(Received for publication, March 24, 1997, and in revised form, May 1, 1997)
From the Wellcome Trust Centre for Cell-Matrix Research, School of
Biological Sciences, University of Manchester, Manchester M13 9PT,
United Kingdom, the § Craniofacial Developmental Biology and
Regeneration Branch, NIDR, National Institutes of Health, Bethesda,
Maryland 20892, the ¶ Department of Vascular Biology, Scripps
Research Institute, La Jolla, California 92037, and the
Nuffield Department of Obstetrics and Gynaecology, John
Radcliffe Hospital, Headington, Oxford, OX3 9DU, United Kingdom
The high affinity interaction of integrin
5
1 with the central cell binding domain (CCBD) of fibronectin
requires both the Arg-Gly-Asp (RGD) sequence (in the 10th type III
repeat) and a second site (in the adjacent 9th type III repeat) which
synergizes with RGD. We have attempted to map the fibronectin binding
interface on
5
1 using monoclonal antibodies (mAbs) that inhibit
ligand recognition. The binding of two anti-
5 mAbs (P1D6 and JBS5)
to
5
1 was strongly inhibited by a tryptic CCBD fragment of
fibronectin (containing both synergy sequence and RGD) but not by GRGDS
peptide. Using recombinant wild type and mutated fragments of the CCBD, we show that the synergy region of the 9th type III repeat is involved
in blocking the binding of P1D6 and JBS5 to
5
1. In contrast,
binding of the anti-
1 mAb P4C10 to
5
1 was inhibited to a
similar extent by GRGDS peptide, the tryptic CCBD fragment, or
recombinant proteins lacking the synergy region, indicating that the
RGD sequence is involved in blocking P4C10 binding. P1D6 inhibited the
interaction of a wild type CCBD fragment with
5
1 but had no
effect on the binding of a mutant fragment that lacked the synergy
region. The epitopes of P1D6 and JBS5 mapped to the NH2-terminal repeats of the
5 subunit. Our results
indicate that the synergy region is recognized primarily by the
5
subunit (in particular by its NH2-terminal repeats) but
that the
1 subunit plays the major role in binding of the RGD
sequence. These findings provide new insights into the mechanisms,
specificity, and topology of integrin-ligand interactions.
Integrins are a large family of heterodimeric receptors,
many of which play critical roles in cell migration, differentiation, and survival. Integrins recognize a large variety of extracellular and
cell surface proteins, and two widespread recognition motifs have been
identified within these ligands: Arg-Gly-Asp (RGD) and Leu-Asp-Val
(LDV) (1, 2).
The extracellular matrix glycoprotein fibronectin contains a number of
domains that mediate its interaction with integrins; the most studied
of these domains lies near the center of the fibronectin subunit and is
known as the central cell binding domain (CCBD).1 This region contains a number of
homologous repeating polypeptide modules termed fibronectin type III
repeats, each about 90 amino acid residues in length. An RGD sequence,
located in the 10th type III repeat, is a key recognition site for
several different integrins, including the prototypic fibronectin
receptor 5
1. Synthetic peptides containing the RGD sequence
completely block cell adhesion to the CCBD (3, 4). However, other data
show that additional sequences outside the RGD region are sometimes required for full adhesive activity. In particular, a region of the 9th
type III repeat which contains the sequence Pro-His-Ser-Arg-Asn (PHSRN)
appears to synergize with RGD for recognition by
5
1 (5).
Replacement of this sequence with a corresponding sequence from the 8th
type III repeat results in a major loss of adhesive activity (5).
Similarly, deletion of this region from the CCBD results in ~20-fold
loss in activity (6). An overlapping, but distinct, sequence plays a
similar role in
IIb
3-fibronectin interactions (7). The mode of
action of such synergy sequences is not currently understood.
Recently, we described the effect of GRGDS peptide and a tryptic
fragment of fibronectin containing the CCBD on the binding of the
inhibitory monoclonal antibody (mAb) 13 to 5
1 (8). Our results
showed that both GRGDS peptide and the CCBD fragment act as allosteric
inhibitors of mAb 13 binding to
5
1. Our data indicated that mAb
13 recognizes an epitope that is strongly attenuated by ligand
occupancy rather than a site directly involved in ligand recognition.
We hypothesized that inhibitory mAbs recognize sites attenuated during
the conformational adaptation of the integrin to ligand and have termed
these ligand-attenuated binding sites (8, 9). Although mAb 13 does not
appear to recognize a sequence that interacts directly with ligand, its
epitope lies very close to regions of the
1 subunit which have been
shown to participate in ligand binding (10, 11).
