(Received for publication, April 5, 1995; and in revised form, July 11, 1995)
From the
We investigated the influence of the activation state of
integrin 5
1 on its dependence on the PHSRN synergy site for
binding to RGD in fibronectin. K562 and MV3 cells lacked
v
3
expression and adhered to fibronectin through
5
1. Mel57 cells
adhered through
v
3 and
5
1. A recombinant
fibronectin polypeptide, containing five type III repeats from the
central cell binding domain 3Fn6-10, and a mutated polypeptide
lacking the synergy site were equally effective in promoting Mel57
adhesion. For K562 and MV3, the mutated polypeptide was not or poorly
active compared to the control polypeptide. Expression of
v
3
in MV3 induced strong adhesion to the mutated polypeptide. TS2/16
stimulatory
1-integrin antibodies or Mn
induced
5
1-mediated adhesion of K562 and MV3 to GRGDSP. In the
presence of TS2/16 or Mn
,
5
1-mediated MV3
adhesion to the mutated polypeptide was equally strong as adhesion to
the control polypeptide. Mn
or TS2/16 induced weak
K562 binding to the mutated polypeptide, and in the presence of a
combination of phorbol 12-myristate 13-acetate, Mn
,
and TS2/16,
5
1-mediated K562 adhesion to the mutated and
control polypeptide was equally strong. Our findings demonstrate that
requirement for the PHSRN synergy site for
5
1-mediated
adhesion to RGD in fibronectin depends on the activation state of the
integrin.
Fibronectin (Fn) ()is an extracellular matrix
glycoprotein that functions in cell adhesion and migration in wound
healing, embryonic development, and malignant
transformation(1, 2) . The Fn molecule is composed of
three types of repeating modules, termed type I, II, and III
repeats(3) , which are organized into functional domains.
Proteolytic cleavage yields several fragments containing domains that
promote cell adhesion, including the carboxyl-terminal HepII
domain(4) , the alternatively spliced type III connecting
segment(5) , and the central cell binding domain (CCBD).
The
CCBD consists of type III repeats, each containing approximately 90
amino acids(6) . Cells bind to the CCBD via receptors of the
integrin family(7) . Integrins are heterodimeric
transmembrane molecules mediating cell-cell adhesion and attachment of
cells to the extracellular matrix(8) . Integrins that bind the
CCBD include
3
1 (9) ,
5
1(10, 11) ,
v
1(12) ,
v
3(13) ,
IIb
3(14, 15) ,
and
v
6(16) .
The Arg-Gly-Asp (RGD) sequence in the
10th type III repeat (3Fn10) is the key attachment site for binding of
these integrins to the CCBD, as demonstrated by inhibition of cell
adhesion with synthetic RGD-containing
peptides(17, 18) . Furthermore, two synergistic
regions in the CCBD besides RGD have been identified that are required
for cell adhesion through IIb
3 (19) and
5
1(20, 21, 22, 23) . For
5
1 binding to Fn, the synergy region in 3Fn9 is the most
important of these two regions(21) , and recently, a short
amino acid sequence Pro-His-Ser-Arg-Asn (PHSRN) was identified in this
repeat that synergistically enhances the cell adhesion promoting
activity of the RGD sequence(24) . This sequence is also
present in an 11-amino acid integrin binding site from 3Fn9 that is
recognized by
IIb
3(25) .
Integrins do not always
constitutively bind to their ligands with high affinity. Integrin
adhesiveness can be stimulated by phorbol esters and other more
physiologically relevant agonists(8, 26) . In
addition, antibodies have been described to integrin
1(27, 28, 29, 30) ,
2(31) , and
3 (32) subunits, which induce a
high affinity state of the integrins. Studies with stimulatory
1
antibodies on hematopoietic cells have demonstrated modulation of
binding to natural ligands (28, 29, 30) ,
modulation of ligand specificity(33) , modulation of binding to
different regions in one ligand(34) , and modulation of the
minimal sequence of a binding site required for adhesion(35) .
