(Received for publication, November 7, 1996)
From the The ninth and tenth type III domains of
fibronectin each contain specific cell binding sequences, RGD in FIII10
and PHSRN in FIII9, that act synergistically in mediating cell
adhesion. We investigated the relationship between domain-domain
orientation and synergistic adhesive activity of the FIII9 and FIII10
pair of domains. The interdomain interaction of the FIII9-10 pair was perturbed by introduction of short flexible linkers between the FIII9
and FIII10 domains. Incremental extensions of the interdomain link
between FIII9 and FIII10 reduced the initial cell attachment, but had a
much more pronounced effect on the downstream cell adhesion events of
spreading and phosphorylation of focal adhesion kinase. The extent of
disruption of cell adhesion depended upon the length of the interdomain
linker. Nuclear magnetic resonance spectroscopy of the wild type and
mutant FIII9-10 proteins demonstrated that the structure of the
RGD-containing loop is unaffected by domain-domain interactions. We
conclude that integrin-mediated cell adhesion to the central cell
binding domain of fibronectin depends not only upon specific
interaction sites, but also on the relative orientation of these sites.
These data have implications for the molecular mechanisms by which
integrin-ligand interactions are achieved.
The regulated adhesion of cells to the extracellular matrix
(ECM)1 is essential for the development and
function of normal tissues, and aberrant regulation of cell adhesion is
often associated with disease. Fibronectins (FNs) are adhesive proteins
that are abundant in the ECM of many cell types and have a critical
role in many biological processes (1). Twenty isoforms of human FN, the expression of which is developmentally regulated, can be generated as a
result of alternative splicing of the primary FN transcript (2-4). The
FN molecules are dimers of disulfide-linked 235-kDa monomers. Each
monomer is composed of type I, type II, and type III domains (FI, FII,
and FIII), identified as repeating amino acid motifs in the primary
structure (5) (Fig. 1). These motifs occur in many
diverse cellular and extracellular proteins (6). Separable, functional
regions of the FN molecule have been identified that contain binding
activities for other components of the ECM, including collagen, fibrin,
and heparin (1). Cells bind to FN via the central cell binding domain
(CCBD) spanning the eighth, ninth, and tenth FIII domains (FIII8-10)
(7) and via the CS1 and CS5 sites in the alternatively spliced IIICS
region (8, 9) (see Fig. 1).
The adhesion of cells to FN in the ECM is mediated by the integrin
family of transmembrane receptors (10-12). The minimal cell recognition sequence RGD in FIII10 has been shown previously to interact with a number of integrins including
An earlier nuclear magnetic resonance (NMR) study of FIII10
demonstrated that the RGD sequence resides in a mobile loop between the
F and G We have shown previously that the structural integrity of the FIII9-10
pair of domains is required for the synergistic activity of the two
domains in supporting cell spreading (36). Here we have investigated
the relationship between domain-domain interactions and the biological
activity of the FIII9-10 pair in specific cell adhesion events. Our
strategy was to modify the strength of the domain-domain interaction
and the relative mobility of the two domains by introducing flexible
polyglycine interdomain linkers of different lengths. The influence of
the linkers on the structure of the FIII9-10 pair was analyzed by NMR
and the affect on biological activity of FIII9-10 was assessed by the
ability of immobilized wild type and mutant GST-FIII fusion proteins to
support events involved in cell adhesion. We discuss the correlation
between our biological data and the underlying structural changes as
examined by NMR and thermodynamic methods.
NMR measurements were performed on
fully 15N-labeled samples of FIII10 and FIII9-10 at
25 °C in 20 mM sodium acetate, 5% D2O, pH
4.8. Protein concentrations were 4 mM (FIII10) or 1.5 mM (FIII9-10). Three-dimensional
15N-correlated 1H,1H NOESY spectra
(37) were recorded on a home-built spectrometer operating at a proton
frequency of 600 MHz.
The constructs
pGEXFIII10 and pGEXFIII9-10 have been described previously (36).
The FIII9-10 linker mutant FIII9-PG-10, containing one additional
proline and one glycine residue between threonine 1415 at the COOH
terminus of FIII9, and valine 1416 at the NH2 terminus of
FIII10 (according to the domain boundaries in Kornblihtt et
al. (38)) was expressed from the construct pGEXFIII9-PG-10. For
construction of pGEXFIII9-PG-10, FIII9, and FIII10 were amplified
separately from FN cDNA (pFHIII (2)) by Pfu polymerase
(Stratagene, La Jolla, CA) in the polymerase chain reaction.