A region of the 5 subunit which plays an important role in
fibronectin recognition has been identified by site-directed
mutagenesis (12). The NH2-terminal portion of the
5
subunit comprises seven homologous repeats, each of ~60 amino acid
residues. Point mutations in a short hydrophobic sequence in the third
NH2-terminal repeat block fibronectin binding. The epitope
recognized by the inhibitory anti-
5 mAb 16 was found to lie near to
this hydrophobic sequence (12). By analogy with our results for mAb 13, these findings suggested to us that fibronectin recognition by
5
1
may affect the binding of inhibitory anti-
5 mAbs and furthermore,
that it might be possible to use inhibitory mAbs as probes to determine whether the
5 and
1 subunits recognize distinct regions of the CCBD.
In this study we tested whether recognition of the CCBD by 5
1
perturbs the binding of inhibitory anti-
5 mAbs and if this perturbation is different from that observed for inhibitory anti-
1 mAbs. We show that the binding of the anti-
5 mAbs P1D6 and JBS5 to
5
1 is inhibited by fibronectin fragments containing both the RGD
sequence and the synergy site but is not inhibited by GRGDS peptide or
by fibronectin fragments lacking the synergy sequence. In contrast, the
binding of the anti-
1 mAb P4C10 is inhibited by GRGDS peptide and
fibronectin fragments containing the RGD sequence alone. We also
present evidence that P1D6 blocks recognition of only the synergy site
by
5
1. Taken together, our data suggest that the
5 subunit is
largely responsible for recognition of the synergy site, whereas the
RGD sequence appears to be recognized mainly by
1 subunit. Based on
an x-ray crystal structure for the CCBD (13), these results allow us to
predict a spatial topology for
5
1-fibronectin interactions.
Rat mAbs 11 and 13 recognizing the human 5 and
1 integrin subunits, respectively, were produced and purified as
described previously (14). Rat anti-mouse
1 mAb 9EG7 was a gift from D. Vestweber (Freiburg, Germany); this mAb also cross-reacts with human
1. Mouse anti-human
1 mAbs P4C10, 4B4, and K20 were purchased from Life Technologies, Inc. (Paisley, Scotland, U. K.), Coulter Corp.
(Miami, FL), and The Binding Site (Birmingham, U. K.), respectively. Mouse anti-human
5 mAbs VC5, P1D6, and JBS5 were purchased from Pharmingen (San Diego, CA), Life Technologies, Inc., and Serotec (Oxford, U. K.), respectively. All antibodies were used as
purified IgG, except P1D6 and P4C10 (as ascites). A recombinant
fragment of fibronectin, H/120, which does not contain the CCBD, was
produced and purified as described (15). The synthetic peptides GRGDS and GRDGS were synthesized using Fastmoc chemistry on an Applied Biosystems 431A peptide synthesizer and purified as outlined
previously (16).
Integrin 5
1 was purified from human placenta
as described previously (8). An 80-kDa fragment of fibronectin
containing the CCBD was purified from a trypsin digest of human plasma
fibronectin as described (17). Recombinant fragments of the CCBD were
produced as before (5, 18, 19). All recombinant proteins, except proteins comprising the 9th or 10th type III repeats, were purified using DEAE-Sephacel (Pharmacia, Milton Keynes, U. K.) and
hydroxylapatite (Bio-Rad, Hemel Hempsted, U. K.) chromatography,
essentially as described previously (19). Recombinant proteins
comprising the individual 9th and 10th repeats were purified as before
(18). All purified proteins solutions were dialyzed against several changes of 150 mM NaCl, 25 mM Tris-Cl, pH 7.4 (buffer A).
Tryptic CCBD fragment of fibronectin or recombinant CCBD fragments (~500 µg/ml in buffer A) were mixed with an equal mass of sulfo-N-hydroxysuccinimido biotin (Pierce, Chester, U. K.) and rotary mixed for 30-40 min at room temperature. The mixture was then dialyzed against several changes of buffer A to remove excess biotin. Dialyzed protein solutions were centrifuged at 13,000 × g for 15 min and stored at 4 °C.
