y178A v 3
mutant.
WT or Y178A v construct was transiently cotransfected
with WT 3 construct into 293 cells. Three different mAbs
specific for 3 LIBS (AP5, anti-LIBS1, and anti-LIBS6)
were employed to assess the LIBS expression, and LM609 (specific for
v 3) was employed to monitor the surface
expression of transfected v 3. (A) LIBS
expression in the absence of RGDW peptide. Closed and open histograms
represent the binding of anti-LIBS antibodies and control mouse
IgG1, respectively. The results are representative of 2 separate experiments. (B) LIBS expression in the presence ( ) or
absence ( ) of 1 mM RGDW peptide. Closed and open histograms
represent the binding of anti-LIBS antibodies in the presence and
absence of RGDW, respectively. The results are representative of 2 separate experiments.
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The Y178A mutation of v inhibits cell adhesion
to immobilized fibrinogen
Because immobilized vitronectin and fibrinogen are
activation-independent ligands for v 3, we
further examined cell adhesion to immobilized ligands in the absence of
integrin activation. Parent 293 cells showed marked adhesion to
vitronectin probably via endogenous v 1
(data not shown),36 whereas they showed only modest
adhesion to fibrinogen even at a concentration of 10 µg/mL.
Therefore, we examined the effect of the
Y178A v 3 on the cell adhesion to
immobilized fibrinogen but not to vitronectin. Transfection of both WT
v and 3 markedly increased the adhesion of the transfectants; transfection of WT 3 alone induced
only a modest increase in adhesion at concentrations of 2.5 and 5 µg/mL (Figure 6). As compared
with the WT 3 transfectant,
Y178A v 3 failed to increase cell adhesion
to immobilized fibrinogen. However, the adhesion of F177A and
W179A v 3 mutants was only slightly impaired, especially at a relatively low concentration of fibrinogen (1.25 µg/mL). Thus, the effect of Y178A on binding of
v 3 to soluble fibrinogen is also observed
with immobilized fibrinogen.

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| Figure 6.
Adhesion of v 3 mutants to
immobilized fibrinogen.
WT or mutant v construct was transiently cotransfected
with WT 3 construct into 293 cells. In WT 3 cells
( ), only WT 3 construct was transfected into cells. WT ( ) or
mutant v 3-transfected cells were incubated for 60 minutes at
37°C with immobilized fibrinogen at serial concentrations. After
washing with PBS, the adherent cells were quantified with a
colorimetric reaction using endogenous cellular acid phosphatase
activity. , Phe177Ala; , Tyr178Ala; , Trp179Ala; ,
untransfected cells. Data represent the mean ± SE of triplicate
measures of optical density at 415 nm.
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|
 |
Discussion |
The aim of this study is to reveal the structural basis for the
interaction between v 3 and its ligands,
especially ligand-binding sites in the v subunit of
v 3. In non-I-domain integrin subunits, particularly the IIb subunit, several
ligand-binding sites have been identified by the characterization of
naturally occurring mutations in patients with Glanzmann thrombasthenia
as well as mutagenesis analyses.15-19 However,
ligand-binding regions of v remain elusive. To clarify
the critical regions for ligand binding in the v
subunit, we focused on the predicted W3 4-1 loop (C142-C155) and W3 2-3 loop (G172-G181) and investigated their role in ligand binding by
alanine-scanning mutagenesis. The results demonstrate that Y178 in the
W3 2-3 loop in v is one of the critical residues for
ligand binding. In sharp contrast to IIb, none of the
mutations in the predicted W3 4-1 loop impaired ligand binding, which
suggests that there are key structural differences in the adhesive
ligand-binding sites of v 3 and
IIb 3. The differences in the locations of the ligand-binding sites between v and
IIb in the -propeller model are summarized and
illustrated in Figure 7.

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| Figure 7.
Critical residues for ligand binding in the subunits
of 3 integrins.
(A) Comparison of critical residues for ligand binding in
v with those in IIb. In
IIb multiple residues (underlined) critical for ligand
binding have been identified in both the W3 4-1 and W3 2-3 loops. In
sharp contrast, in v only Y178 within the W3 2-3 loop is
critical for ligand binding. The figures in panels B and C are adapted
from a -propeller model proposed by Springer,20 and
they show the location of these critical residues. The view is shown
from the top (B) and from the side (C).
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There is mounting evidence that the predicted loops between N-terminal
repeats 2 and 3 (W3 4-1 loop) and within repeat 3 (W3 2-3 loop) are
important for ligand binding in non-I-domain
subunits.18,40-43 Previous studies have shown that the
N-terminal one-third of the subunit regulates ligand-recognition
specificity of 3 integrins44 and that
residues 139-167 in v corresponding to the W3 4-1 loop are a cross-linking site for RGD peptides.21 However, the
v Y178 identified in this study is located in the
predicted W3 2-3 loop, and none of the alanine substitutions within the
W3 4-1 loop examined impaired ligand binding. The inhibition of ligand binding by alanine substitutions at the residues adjacent to Y178 (F177
and W179) and the failure of the induction of 3 LIBS
with RGDW, even at high concentrations, provide further support for the
critical role of Y178 in ligand binding. Although the W3 4-1 loop and
W3 2-3 loop are separated in the primary structure, the proposed
propeller model suggests that these regions are close to each other in
3-dimensional space and may explain the apparent discrepancy between
the previous cross-linking study and the present study (Figure 7).
Interestingly, many critical residues identified in non-I-domain subunits have aromatic side chains, suggesting that in addition to
oxygenated residues such as D163 in
IIb,16 aromatic residues are
important for ligand binding.45 The corresponding residues
to the v Y178 in IIb
(Y190),18 4 (Y187),42,43 and
5 (F187),43 and the adjacent residues Y186
and W188 in 340 have been demonstrated to be
critical for ligand binding, although the role of W3 2-3 loop in
3 is still controversial.41 In contrast,
the role of W3 4-1 loop appears to be different between integrin subunits. D163 in IIb and threonine (T)162 and G163 in
3, which are located in the predicted W3 4-1 loop,
appear critical for ligand binding, whereas the W3 4-1 loop in
4 and in v (this study) do not. More
recently it has been demonstrated that the replacement of the W3 4-1 loop in v with the corresponding loop in
5 did not disturb ligand binding but changed
ligand-recognition specificity.46 Our swapping mutagenesis
of the W3 4-1 loop of v with the corresponding region of
IIb did not abolish WOW-1 Fab binding, suggesting that
the W3 4-1 loop is not critical for ligand-recognition specificity
between v 3 and
IIb 3.
WOW-1, a monovalent ligand-mimetic antibody, was created by replacing
the H-CDR3 of PAC-1 Fab with a single integrin-binding domain of the
adenovirus penton base, a viral coat protein that consists of 5 subunits, each containing an integrin-binding RGD motif. Penton base is
known to facilitate adenovirus internalization through v
integrins, particularly v 3 and
v 5.47 WOW-1-like penton
base recognizes that activated state of
v |