Oligonucleotide primers were designed that introduced a
BglII site into the 5 The mutant pGEXFIII9-P[G]5-10, encoding the fusion
protein GFIII9-P[G]5-10, was generated by the
introduction of the sequence GGAGGCGGAGGC encoding three glycine
residues. Annealed oligonucleotides were inserted into the
SmaI site of pGEXFIII9-PG-10. The sequence of the
recombinant clones was confirmed as above. The position of insertion of
the extended linkers in the mutant FIII9-10 proteins is shown in Fig.
2.
GST fusion proteins (GFIII9, GFIII10,
GFIII9-10, GFIII9-PG-10, GFIII9-P[G]5-10) were expressed
as described previously (36) with some modifications. Cultures of
Escherichia coli transformed with pGEXFIII constructs were
grown overnight in 8 × TY (6.4% tryptone T, 4.0% yeast extract,
0.5% NaCl), diluted 1 in 10 into fresh 8 × TY, and incubated
with shaking for 1 h at 37 °C. Induction of protein expression
was achieved by addition of 0.1 M
isopropyl- Baby hamster kidney
(BHK) cells and human endometrial stromal fibroblasts (hESF) were used
in cell attachment and spreading assays. Pure cultures of hESF were
prepared by collagenase digestion of endometrial tissue followed by
centrifugation through Percoll as described elsewhere (39). Cell
adhesion and spreading assays were carried out as described previously
(36). Essentially, the surface of duplicate wells of 96-well
flat-bottomed, tissue culture-grade plates (Becton Dickinson, Oxford,
UK) was coated with doubling dilutions of 100 µg ml For the inhibition assays, the surface of duplicate wells of a 96-well
plate was coated with 5 µg ml The
surface of duplicate wells of a 96-well plate were coated with 10 µg
ml BHK cells
were allowed to spread for 1 h on tissue culture plastic-coated
with 100 µg ml Detailed NMR and thermodynamic studies of the five
cleaved constructs (FIII9, FIII10, FIII9-10, FIII9-PG-10, and
FIII9-P[G]5-10) are presented in a separate publication
(45). Briefly, introduction of additional linker residues leads to a
gradual increase in the mobility of the domains relative to each other,
as indicated by increasing T2 relaxation times
of the backbone amide 15N-nuclei and to decreasing
thermodynamic stabilization energies. Our results indicate that
domain-domain interactions are significantly perturbed, but not
completely abolished, by introduction of additional linker residues.
However, perturbation of the domain-domain interactions does not affect
the structure of the RGD site in solution. Fig. 3 shows
that the RGDS peptide segment in FIII10 and FIII9-10 has near
identical chemical shifts of backbone and side chain resonances and a
completely conserved pattern of proton-proton NOEs. These data indicate
that the presence of FIII9 does not introduce any additional structural
or motional constraints into the highly flexible RGDS peptide.
The
receptors that mediate the attachment of BHK cells to the FIII9-10
region of FN were defined by the use of function blocking monoclonal
antibodies (Fig. 4). Attachment of BHK cells to FN was
inhibited in the presence of anti-
The FIII domain fusion proteins GFIII10, GFIII9-10, the two modified
fusion proteins GFIII9-PG-10 and GFIII9-P[G]5-10, and FN
were assayed for their ability to support BHK cell attachment, as
assessed by measurement of crystal violet incorporation into adherent
cells on plastic coated with the immobilized proteins (Fig.
5A). The fusion proteins exhibited similar
binding capacities to plastic as assessed by enzyme-linked
immunosorbent assay (data not shown). At high coating concentration
(100 µg ml
The requirement for structural integrity of FIII9-10 for cell
attachment via integrins in addition to
The
morphology of BHK cells plated onto FN, GFIII9-10, GFIII9-PG-10,
GFIII9-P[G]5-10, GFIII10, or GST is shown in Fig.
6. Cells plated onto FN (Fig. 6A) exhibited a
spread morphology. Cells plated onto GFIII9-10 were also spread, but
not to the same extent as on FN (Fig. 6, compare B with
A). The cells spread less on GFIII9-PG-10 (Fig.
6C), and very few cells exhibited a spread morphology on
GFIII9-P[G]5-10 (Fig. 6D) or GFIII10 (Fig.
6E). Very few cells attached to GST (Fig. 6F) and
those that did attach remained rounded.