Effect of Ligand on the Binding of mAbs P1D6, JBS5, and P4C10 toPurified 5
1 (at a concentration of ~500 µg/ml)
was diluted 1:500 with phosphate-buffered saline containing 1 mM Ca2+ and 0.5 mM
Mg2+, and 100-µl aliquots were added to the wells of a
96-well ELISA plate (Dynatech Immulon 3). Plates were incubated
overnight at room temperature, and wells were blocked for 1-3 h with
200 µl of 5% (w/v) BSA, 150 mM NaCl, 0.05% (w/v)
NaN3, 25 mM Tris-Cl, pH 7.4. Wells were then
washed three times with 200 µl of 150 mM NaCl, 1 mM MnCl2, 25 mM Tris-Cl, pH 7.4, containing 1 mg/ml BSA (buffer B). 100-µl aliquots of mAbs (0.3 µg/ml or 1:10,000 dilution of ascites in buffer B) were added to the
wells in the presence or absence of varying concentrations of tryptic
CCBD fragment or GRGDS peptide. The H/120 fragment of fibronectin and the peptide GRDGS were used as controls. The plate was then incubated at 30 °C for 2 h. Unbound antibody was aspirated, and the wells were washed three times with buffer B. Bound antibody was quantitated by addition of 1:1,000 anti-mouse peroxidase conjugate (Dako A/C, Denmark) in buffer B for 20 min. Wells were then washed four times with
buffer B, and color was developed using ABTS substrate (Sigma, Poole,
U. K.). Measurements obtained were the mean ± S.D. of four replicate wells.
To test if ligand behaved as a direct competitive inhibitor or an allosteric inhibitor of mAb binding, the inhibition of mAb binding at different concentrations of tryptic CCBD fragment was measured as described above for 10-fold different concentrations of mAb (0.3 and 3 µg/ml or 1:10,000 and 1:1,000 dilution of ascites). The concentration of ligand required to inhibit antibody binding half-maximally and the maximal extent of inhibition were estimated by nonlinear regression analysis as described previously (8).
Solid Phase AssaysSolid phase ligand-receptor binding was
performed essentially as described previously (20). ELISA plate wells
were coated with 5
1, blocked as described above, and washed three
times with 200 µl of buffer B. In experiments examining the ability of recombinant proteins and GRGDS peptide to inhibit competitively the
binding of the tryptic CCBD fragment to
5
1, 100-µl aliquots of
biotinylated tryptic CCBD fragment (0.1 µg/ml) in buffer B were added
to the wells, with or without varying concentrations of competing
peptide or proteins. The plate was then incubated at 30 °C for
3 h. Biotinylated ligand was aspirated, and the wells were washed
three times with buffer B. Bound ligand was quantitated by the addition
of 1:200 ExtrAvidin-peroxidase conjugate (Sigma) in buffer B for 10 min. Wells were then washed four times with buffer B, and color was
developed using ABTS. Measurements obtained were the mean ± S.D.
of four replicate wells. To estimate the concentrations of peptide or
proteins required for half-maximal inhibition of CCBD fragment binding,
nonlinear regression analysis of the data was performed as described
previously (15).
In experiments examining the direct binding of biotinylated recombinant
CCBD fragments to 5
1, ELISA plate wells were coated with
5
1, blocked, and washed as described above. 100-µl aliquots of
biotinylated proteins (0.05-30 µg/ml) diluted in buffer B were added
to the wells, and the plate was then incubated at 30 °C for 3 h. Bound ligand was then quantitated as described above. To estimate
apparent KD values, nonlinear regression analysis of
the data was performed (15). For experiments examining the effect of
anti-
5 and anti-
1 mAbs on the binding of biotinylated recombinant
CCBD fragments to
5
1, assays were performed in the presence of 10 µg/ml purified mAbs or 1:500 dilution of ascites.
In all of the assays described above, the amount of nonspecific binding was measured by determining the level of antibody or ligand binding to wells coated with BSA alone; these values were subtracted from the corresponding values for receptor-coated wells. Each experiment shown is representative of at least three separate experiments. Under the conditions used in these assays, the ELISA signal appeared to be directly proportional to the amount of bound antibody or ligand because plots of 1/absorbance versus 1/(free antibody or ligand) did not deviate from linearity at high concentrations. Note, however, that binding constants cannot be determined precisely in these assays and that only apparent KD values are quoted.