In the present study, we have investigated the role of the PHSRN
synergy site in 5
1- and
v
3-mediated cell adhesion
to the CCBD in Fn. We show that requirement for the PHSRN synergy site
for cell adhesion to the CCBD depends on the integrins expressed and on
the activity of the integrins involved.
Figure 2: Recombinant FN polypeptides. A, schematic representation of a recombinant Fn polypeptide 3Fn6-10 consisting of five type III Fn repeats from 3Fn6 through 3Fn10 and of a mutated Fn polypeptide 3Fn6-10(SPSDN) where the region containing PHSRN in 3Fn9 has been substituted by the corresponding region from 3Fn8 (hatchedbar). B, in 3Fn6-10(SPSDN), 16 amino acid residues from 3Fn9 were substituted with the corresponding residues from 3Fn8 to disrupt the PHSRN site in 3Fn9. Boxedsequences are from 3Fn6-10(SPSDN). The region with italicizednucleotides differs from the original sequence in 3Fn8 for technical reasons to alter restriction sites, but this does not alter the amino acid sequence.
Protein expression was
induced by 1 mM
isopropyl-1-thio--D-galactopyranoside treatment of Escherichia coli strain BL21 (DE3, pLysS) containing the
expression plasmid. The expressed recombinant polypeptides were
purified by sequential DEAE and hydroxyapatite column chromatography.
The polypeptide was eluted from a DEAE column (DE52, Whatman) using a
linear gradient of 0-0.5 M NaCl in 10 mM sodium
phosphate (pH 7.4), 1 mM EDTA, 0.02% sodium azide, applied to
a hydroxyapatite column (Bio-Rad), and eluted using a linear gradient
from 5 mM sodium phosphate (pH 6.5), 0.4 mM EDTA,
0.02% sodium azide to 250 mM sodium phosphate (pH 6.5), 0.4
mM EDTA, 0.02% sodium azide. The fractions with peak
absorbance were evaluated for purity by SDS-polyacrylamide gel
electrophoresis, pooled, dialyzed against phosphate-buffered saline
without Ca
or Mg
and with 0.02%
sodium azide, and stored at -80 °C.
To exclude influences from domains outside the
CCBD that are known to have cell adhesive activity (HepII, IIICS), we
used a 120-kDa Fn fragment that lacks the heparin-binding domain and
the V region but includes the CCBD. K562 adhered weakly to Fn120 kDa,
whereas MV3 and Mel57 both adhered strongly (Fig. 1). As
expected from the surface expression data, adhesion of K562 was
completely blocked by mAbs to 5 or
1. Even though MV3
expressed several Fn-binding integrins, adhesion was fully blocked by
mAbs to
5, whereas mAbs to
3 or
4 or polyclonal
anti-
v had no effect. Adhesion of Mel57 was inhibited
approximately 35% by mAbs to
5 or
1 and about 50% by mAbs to
3 or
v
3 or by polyclonal
v. The combination of mAbs
to
5 and
v
3 completely blocked adhesion of Mel57.
Figure 1: Inhibition of adhesion to Fn120 kDa with integrin mAbs. Cells were allowed to adhere to wells coated with 20 µg/ml of a 120-kDa fragment of Fn (Fn120 kDa) in the absence (no) or in the presence of inhibitory mAbs to integrin subunits as indicated. Adhesion to BSA was less than 5%. One representative experiment of four is shown.
Thus, K562 adheres weakly to the CCBD through 5
1, MV3
binds strongly through
5
1, and Mel57 binds strongly through
5
1 and
v
3.
Figure 3: Adhesion to recombinant Fn polypeptides. K562, MV3, or Mel57 cells were allowed to adhere to wells coated with increasing concentrations of 3Fn6-10(SPSDN) (dottedline) or 3Fn6-10 (line) as indicated. Adhesion to 0.1 mg/ml BSA was less than 4%. One representative experiment of four is shown.