Cells attached to plates coated with the GST-FIII fusion proteins and
FN were scored for spreading, expressed as the percentage of attached
cells that exhibited a spread morphology. Both BHK (Fig.
7A) and hESF (Fig. 7B) cells
plated onto GFIII9-10 at a coating concentration of 100 µg
ml
Spreading of BHK cells on GFIII9-PG-10 coated at 100 µg
ml The number of hESF cells that spread on GFIII9-10 coated at 100 µg
ml The capacity of purified wild type and mutant FIII domains
to inhibit adhesion of BHK cells to fibronectin was also assessed in
inhibition assays. The FIII domains were purified from GST adsorbed to
glutathione-Sepharose beads by cleavage with thrombin and were tested
for their capacity to inhibit attachment of BHK cells to immobilized FN
coated on plastic at 5 µg ml
Given that perturbation of the FIII9-10
had a profound effect on cell spreading, and given that
integrin-mediated cell spreading appears to be preceded by FAK
phosphorylation (11), we examined the relationship between cell
spreading and FAK phosphorylation in response to adhesion to the wild
type and mutant FIII9-10 proteins. The relative amounts of
phosphorylated FAK in BHK cells plated onto immobilized wild type or
mutant GFIII9-10 domains were determined by immunoprecipitation of FAK
and subsequent Western blotting with anti-phosphotyrosine antibodies as
shown in Fig. 9A. The intensity of bands
identified with anti-phosphotyrosine antibodies and representing
phosphorylated FAK was expressed relative to total FAK as shown in Fig.
9B. Introduction of the short interdomain linker in
GFIII9-PG-10 reduced levels of FAK phosphorylation by one-third. The
longer linker in GFIII9-P[G]5-10 led to a further reduction in FAK phosphorylation, giving levels approximately 30% of
that obtained with cells plated onto GFIII9-10 and similar to that of
cells plated on GFIII10.
For comparison of cell attachment, cell spreading and FAK
phosphorylation in response to GFIII10 and the mutant FIII9-10
proteins, the levels of activity obtained in each assay were expressed
relative to those obtained for FIII9-10 (Table I).
Phosphorylation of FAK and cell spreading activities were tightly
correlated and affected to the same degree by insertion of additional
linker sequence, whereas levels of cell attachment were consistently higher.
Comparison of FAK phosphorylation, cell spreading, and cell attachment
on wild type and mutant FIII9-10 proteins
Nuffield Department of Obstetrics and
Gynaecology,
Fig. 1.
Domain structure of fibronectin. The
diagram illustrates the organization of domains within a FN monomer.
Binding regions for other ECM components are indicated below the
molecule and the alternatively spliced EDIIIA, EDIIIB, and IIICS
regions above. The central cell binding domain (CCBD). The
three types of FN structural domains are represented by symbols: ,
FN type I domain;
, FN type II domain; open box with number
inside, FN type III domain. The site of the disulfide bridges
linking two monomers is indicated by SS.
[View Larger Version of this Image (8K GIF file)]
5
1 (13),
3
1 (14),
v
1 (15, 16),
v
3 (17),
v
6
(18, 19),
8
1 (20, 21), and
IIb
3 (20, 21). Integrin-mediated cell adhesion to FN results in phosphorylation of focal adhesion kinase (FAK, also known as pp125FAK), organization of the actin
cytoskeleton, and cell spreading (22-26). Given the ubiquity of
expression of FNs and their extensive role in cell adhesion, the
molecular mechanisms by which FN supports cell binding and induces the
integrin signaling pathway are of great interest.
strands of the domain (27). Synthetic peptides that contain
the RGD sequence exhibit some cell adhesive activity but do not mimic
the full adhesive function of the FN molecule (7, 28, 29). Additional
sites have been identified recently that are required for maximal
adhesive activity. One site has been mapped to the loop between the C
and E
strands in the ninth FIII domain and contains the peptide
sequence PHSRN (30-34). The PHSRN site has been shown to act
synergistically with RGD in cell adhesion mediated by
5
1,
v
3, and
IIb
(32, 34, 35).