Flow Cytometric AnalysisK562 erythroleukemia cells were
grown in RPMI 1640 medium containing 10% (v/v) fetal calf serum, as
described previously (24). Cells were washed with 150 mM
NaCl, 25 mM HEPES, pH 7.4, incubated at 37 °C for 15 min
in 150 mM NaCl, 25 HEPES, 2 mM EDTA, pH 7.4, washed twice in 150 mM NaCl, 1 mM
MnCl2, 25 mM HEPES, pH 7.4, containing 1 mg/ml
BSA (buffer C), and resuspended in buffer C to a concentration of
1 × 107/ml. Aliquots of buffer C (50 µl) containing
2 × the final concentration of mAbs (1:1,000 diution of P1D6, 0.6 µg/ml JBS5, and 1:5,000 dilution of P4C10) and 2 × the final
concentration of CCBD fragment (50 µg/ml) or GRGDS peptide (200 µg/ml) were added to 50-µl aliquots of cells. Samples were then
incubated at room temperature for 1 h. Cells were washed three
times in buffer C, and 50 µl of fluorescein isothiocyanate-conjugated
F(ab)2 anti-mouse Fc secondary antibody (Serotec) diluted
1:200 in buffer C with 1% (v/v) normal human serum was added to each
sample, and the samples were incubated at room temperature for a
further 30 min. (The secondary antibody conjugate does not cross-react
with rat mAbs.) Cells were then washed twice in buffer C, once in
phosphate-buffered saline, and fixed in phosphate-buffered saline
containing 0.2% (w/v) formaldehyde. 20,000 cells were analyzed from
each sample using a FACscan flow cytometer (Becton Dickinson, Cowley,
Oxford, U. K.), and mean fluorescence intensity values were
calculated. In repeated measurements, standard errors were <5% of the
mean fluoresence intensity values.
To estimate the amount of nonspecific fluoresence, the level of binding of an irrelevant mouse mAb 9E10 (anti-myc tag) to K562 cells was measured under the same conditions.
Cell Attachment AssayK562 cells were washed with 150 mM NaCl, 25 mM HEPES, pH 7.4, incubated at
37 °C for 30 min in the same buffer, and resuspended in buffer C to
a concentration of 1 × 106/ml. Assays were performed
in 96-well microtiter plates (Costar, High Wycombe, U. K.). Wells were
coated for 60 min at room temperature with 100-µl aliquots of
recombinant proteins (0.2-100 µg/ml) diluted with Dulbecco's
phosphate-buffered saline, and then sites on the plastic for
nonspecific cell adhesion were blocked for 40-60 min at 37 °C with
100 µl of 10 mg/ml heat-denatured BSA. The BSA was removed by
aspiration, and the wells were then washed once with buffer C. Aliquots
of the cells (100 µl) in buffer C were then added to the wells and
incubated for 20 min at 37 °C in a humidified atmosphere of 5%
(v/v) CO2. For experiments examining the effect of
anti-5 and anti-
1 mAbs on cell attachment, cells were
preincubated with mAbs (10 µg/ml or 1:500 dilution of ascites) for 30 min at room temperature before being added to the wells. To estimate the reference value for 100% attachment, cells in quadruplicate wells
coated with poly-L-lysine (500 µg/ml) were fixed
immediately by direct addition of 100 µl of 5% (w/v) glutaraldehyde
for 30 min at room temperature. Loosely adherent or unbound cells from experimental wells were removed by aspiration, the wells washed once
with 100 µl of buffer C, and the remaining bound cells were fixed as
described above for reference wells. The fixative was aspirated, the
wells were washed three times with 200 µl of H2O, and
attached cells were stained with Crystal Violet (Sigma) as described
previously (20). The absorbance of each well at 570 nm was then
measured using a multiscan ELISA reader (Dynatech, Billingshurst,
U. K.). Each sample was assayed in quadruplicate, and attachment to
BSA (<3% of the total) was subtracted from all measurements.
Wild type human 5 cDNA was subcloned
into the pBJ-1 vector. Putative surface loops in the
NH2-terminal repeats of
5 were swapped with the
corresponding loops in the human
4 sequence. The positions of the
swapping mutations are residues 41-48 in repeat 1 (R1), 116-134 in
repeat 2 (R2), 179-191 in repeat 3 (R3), 252-259 in repeat 4 (R4),
308-321 in repeat 5 (R5), and 375-380 in repeat 6 (R6). The
expression vector of each mutant was constructed using the overlap
extension polymerase chain reaction (21). The presence of the mutation
was verified by DNA sequencing. Twenty µg of wild type or mutant
5
cDNA construct was transfected into Chinese hamster ovary-B2 cells
(22) (8 × 106 cells), together with 1 µg of pFneo
DNA containing a neomycin resistance gene, by electroporation.
Transfected cells were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum at 37 °C in 6%
CO2 for 2 days. Then the cells were transferred to the same
medium containing 700 µg/ml G418 (Life Technologies, Inc.). After
10-14 days, G418-resistant colonies were harvested. The expression
level of wild type and mutant
5 was confirmed by flow cytometric
analysis in FACScan with VC5, a mAb that recognizes a nonfunctional
epitope on the
5 subunit. Chinese hamster ovary cells expressing
mutant
5 were then cloned to obtain cells expressing high levels of
mutant
5 by cell sorting in FACStar (Becton Dickinson). The cloned
cells expressed mutant
5 at levels comparable to that of cells
expressing wild type
5. Characteristics of these cells will be
described in more detail elsewhere.2 To
localize the epitopes for P1D6, JBS5, and mAb 11, the reactivity of the
wild type and mutant
5-expressing cells with these mAbs was examined
by flow cytometry in FACScan.