Figure 4:
Expression of v
3 on MV3 induces
adhesion to 3Fn6-10(SPSDN). A, MV3 cells were
untransfected (dottedline), transfected with pBJ1neo
alone (thinline), or transfected with pBJ1neo
including integrin
3 cDNA followed by sorting with LM609
anti-
v
3 mAbs (thickline). Shown is the
relative fluorescence after incubation with LM609 and a
fluorescein-isothyocyanate-labeled second antibody. B, MV3neo
or MV3-
3 cells were allowed to adhere to wells coated with
increasing concentrations of 3Fn6-10(SPSDN) (dottedline) or 3Fn6-10 (line) as indicated. Filledbars represent remaining adhesion to wells
coated with 32 µg/ml 3Fn6-10(SPSDN) in the presence of
inhibitory anti-integrin mAbs as indicated. Adhesion to BSA was less
than 3%. One representative experiment of three is
shown.
From these results, we conclude
that the differential requirement for the PHSRN synergy site for
adhesion to RGD in the CCBD of MV3 versus Mel57 is due to the
different binding mechanisms of 5
1 versus
v
3.
Figure 5:
Stimulation of 5
1-mediated
adhesion to GRGDSP. A, Cells were incubated in the absence (no) or in the presence of PMA, 8A2 or TS2/16 stimulatory
1 mAbs, or manganese (Mn) and allowed to adhere to wells coated
with 20 µg/ml BSA-GRGDSP. B, cells that had been
previously incubated in the absence (x) or in the presence of
8A2 stimulatory
1 mAbs (8A2) were incubated in the
absence (no) or in the presence of inhibitory anti-integrin
mAbs as indicated and allowed to adhere to wells coated with 20
µg/ml BSA-GRGDSP. Adhesion to BSA was less than 5%. One
representative experiment of three is
shown.
Thus, the strength of 5
1
binding to RGD can be increased by Mn
and by
activating
1 antibodies.
Figure 6:
Stimulation of adhesion to recombinant Fn
polypeptides. Cells were incubated in the absence () or in the
presence of PMA (*), TS2/16 (
), or manganese (
) and
allowed to adhere to wells coated with increasing concentrations of
3Fn6-10(SPSDN) (dottedline) or 3Fn6-10 (line) as indicated. Adhesion to BSA was less then 4%. One
experiment of three is shown.
The fact that in the presence of TS2/16
or Mn no difference was observed between the mutated
and control polypeptide regarding adhesion of MV3 cells, whereas for
K562 the mutated polypeptide was still poorly active, could suggest 1)
that stimulation of MV3 cells resulted in recruitment of other
RGD-binding integrins or 2) that
5
1 on K562 cells was not
maximally activated by these agents. To exclude possibility 1, we used
mAbs to
3,
4,
5,
v,
1,
3,
v
3,
or the combination of these mAbs in the absence of anti-
5 for
inhibition of TS2/16-stimulated adhesion of MV3 cells to the mutated
polypep-tide. Stimulated adhesion was blocked by the anti-
5 mAb
and not by any of the other mAbs or their combination (Fig. 7),
suggesting that induction of adhesion to the mutated polypeptide of MV3
by TS2/16 was due to activation of
5
1 and not to recruitment
of other integrins.
Figure 7: Inhibition of stimulated adhesion of MV3 to 3Fn6-10(SPSDN) with integrin mAbs. MV3 cells were incubated in the absence or in the presence of TS2/16 and allowed to adhere to wells coated with 32 µg/ml 3Fn6-10(SPSDN). Inhibitory mAbs to integrin subunits were added as indicated. Adhesion to BSA was less then 3%. One experiment of three is shown.
To investigate possibility 2, we incubated K562
cells with PMA, TS2/16, or Mn and the various
combinations and allowed the cells to adhere to the mutated and control
polypeptide. In the presence of the combination of TS2/16 and
Mn
, adhesion to the mutated polypeptide was more than
half the level of adhesion to the control polypeptide (Fig. 8).
PMA had no effect by itself on adhesion to the mutated polypeptide but
enhanced adhesion to the control polypeptide more than 2-fold. Finally,
in the presence of the combination of PMA, TS2/16, and
Mn
, the control and the mutated polypeptide were
equally effective in promoting adhesion of K562 cells. This adhesion
was blocked by
5 mAbs (not shown).