Biophysical Studies
end of the amplified FIII9 cDNA,
an XhoI site into the 3
end of FIII10, and a
SmaI site, CCCGGG, encoding the extra proline and glycine
residues, into the FIII9-10 linker. The primers used were: FIII9 5
,
TGAAGATCTGGTCTTGATTCCCCAACT, FIII9 3
, GGGTGTTGATTGTTGGCCAATCAATA;
FIII10 5
, GGGGTTTCTGATGTTCCGAG GGA, FIII10 3
,
TCACTCGAGTCATGTTCGGTAATTAATGGA. The polymerase chain reaction products
were cleaved with either BglI (FIII9) or XhoI
(FIII10), gel-purified using QIAEX II (Qiagen Ltd., Dorking, United
Kingdom (UK)) according to the manufacturer's instructions, and cloned
into the BamHI/XhoI sites of pGEX4T (Pharmacia,
Uppsala, Sweden). The sequence of the inserts in the recombinant clones was confirmed using the Sequenase 2.0 kit (Amersham International plc,
Amersham, UK).
Fig. 2.
Crystal structure of the FIII9-10 pair
showing linker insertion point. Ribbon structure of FIII9-10 pair
from Leahy et al. (44). Linkers were inserted immediately
before valine 1416 (arrow), the first valine of FIII10. The
sequences GRGDS and PHSRN are displayed in ball-and-stick
format.
[View Larger Version of this Image (28K GIF file)]
-D-thiogalactopyranoside and further
incubation for 3 h at 37 °C. Cells were pelleted by centrifugation, resuspended in 0.02 volumes ice-cold PBS, and lysed by
freeze-thawing and sonication. The cell debris was pelleted by
centrifugation and the supernatant filtered through a 0.22-µm pore
filter and nutated with 50% glutathione-Sepharose beads (Pharmacia Biotech Inc.) for 10 min at 20 °C. The fusion proteins were eluted from the glutathione-Sepharose beads with 10 mM
glutathione, 50 mM Tris-Cl, pH 8, and dialyzed against PBS
at 4 °C. Cleaved FN fragments FIII9, FIII10, FIII9-10, FIII9-PG-10,
and FIII9-P[G]5-10 were produced by the addition of
thrombin (2.5 units of thrombin/mg of protein) to GST-FIII fusion
proteins adsorbed to the glutathione-Sepharose matrix. Purity of the
proteins was assessed by SDS-PAGE and mass spectroscopy.
1
GST-FIII fusion proteins in PBS for 16 h at 4 °C and washed
with PBS. Uncoated plastic was blocked by incubation in 1% bovine
serum albumin in PBS for 1 h at 37 °C. Either BHK or hESF cells
(104 in 100 µl in GMEM or Dulbecco's modified Eagle's
medium, respectively) were inoculated into each well and incubated in
5% CO2 for 1 h at 37 °C. Cells were washed gently
in PBS, fixed in 4% formaldehyde, 4% glutaraldehyde in PBS, and
scored for spreading as described previously (36). Total cell
attachment was assessed by staining with 0.1% crystal violet as
described elsewhere (40). Bound dye was measured at
A570 nm with a Titertek Multiscan plate reader.
1 FN and washed and
blocked as described above. Test protein diluted in 25 µl of PBS was
incubated with 104 BHK cells in 25 µl of GMEM for 2 min.
Cells and protein were then inoculated into the precoated wells
equilibrated with 50 µl of GMEM. Cells were incubated, fixed, and
analyzed as described above.
1 FN and blocked with bovine serum albumin as described
above. GMEM (30 µl) was added to each well and the plate incubated
for 40 min at 37 °C. BHK cells (104 cells in 50 µl of
GMEM) were incubated with 1 µg of anti-
5 (clone SAM-1),
2 (clone AK7; both from Serotec, Kidlington,
UK),
3 (clone P1B5) or
v (clone VNR147);
both from Life Technologies, Inc., Paisley, UK) antibodies in 20 µl
of PBS for 2 min at 20 °C. Cells in antibody solution and GMEM were
then added to the wells and the attachment assays performed as descibed
above. The results were expressed as the mean of three independent
experiment plus or minus the standard error of the mean (S.E.).