We showed previously that the binding of the inhibitory
anti-1 mAb 13 to
5
1 was perturbed by both GRGDS synthetic
peptide and a tryptic CCBD fragment (8). We therefore investigated whether the binding of two inhibitory anti-
5 mAbs to
5
1 was affected by these ligands. The results (Fig. 1,
A and B) showed that the binding of P1D6 and JBS5
was not inhibited by GRGDS peptide; in fact, we reproducibly observed a
slight stimulation (1.2-1.4-fold) of P1D6 binding to
5
1 by GRGDS
peptide. However, binding of both P1D6 and JBS5 was strongly attenuated
by the CCBD fragment. In correspondence with our previous results for
mAb 13 (8), binding of the inhibitory anti-
1 mAb P4C10 to
5
1
was partially blocked by both GRGDS peptide and the CCBD fragment (Fig.
1C).
In control experiments (not shown) the binding of the noninhibitory
anti-5 mAb 11 and the noninhibitory anti-
1 mAb K20 to
5
1
was not affected by either GRGDS peptide or the CCBD fragment. We used
1 mM Mn2+ in all the above experiments because
ligand recognition by
5
1 is optimal under these conditions (20).
The binding of P1D6, JBS5, and P4C10 to
5
1 was not influenced by
GRGDS peptide or CCBD fragment when Mn2+ in the assay
buffer was replaced with 1 mM EDTA (data not shown), i.e. conditions under which ligand recognition does not
occur (20). These findings strongly suggest that the altered binding of
P1D6, JBS5, and P4C10 seen in the above experiments (Fig. 1, A-C) is a consequence of ligand recognition by
5
1.
GRGDS peptide was also ineffective at blocking the binding of P1D6 and
JBS5 to 5
1 on K562 cells (Fig. 1D), whereas the CCBD fragment was strongly inhibitory. In contrast, P4C10 binding was attenuated by both GRGDS peptide and the CCBD fragment. (These experiments were performed in the presence of the activating anti-
1 mAb 9EG7 because GRGDS peptide recognition by
5
1 on K562 cells does not occur unless the integrin is stimulated using activating mAbs
(8).)
To examine if the tryptic CCBD fragment
acts as a direct competitive inhibitor or as an allosteric inhibitor of
P1D6, JBS5, and P4C10 binding to 5
1, we examined the inhibitory
effect of the CCBD fragment at 10-fold different mAb concentrations.
The results showed that the concentration of CCBD fragment required for
half-maximal inhibition of P1D6 (Fig. 2A) and
P4C10 (Fig. 2C) binding was not significantly different over
this range of antibody concentrations. However, the concentration of
CCBD fragment required for half-maximal inhibition of JBS5 (Fig.
2B) binding to
5
1 was increased approximately 4-fold.
For all the mAbs, the maximal extent of inhibition decreased with
increasing antibody concentration. If ligand behaved as a direct
competitive inhibitor of mAb binding, the concentration of ligand for
half-maximal inhibition of antibody binding should increase in parallel
with the antibody concentration, and the maximal extent of inhibition
should be unchanged. For P1D6 and P4C10, the results are instead
consistent with an allosteric inhibition, in which ligand does not
compete directly with mAb for binding to
5
1 but instead binds to
a separate site and decreases the affinity of mAb binding by an
allosteric effect on the conformation of the integrin. However, for
JBS5 the inhibition is not purely allosteric in nature; the increased concentration of ligand for half-maximal inhibition of antibody binding
with increased mAb concentration indicates that some direct competitive
inhibition may also be involved. For each mAb, plots of 1/(antibody
binding) versus ligand concentration were hyperbolic (not
shown), which is diagnostic of a mainly allosteric type of inhibition
(23). Hence, as observed for mAb 13 (8), P1D6, JBS5, and P4C10 appear
to recognize sites on
5
1 which are attenuated as a consequence of
ligand occupancy.
The Region of the CCBD Responsible for Perturbing the Binding of the Anti-
The above results show that the binding of P1D6 and JBS5
to 5
1 is decreased markedly when the CCBD of fibronectin is
recognized by
5
1. However, recognition of the RGD sequence alone
does not decrease the affinity of P1D6 and JBS5 binding. To narrow down which region of the CCBD is involved in blocking P1D6 and JBS5 binding,
recombinant wild type and mutant fragments from the CCBD were produced
(Fig. 3). Initially, the activity of these fragments was
assessed by testing their ability to inhibit competitively the binding
of the tryptic CCBD fragment to
5
1 in a solid phase assay (Fig.