Figure 8:
Stimulation of adhesion of K562 to
recombinant Fn polypeptides. K562 cells were incubated in the absence
or in the presence of various combinations of PMA, TS2/16, and
Mn as indicated and allowed to adhere to wells coated
with 32 µg/ml 3Fn6-10(SPSDN) (dottedbars)
or 3Fn6-10 (filledbars). Adhesion to BSA was
less then 4%. One experiment of three is
shown.
From these results, we
conclude that requirement of the PHSRN synergy site for
5
1-mediated adhesion to RGD in the CCBD depends on the
activation state of
5
1.
In line with earlier reports, we find that v
3 does
not require the PHSRN site. We base this conclusion on 2 observations.
First, Mel57 cells express
v
3 and adhere equally well to all
molecules tested containing RGD, i.e. GRGDSP, the mutated
polypeptide lacking the synergy site 3Fn6-10(SPSDN), the control
polypeptide 3Fn6-10, and Fn120 kDa. Second, the
v
3
negative MV3 cells do not adhere to RGD-containing ligands that lack
the PHSRN site, and transfection with
3 cDNA resulting in
v
3 surface expression leads to binding of these cells to
GRGDSP and 3Fn6-10(SPSDN).
These findings confirm and extend
the observations that v
3 can be retained on an RGD column (56) whereas
5
1 cannot(11) . Furthermore,
these data are in agreement with the recent report that
v- and
3- but not
5-containing integrins are bound by a column
containing a Fn fragment lacking the synergy region(57) .
Similarly, it has been reported that
IIb
3 but not
v
3 binding to Fn can be inhibited by an 11-amino acid peptide
from 3Fn9 that also contains the PHSRN sequence(25) . Thus, RGD
is sufficient for binding to Fn through
v
3, whereas
5
1 and
IIb
3 require the synergy region for
efficient binding to Fn(24, 25) .
Parenthetically,
it has been reported that cross-talk between v
3 and
5
1 can occur(58, 59) . Therefore, the
induced adhesion to 3Fn6-10(SPSDN) upon expression of
v
3 in MV3 cells did not necessarily have to be due to
v
3-mediated adhesion. Even though Blystone et al.(59) show that
v
3 regulates only
5
1-mediated phagocytosis, in our system
v
3 might
influence
5
1-mediated adhesion. Ligation of
v
3 with
LM609 mAbs might induce a signal that inhibits
5
1. To exclude
this possibility, we used C17 anti-
3 for adhesion inhibition
assays. The fact that these mAbs inhibit adhesion of
3-transfected
MV3 cells to 3Fn6-10(SPSDN) suggests that direct binding through
v
3 rather than signaling to
5
1 is involved.
The
major conclusion from this study is that the requirement for the PHSRN
synergy site for 5
1-mediated adhesion to the CCBD depends on
the activation state of
5
1. This is based on three findings.
First, stimulation of K562 cells that express only
5
1, with
Mn
or stimulatory
1-integrin mAbs, induces
adhesion to GRGDSP and 3Fn6-10(SPSDN). Second, in the presence of
the combination of PMA, TS2/16, and Mn
, the mutated
and control polypeptide are equally effective in promoting K562 cell
adhesion. Third, treatment of MV3 cells with these agents induces
adhesion to GRGDSP and enhances adhesion to 3Fn6-10(SPSDN) to the
level of adhesion to 3Fn6-10, and this effect is completely
blocked by antibodies to
5 but not by mAbs to
3,
4, or
v or the combination.
Even though the v
3-negative
K562 and MV3 cells express similar levels of
5
1, they differ
dramatically in binding to Fn120 kDa through this receptor. The view of
cell type-specific regulation of
5
1 affinity proposed by
O'Toole et al.(60) suggests that the default
low affinity state of the integrin as observed in K562 is switched to a
high affinity state in MV3. As a result, MV3 but not K562 cells bind
strongly to Fn120 kDa. Our finding that K562 cells bind poorly to Fn120
kDa and that 8A2 increases that adhesion two to three times is in line
with earlier findings(61) . As expected, Mn
and stimulatory
1 mAbs do not affect the strong adhesion of
MV3 to Fn120 kDa. However, our findings demonstrate that these agents
do in fact alter the avidity of
5
1 in MV3 cells but that this
change can only be observed in the absence of the PHSRN synergy site.