1 GST-FIII fusion proteins as descibed
above. The cells were washed three times with PBS, lysed with 1% SDS,
10 mM Tris, pH 7.4, 1 mM phenylmethylsulfonyl
fluoride, 10 µg ml
1 leupeptin for 20 min at 0 °C,
and passed several times through a 25-gauge needle. Cleared lysates
containing equal amounts of total cell protein were incubated with
anti-FAK antibody (Transduction Laboratories, Lexington, KY) in
immunoprecipitation buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1% (w/v) Triton X-100, 10% (w/v) glycerol, 1 mM EDTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg ml
1
leupeptin) for 1 h at 4 °C. Agarose-conjugated secondary
antibody was added and incubated for 1 h at 4 °C. Precipitates
were washed three times with immunoprecipitation buffer, resuspended in
SDS-PAGE sample buffer, and boiled for 3 min. Duplicate samples of
proteins were subjected to 10% SDS-PAGE (41) and Western blotted onto nitrocellulose membranes (42). Membranes were incubated in either anti-phosphotyrosine antibody (PY20; Transduction Laboratories) or
anti-FAK antibody followed by horseradish peroxidase-conjugated secondary antibody (Sigma, Poole, UK). Proteins were visualized by
enhanced chemiluminescence (ECL) using the ECL kit (Amersham International plc) according to the manufacturer's instructions. The
bands were quantified by scanning densitometry and Intelligent Quantifier software (BioImage, Ann Arbor, MI), and the level of FAK
phosphorylation was expressed as relative to total FAK.
Extension of the FIII9-10 Linker Leads to Reduced Interdomain
Interaction
Fig. 3.
NMR spectroscopy of the RGD site of FIII10
and FIII9-10. For each residue a pair of strips from
1H,1H NOESY planes of three-dimensional
15N-correlated NOESY spectra of uniformly
15N-labeled FIII10 and FIII9-10 is shown. Strips were
taken at the 15N frequency of the
15N,1H direct correlation peak.
Left- and right-hand strips of each pair
correspond to the spectra of FIII10 and FIII9-10, respectively. The
positions of NH and CH cross-peaks are indicated by
rectangles and circles, respectively.
[View Larger Version of this Image (37K GIF file)]
5 and
v
antibodies by 80 and 25%, respectively, indicating that binding of
these cells to FN is primarily a function of integrin
5
1, with some
v
3 activity.
Fig. 4.
Inhibition of cell attachment by
function-blocking anti-integrin antibodies. Adhesion of BHK cells
to 100 µg ml1 FN (shaded columns)- or
GFIII9-10 (white columns)-coated plastic in the presence of
10 µg ml
1 anti-
2, anti-
3,
anti-
5, or anti-
v antibody.
[View Larger Version of this Image (50K GIF file)]
1) FN and GFIII9-10 exhibited similar levels
of cell attachment activity. Attachment activities of GFIII10,
GFIII9-PG-10, and GFIII9-P[G]5-10 were 50, 65 and 60%,
respectively, that of GFIII9-10. Thus attachment activity of both the
mutant FIII9-10 proteins was reduced to a level similar to that of
GFIII10. At lower coating concentrations (e.g. 6 µg
ml
1, Fig. 5A) the differences in attachment
activity of all GST-FIII fusion proteins were more pronounced.
GFIII9-10 exhibited 65% activity relative to FN, GFIII9-PG-10, 38%,
GFIII9-P[G]5-10, 28%, and GFIII10, 10% that of FN.
Fig. 5.
Cell attachment to wild type and mutant
linker FIII9-10 proteins. BHK (A) and hESF
(B) cell attachment at 100 µg ml1
(shaded columns) and 6.25 µg ml
1
(white columns) coating concentration. Cell attachment to
each of the test proteins was assessed by measurement of crystal violet incorporation at A570 nm and expressed relative
to attachment on 100 µg ml
1 FN. The data shown
represent the mean ± S.E. of at least four experiments.
[View Larger Version of this Image (28K GIF file)]
5
1 was also investigated in cell
attachment assays using hESF cells, attachment of which is mediated by
multiple integrins including
5
1.2 The data
for these experiments are shown in Fig. 5B. At high coating
concentrations (100 µg ml
1) the level of attachment to
wild type GFIII9-10 was similar to that on FN. In addition, hESF cells
attached efficiently to both GFIII9-PG-10,
GFIII9-P[G]5-10, and GFIII10 coated at 100 µg
ml
1, at a similar level to FN and GFIII9-10. At lower
coating concentrations (e.g. 6 µg ml
1, Fig.
5B), while GFIII9-10 supported maximal attachment of
stromal cells there was a stepwise decrease with the mutant FIII9-10
proteins with increase in the length of the interdomain linker.
Immobilized GFIII10 supported approximately 10% the attachment on FN
or GFIII9-10 at this concentration.
Fig. 6.
BHK cell adhesion to wild type and mutant
linker proteins. Phase contrast microscopy of BHK cells plated on
FN (A) GFIII9-10 (B), GFIII9-PG-10
(C), GFIII9-P[G]5-10 (D), GFIII10 (E), or GST (F) coated at 100 µg
ml1 protein. Bar = 25 µm.