3). In close agreement with a previous study using intact cells (5),
mutation of the synergy region led to a 50-200-fold loss of activity
in this assay. However, when used at sufficiently high concentrations,
such fragments did fully inhibit tryptic CCBD fragment binding. In
contrast, fragments lacking the RGD sequence had little or no activity.
Since the synergy region and the RGD sequence lie in the 9th and 10th
type III repeats, respectively, we first tested whether these repeats
were involved in blocking P1D6 and JBS5 binding to
5
1. The
results (Fig. 4, A and B) showed that a recombinant protein comprising only the 10th type III repeat (III10) did not perturb P1D6 and JBS5 binding; however, a
recombinant protein containing the 9th and 10th type III repeats
(III9-10) strongly inhibited the binding of these mAbs. In
contrast, binding of P4C10 to
5
1 was inhibited by both
III10 and III9-10 (Fig. 4C). A
recombinant protein comprising only the 9th type III repeat
(III9) had no effect on the binding of any of the mAbs to
5
1. A mixture of III9 and III10 also
failed to perturb P1D6 and JBS5 binding (data not shown). These results
indicate that the region of the CCBD responsible for blocking P1D6 and
JBS5 binding lies in the 9th type III repeat; however, the activity of
this region is only observed when the 10th type III repeat is also
present in the same protein. In contrast, consistent with the effect of
GRGDS peptide on P4C10 binding to
5
1, the region of the CCBD
responsible for blocking the binding of this mAb appears to lie mainly
within the RGD-containing 10th type III repeat.
The PHSRN Site in the 9th Type III Repeat Is Involved in Perturbing the Binding of the Anti-
To define further
the region of the CCBD involved in blocking P1D6 and JBS5 binding, we
examined the effect of mutations in the 9th type III repeat on the
capacity of the III9-10 protein to block P1D6 and JBS5
binding. The results (Fig. 5, A and
B) showed that replacement of the synergy sequence region
(PHSRN) in the 9th type III repeat with an inactive sequence (SPSDN)
from the 8th type III repeat resulted in a marked loss of activity for
perturbing P1D6 and JBS5 binding. Replacement of the entire 9th type
III repeat with the 8th caused a further loss of activity. In contrast,
mutations in the 9th type III repeat had little effect on the ability
of the recombinant proteins to inhibit P4C10 binding (Fig.
5C) (although in agreement with the lower affinity of
binding of the mutant proteins to 5
1, much higher concentrations
of the mutant proteins were required to inhibit P4C10 binding maximally compared with the wild type III9-10 protein). These data indicate that the synergy region of the 9th type III repeat
(particularly the PHSRN sequence) is required for blocking P1D6 and
JBS5 binding to
5
1; however, this region plays little or no role
in blocking P4C10 binding.
Binding of a CCBD Fragment Lacking the Synergy Site to
Since binding of the
synergy region of the 9th type III repeat to 5
1, but not the RGD
sequence of the 10th type III repeat, causes attenuation of the
epitopes recognized by P1D6 and JBS5, we hypothesized that the binding
of these mAbs to
5
1 should inhibit recognition of the synergy
sequence but not the RGD sequence. To test this hypothesis we produced
recombinant fragments of the CCBD containing wild type and mutant
versions of type III repeats 6-10. The use of these longer fragments
of the CCBD allowed us to compare results in solid phase and cell
attachment assays. The wild type protein (III6-10) bound
with high affinity to
5
1 in solid phase assays. In correspondence
with the results shown in Fig. 3, a mutant protein in which 16 amino
acid residues in the synergy region of the 9th type III repeat were
replaced with corresponding sequence from the 8th type III repeat
(III6-10(SPSDN)) also bound to
5
1, but with
>100-fold lower apparent affinity than the wild type protein. A second
mutant protein in which the RGD sequence in the 10th type III repeat
was mutated to KGE (III6-10KGE) bound very poorly to
5
1 (Fig. 6A). None of the proteins
bound to
5
1 in the presence of EDTA (data not shown). Parallel
results were obtained in K562 cell attachment assays (Fig.
6B).