One interpretation of these findings is that intracellular factors
(induced by PMA for K562 and factors already present in MV3) can
increase the affinity of
5
1 to a level that RGD is recognized
in the Fn molecule and that additional extracellular events are
required for the final activation of
5
1, leading to full
adhesion to RGD in Fn. The synergy site could be involved in the last
step by locking the RGD site in the
5
1 binding pocket, and in
the presence of TS2/16 or Mn
that last step seems to
be no longer required. Our finding that PMA enhances K562 adhesion to
the control polypeptide, whereas by itself it has no effect on adhesion
to the mutated polypeptide, is in line with this idea. Furthermore, the
fact that K562 cells in the presence of TS2/16 bind strongly to the
control polypeptide without the need for PMA demonstrates that optimal
extracellular stimulation (the synergy site plus stimulatory
1
mAbs) can abbrogate the need for intracellular activation (PMA).
It
is of interest that comparable observations have been reported for
4
1(35) . Even though Jurkat and Ramos cells express
an active form of
4
1 in the sense that they are capable of
binding to the CS1 domain of Fn, they only bind to a peptide containing
the EILDV recognition sequence from CS1 in the presence of stimulatory
1 mAbs. The authors suggest that sequences may be present in the
NH
-terminal portion of CS1 that strengthen
4
1
binding to EILDV, although none have yet been identified. Thus, the
presence of sites that synergistically enhance binding of integrins to
their recognition sequence might be a general mechanism, and activation
by stimulatory
1 mAbs and Mn
may bypass the
dependence on such sites. Our report, however, provides the first
example of substitution of the function of a well characterized synergy
site by agents that activate the integrins involved.
For leukocytes,
stimulatory 1 mAbs also increase the affinity of
4
1 for
CS1 (35, 62) and for VCAM-1(29) , and they can
even induce
4
1 binding to the RGDS sequence(34) . MV3
cells adhere to CS1 and tumor necrosis factor
-stimulated
endothelial cells in the absence of stimuli (not shown), indicating the
expression of active
4
1 on these cells. For MV3 cells in the
absence or presence of stimuli, we do not observe any inhibition of
binding to the CCBD with HP2/1 anti-
4 mAbs, whereas these mAbs
inhibit binding to CS1 (not shown). Thus, the reported recognition of
RGD by stimulated
4
1 does not play a role in our assay. This
difference may be explained by the fact that Ramos cells, as used by
Sanchez-Aparicio et al.(34) , do not express
5
1. In MV3, the effect of TS2/16 on
4
1 may be
masked by the binding to RGD through
5
1. Alternatively, as
previously reported(63) , stimulatory
1 mAbs may
selectively activate
5
1 while leaving
4
1
unaffected.
A possible interpretation of our findings may be that
the PHSRN synergy site binds to the same epitope as recognized by the
stimulatory 1 mAbs. It has been previously suggested that the
epitope where these mAbs bind may physically interact with
extracellular proteins(55) . However, the fact that binding of
K562 to 3Fn6-10 can be enhanced in the presence of 8A2 or TS2/16
when PMA is absent indicates that the synergy site and stimulatory mAbs
can have additional combined stimulatory effects. Therefore, these data
seem to support a model where the synergy site and stimulatory mAbs
have different binding sites on
5
1. This is in agreement with
the recent report that the mechanism of binding of integrin
5
1 to Fn seems to be through binding of the
5 subunit to
the synergystic regions and of the
1 subunit to RGD(64) .
In conclusion, our data demonstrate that 5
1 but not
v
3 requires the PHSRN synergy site for cell adhesion to RGD
in the CCBD of Fn but that induction of a high affinity state of
5
1 with PMA, stimulatory mAbs, and/or Mn
abrogates this dependence on the PHSRN sequence.