[View Larger Version of this Image (141K GIF file)]
1 exhibited similar spreading to cells on FN. However,
50% spreading activity was obtained at a much lower coating
concentration of FN (~0.2 µg ml
1) compared with 50%
spreading activity on GFIII9-10 (12.5 µg ml
1). Both
BHK and hESF cells spread poorly on GFIII10, which for both cell types
supported less than 10% the spreading activity of GFIII9-10 at a
coating concentration of 100 µg ml
1.
Fig. 7.
Cell spreading on wild type and mutant linker
proteins. BHK cells (A, C) and hESF cells (B,
D) were assayed for spreading on FN (), GFIII9-10 (
),
GFIII10 (×), GFIII9-PG-10 (
), GFIII9-P[G]5-10 (
),
or GST (
) on doubling dilutions of each test protein (A and B). The percentage of cells spread on proteins coated at
100 µg ml
1 (shaded columns), and 6.25 µg
ml
1 (white columns) is shown in C
and D. The data shown represent the mean ± S.E. of at
least four experiments.
[View Larger Version of this Image (35K GIF file)]
1 (Fig. 7C) was reduced to 70% compared
with GFIII9-10, similar to the relative levels of cell attachment
observed for these proteins. Spreading on the mutant
GFIII-P[G]5-10, containing the longer linker, was
substantially reduced at 100 µg ml
1, exhibiting levels
of spreading activity less than 25% that of GFIII9-10. These
differences were more pronounced at lower coating concentrations. At a
coating concentration of 6.25 µg ml
1 (Fig.
7C) spreading activity of BHK cells on GFIII9-10 was
approximately 30% that of FN, spreading on GFIII9-PG-10 was ~25%
that of GFIII9-10, and there was no spreading on
GFIII9-P[G]5-10.
1 was similar to BHK cells (Fig. 7, compare
D with C). The number of hESF cells spread on
GFIII9-PG-10 or GFIII9-P[G]5-10 was approximately 90 and
30%, respectively, of the number of cells spread on GFIII9-10. At a
coating concentration of 6.25 µg ml
1, spreading of hESF
cells on GFIII9-10 was 10% that of cells on FN. Cells plated onto
GFIII9-PG-10 and GFIII9-P[G]5-10 exhibited 35 and 10%,
respectively, levels of spreading on GFIII9-10. Thus spreading
activity of hESF cells on FIII9-PG-10 was elevated compared with BHK
cells. Spreading of hESF cells on GFIII10 at 100 µg ml
1
and at 6.25 µg ml
1 was 19 and 5%, respectively,
relative to spreading on GFIII9-10, similar to levels found for BHK
cells.
1. These data are shown in
Fig. 8. Attachment of BHK cells to FN was completely
abolished by the presence of 125 µM FIII9-10 in the
culture. At a concentration of 125 µM, FIII9-PG-10
inhibited attachment of BHK cells to FN by 20%, and no significant
inhibition was observed with FIII9-P[G]5-10 within the
molar range used in these experiments. Relative to the controls, in
which no protein was added to the cells prior to plating out, 50%
inhibition of spreading of BHK cells on FN was achieved in the presence
of 12.5 µM FIII9-10 and 125 µM
FIII9-PG-10. No inhibition of cell spreading was observed with
FIII9-P[G]5-10. At a concentration of 125 µM, FIII10 and GRGDS peptide both inhibited attachment of
BHK cells to FN by 20 and 30%, respectively, and had a small affect on
cell spreading. Thus insertion of two amino acids in the interdomain linker of FIII9-10 both immobilized and in solution had different affects on cell attachment and cell spreading.
Fig. 8.
Inhibition of BHK cell adhesion by wild type
and mutant FIII9-10 domains. Attachment (A) and
spreading (B) of BHK cells to FN (coated at 10 µg
ml1) was assessed in the presence of doubling dilutions
of FIII10 (×), FIII9-10 (
), FIII9-PG-10 (
),
FIII9-P[G]5-10 (
), or GRGDS peptide (
).
[View Larger Version of this Image (17K GIF file)]
Fig. 9.