The effects of inhibitory anti-5 and anti-
1 mAbs on
III6-10 and III6-10(SPSDN) binding to
5
1 were then tested (Fig. 7A), using
concentrations of these proteins which gave similar levels of binding
in the absence of mAbs. The results showed that, as predicted, P1D6
inhibited binding of III6-10 but not III6-10(SPSDN) to
5
1. However, JBS5
inhibited both III6-10 and III6-10(SPSDN)
binding. All of the inhibitory anti-
1 mAbs tested (P4C10, 13, and
4B4) blocked both III6-10 and III6-10(SPSDN) binding. There was no
inhibition of either III6-10 or
III6-10(SPSDN) binding by control anti-
5 or anti-
1
mAbs. Again, parallel results were obtained in cell attachment assays
(Fig. 7B). Taken together, these data show that P1D6
inhibits recognition of the synergy region but not the RGD sequence by
5
1. However, it appears that JBS5 does affect binding of the RGD
sequence to
5
1.
Epitopes of P1D6 and JBS5 Lie within the NH2-terminal Repeats of the
For the integrin 4
1 it has been
shown that the epitopes of inhibitory anti-
4 mAbs map to sites
mainly within the third NH2-terminal repeat (24, 25) and
are probably close to, or within, predicted loop structures (12, 26,
27). To localize the epitopes of P1D6 and JBS5, putative loop regions
within the NH2-terminal repeats of the
5 subunit were
swapped with the corresponding loops from the
4 subunit. Mutant
5
subunits (which formed heterodimers with endogenous hamster
1) were
expressed on the surface of Chinese hamster ovary-B2 cells and tested
for their ability to bind P1D6 and JBS5 using flow cytometric analysis.
The results (Table I) showed that swapping of loops in
the NH2-terminal repeats, particularly in repeats 2 and 3, perturbs P1D6 and JBS5 binding. Hence it appears that the epitopes of
these mAbs comprise several noncontiguous residues in these repeats.
Binding of the noninhibitory anti-
5 mAb 11 to
5
1 was not
affected by any of the loop swaps, suggesting that its epitope lies
outside the NH2-terminal repeats. The epitopes of the
inhibitory anti-
1 mAbs used in this study (P4C10, 13, and 4B4) have
been shown previously to map to a region of the
1 subunit which
contains amino acid residues 207-218 (10).
|
The NH2-terminal portions of both integrin and
subunits appear to cooperate in ligand recognition; however, it is not yet understood how this cooperativity arises, how specificity is
generated, and whether the
and
subunits recognize the same or
distinct sequences in the ligand. The prototypical interaction of
5
1 with the CCBD of fibronectin studied here allows us to begin
to address these questions.
The main findings of this report are as follows. (i) Binding of the
anti-5 mAbs P1D6 and JBS5 to
5
1 is strongly inhibited by CCBD
fragments containing both synergy and RGD sequences, but not by GRGDS
peptide or CCBD fragments containing only the RGD site. In contrast,
binding of the anti-
1 mAb P4C10 to
5
1 is inhibited to a
similar extent by GRGDS peptide, CCBD fragments containing both synergy
and RGD sequences, or CCBD fragments containing only the RGD site. (ii)
P1D6 inhibits the interaction of a wild type recombinant CCBD fragment
with
5
1 but has no effect on the binding of a mutant CCBD
fragment that lacks the synergy sequence. In contrast, the binding of
both wild type and mutant fragments to
5
1 is inhibited by P4C10
(and by other anti-
1 mAbs). (iii) The epitopes of P1D6 and JBS5 lie
in the NH2-terminal repeats of the
5 subunit. Taken
together, our results indicate that the synergy region is recognized
primarily by the
5 subunit (in particular by its
NH2-terminal repeats) but that the
1 subunit plays the major role in binding of the RGD sequence.
Although our data suggested that ligand acted mainly as an allosteric
inhibitor of antibody binding, we believe that this should not be taken
as evidence that these antibodies recognize epitopes that are spatially
distant from the ligand binding sites. Inhibitory mAbs probably
recognize sites proximal to the ligand binding domains because they lie
in the same regions of the subunits implicated by other techniques
(such as cross-linking and site-directed mutagenesis) as containing
ligand binding sequences (9). Our epitope mapping results indicate that
the epitopes of JBS5 and P1D6 include a putative -turn sequence in
the third NH2-terminal repeat of
5, a region that has
previously been shown to pay a role in fibronectin recognition (12).
These findings are consistent with the concept that inhibitory mAb
epitopes are at sites that are attenuated as a result of the
conformational changes that take place in the integrin upon ligand
recognition and that these changes take place close to the ligand
binding sites. Furthermore, our results suggested that the inhibition
of JBS5 binding to
5
1 by ligand was not purely allosteric but
also involved a direct competition; hence the epitope recognized by
this mAb appears to be partly overlapping with the ligand binding
pocket.
P1D6 only inhibited binding of the wild type CCBD fragment that
contained both synergy sequence and RGD to 5
1 but had no effect
on the binding of a mutant fragment lacking the synergy site but
retaining the RGD sequence. These results imply that P1D6 only blocks
recognition of the synergy sequence by
5
1. A similar conclusion
was reached in a study examining the effect of P1D6 on epithelial cell
adhesion to mutant fibronectin fragments (28). In contrast, however, we
found that the other anti-
5 mAb used in this study (JBS5) inhibited
binding of both wild type and mutant CCBD fragments to
5
1. Since
not all
1 integrins recognize RGD sequences, it is likely that the
5 subunit plays some role in RGD binding, and thus it is possible
that the JBS5 epitope may be spatially close to a region of the
5
subunit involved in recognition of RGD. In agreement with this
suggestion, mutations in the third NH2-terminal repeat of
the
IIb subunit blocked binding of mAbs containing an RGD-type motif
to
IIb
3 (29). Alternatively, as seen with the anti-
1 mAb 13 (8), JBS5 may induce a conformational change in
5
1 which
precludes binding of the RGD sequence. All of the inhibitory anti-
1
mAbs tested blocked the binding of both wild type and mutant fragments,
as would be predicted if they allosterically affect an RGD-binding
region of the
1 subunit. Our proposal that the
1 subunit contains
the main site involved in RGD recognition is consistent with studies
examining the cross-linking of RGD-containing peptides to
IIb
3
and
V
3, which showed that the
3 subunit contained the major
site of cross-linking (30, 31). A possible RGD binding motif in
integrin
subunits has recently been identified from a phage display
library (32).
Our observation that the 9th type III repeat appears unable to bind to
5
1 in the absence of the 10th type III repeat is consistent with
a previous report showing that the 9th repeat alone cannot support
5
1-mediated cell attachment (18). It has also been shown
previously that a peptide containing the PHSRN sequence is unable to
inhibit
5
1-mediated cell adhesion to fibronectin; however, when
the same peptide was positioned via a covalent bond in the
corresponding site of the normally inactive 8th type III repeat in a
recombinant fragment containing the 8th and 10th repeats, adhesive
activity was partially restored (5). Hence it was proposed that the
PHSRN sequence may need to be located a specific distance from the RGD
sequence to be recognized by
5
1. Our data suggest an explanation
for this observation, i.e. the recognition site for the
PHSRN sequence on the
5 subunit is positioned a specific distance
from the RGD recognition site on the
1 subunit. The synergistic
activity of the PHSRN site can also be readily explained if it provides
a second (weaker) site for integrin recognition.
5
1 recognizes
fibronectin with high affinity but binds only poorly, or not at all, to
other extracellular matrix proteins containing an RGD sequence. The
interaction of
5
1 with the synergy region provides a mechanism
for achieving higher affinity and higher fidelity binding that could be
conferred by the RGD site alone. By analogy, the binding of an RGD/LDV
site in an integrin ligand to the
subunit and the binding of a
second (synergistic) sequence to the
subunit may constitute a
widespread paradigm for generating specificity of integrin-ligand
interactions.
The x-ray crystal structure of a recombinant protein spanning type III
repeats 7-10 of the CCBD revealed that the synergy sequence and the
RGD site are located on the same face of the fibronectin molecule,
separated by 3-4 nm (13). Since the large globular head of an integrin
(about 12 nm across) can easily span this distance, it is possible that
an integrin could interact simultaneously with both sites. Based on the
structure of the CCBD (13), the recognition site for the PHSRN sequence
on the 5 subunit is probably positioned 3-4 nm away from the RGD
binding site on the
1 subunit. The NH2-terminal repeats
of integrin
subunits have recently been proposed to form a
-propeller structure in which the sequences implicated in ligand
recognition are within loops on the upper surface of the propeller
(27). The NH2-terminal portion of integrin
subunits has
been proposed to contain a region with an A-domain-like fold (11,
33-35), with the ligand binding site on the top face of this domain.
Since the synergy site and the RGD sequence lie on the same side of the
fibronectin molecule, our results imply that the top surface of the
subunit
-propeller and the top face of the
subunit A-domain must
be approximately coplanar in the ligand-occupied state of
5
1.
This arrangement would also place sites involved in ligand binding and
the epitopes of inhibitory mAbs near the
subunit/
subunit interface, as proposed in models of integrin activation (9, 36,
37).
We thank J. Aplin for providing human placenta, and D. Vestweber for supplying mAb 9EG7. We are grateful to D. S. Tuckwell, P. Newham, R. C. Liddington, and A. G. Lowe for helpful discussions.