Phosphorylation of FAK in BHK cells on wild
type and mutant FIII9-10 fusion proteins. Total FAK and
phosphorylated FAK (coprecipitated with anti-FAK antibodies) from BHK
cells plated on plastic coated with 100 µg ml1 fusion
proteins were detected by Western blotting (A). The
percentage of phosphorylation of FAK on the mutant proteins relative to
phosphorylation on GFIII9-10 (100%) was determined by densitometric
analyses of the Western blots in A (B).
[View Larger Version of this Image (28K GIF file)]
Protein
FAK phosphorylation
Cell spreading
Cell attachment
GFIII9-10
1.00
1.00
1.00
GFIII9-PG-10
0.67
0.65
0.73
GFIII9-P[G]5-10
0.29
0.29
0.65
GFIII10
0.14
0.10
0.55
Previous studies have identified the FN peptide sequences RGD in FIII10 and PHSRN in FIII9 as integrin binding sites (32, 34) and have demonstrated the importance of structural integrity and contiguity of the FIII9-10 pair for synergistic cell adhesive activity of the two domains (36). In this study we have further dissected the intra- and intermolecular interactions involved in the cell adhesion activity of the FIII9-10 pair. We show that 1) precise spatial positioning of the two binding sites in FIII9 and FIII10 is critical for synergistic activity, and 2) downstream cell adhesion events of FAK phosphorylation and cell spreading are more sensitive than cell attachment to disruption of the spatial relationship of FIII9 and FIII10.
A number of experimental approaches have been used to dissect the cell
adhesive activity of FN. The synergistic site containing PHSRN was
recently identified in two important experiments (32, 34). In the case
of 5
1-mediated cell adhesion, Aota
et al. (32) used hybrid constructs consisting of the eigth
and tenth FIII domains. In this experiment putative synergy regions
from FIII9 were swapped into the corresponding position in FIII8 in the
FIII8-10 hybrid. The synergistic site was mapped in this way to the
loop between the C
and E
strands in FIII9. In a second study by
Bowditch et al. (34) the sequence DRVPHSRNSIT was identified as the candidate synergistic site for
IIb
3-mediated cell adhesion, based upon
the ability of the peptide to inhibit binding of a 20-kDa fragment of
FN containing FIII9 and FIII10 to the platelet-specific integrin
IIb
3.
Akiyama et al. (35) recently reported that cell adhesive capacity of FN fragments derived from the CCBD is enhanced when the fragments are presented in solution by nonfunction blocking antibodies adsorbed onto plastic. In consideration of the possible steric and conformational effects of the presenting antibody molecule (the mass of which is up to one order of magnitude larger than the FN fragments we tested) on the adhesive properties of the FN fragments, we chose an alternative method in which the GST-FIII fusion proteins were adsorbed directly onto the plastic. A further consideration was that in situ, FN exists as one of many closely associated components of the ECM, and dimeric FN is presumably immobilized within the matrix, by virtue of its interaction with other ECM components, rather than being mobile above the surface. Ugarova et al. (43). reported that in solution only one of the two FIII10 cell binding sites in dimeric FN is accessible to a monoclonal antibody specific for that site, whereas adsorption of dimeric FN to polystyrene results in a conformational change in the molecule resulting in accessibility of both sites to the antibody, thus having possible implications for our interpretation of solid and soluble phase assays.
The structure of the proteins tested was also taken into account. Recombinant FIII9-10 fragments, in which the domain boundaries were strictly maintained as defined previously (27, 38), were used in this study. The strategy we adopted to investigate the molecular mechanisms involved in the adhesion of cells to FIII9-10 was thus chosen so that the nature of the synergistic interaction between FIII9 and FIII10 could be examined under conditions that mimic as closely as possible, within the limitations of the assays, the molecular configuration of the sites as they exist within the native FN molecule in the ECM.
We dissected the molecular mechanisms involved in the adhesive activity of FIII9 and FIII10 by assaying cell attachment as an indicator of primary, integrin-ligand interaction, and FAK phosphorylation and cell spreading as indicators of downstream cell adhesion events, in response to wild type and mutant linker FIII9-10 proteins. The introduction of just two amino acid residues in the interdomain linker between FIII9 and FIII10 in the mutant protein GFIII9-PG-10 resulted in some decrease in cell attachment but substantial decreases in phosphorylation of FAK and cell spreading. The inclusion of four more amino acids in the interdomain linker had little additional effect on cell attachment. Levels of FAK phosphorylation and cell spreading were, however, reduced even further. The data presented here show that in contrast to cell attachment, signaling via FAK phosphorylation and cell spreading are highly dependent upon the physical interaction between FIII9 and FIII10. Furthermore, the close correlation between FAK phosphorylation and cell spreading activities on the wild type and mutant proteins suggests that these events are tightly coupled. While FIII10 is sufficient for integrin binding, our data further demonstrate that the precise structural relationship between FIII9 and FIII10 must be maintained for facilitation of downstream cell adhesion events. The foregoing arguments suggest that there is a temporally resolvable sequence of FN-mediated adhesion events that can be tested experimentally.
There are a number of possible physical effects of the interdomain
linkage that might account for reduced biological activity of the
mutant linker FIII9-10 proteins. One reason could be that the mutant
FIII protein structure may be incorrectly folded. Thermodynamic data
(described in further detail in Spitzfaden et al. (45)) show
that FIII10 has a significant stabilizing effect on FIII9. This effect
is greatest in FIII9-10 and is progressively reduced as the
interdomain linker is extended, but the change in
Geq from FIII9-10 to
FIII9-P[G]5-10 is relatively small compared with the
change from FIII9-P[G]5-10 to free FIII9. Additionally,
comparison of 1H,15N NMR spectra shows that the
polypeptide backbones of FIII9 and FIII10 in the wild type and mutant
domain pairs are almost identical (45). These data indicate that the
mutant FIII9-10 proteins have a three-dimensional, folded structure
comparable with the wild type FIII9-10.
Another possible explanation for reduced biological activity of the
mutants could be possible steric interference between the
RGD-containing loop in FIII10 and another part of the molecule. Whereas
the overall mobility of FIII10 increases in the mutant proteins
compared with the wild type (T2 data (45)),
there are no significant changes in the chemical shifts and
proton-proton NOE effects of backbone amides and side chain -carbons
in the RGDS peptide (Fig. 3). Therefore, the RGD loop appears to form no new atomic contacts as FIII9 and FIII10 are separated, neither are
existing contacts broken, which rules out the possibility of steric
interference.
The most likely explanation for the strong influence of the linkers upon biological activity is that synergy of the integrin-binding sites within the FIII9-10 pair is absolutely dependent upon specific interdomain interactions and therefore maintenance of the relative orientation of the RGD and PHSRN peptides. The data we present here are consistent with previous reports (34, 44) that the RGD and PHSRN sites in FIII9 and FIII10 are not in physical contact. The importance of the FIII9-10 interdomain interface is surprising considering that in the crystal structure the interdomain area is only 333 Å2 (44) and relatively small in comparison with interface areas between other FN FIII domains. Furthermore, solution NMR measurements revealed that specific contacts between the two modules are scarce. Our data nevertheless show that synergistic activity not only requires a specific distance between RGD and PHSRN, but also precise spatial positioning of the two sites.
In the wild type FIII9-10 domain pair, the orientation of the integrin binding sites with respect to each other is dependent upon the twist and tilt angles between FIII9 and FIII10. These angles are maintained by specific interdomain interactions at the interface between the two domains and by the torsional restraints of the interdomain amino acid residues. Partial disruption of this interface results in random flexing and rotating of the two domains between active and inactive conformations, and this effect is amplified in the longer linker mutant. It is therefore possible that the incremental nature of the changes observed in cell adhesion on immobilized FIII9-10 wild type and mutant proteins is a result of stabilization of a subset of active conformations. However, our data showing lower activity of the mutant in inhibition assays is in agreement with Aota et al. (32) who found that substitution mutants of PHSRN also showed lower activity in inhibition assays than in direct adhesion assays. Our biophysical data clearly show that there is a significant breakdown of the specific interdomain contacts after the addition of two extra linker residues, resulting in increased mobility of the two domains relative to each other. The differences in biological activity of the mutant proteins in the direct adhesion and inhibition assays can be explained if, in the direct adhesion assays, the two integrin-binding peptides are fixed in the optimal relative orientation for binding the integrin, whereas in solution the mutants oscillate between optimal and suboptimal orientation. Thus it would be expected that immobilized fibronectin competes more effectively for the integrin receptor than does soluble protein. This is not surprising in view of the proposed effect on FN of adsorption to surfaces (43).
In conclusion, our findings demonstrate a requirement for the precise positioning of the RGD loop and the synergy site in FIII9-10 for cell adhesion and strongly suggest that the different synergistic regions of the CCBD have separable, and possibly sequential, functions in cell adhesion. These data have implications for our understanding of the inter- and intramolecular mechanisms involved in integrin-mediated adhesion and signaling.