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J. Biol. Chem., Vol. 276, Issue 30, 27913-27922, July 27, 2001
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From the
Received for publication, October 18, 2000, and in revised form, May 8, 2001
The recognition of extracellular matrix
components can be regulated by conformational changes that alter the
activity of cell surface integrins. We now demonstrate that
conformational regulation of the matrix glycoprotein thrombospondin-1
(TSP1) can also modulate its binding to an integrin receptor. F18 1G8
is a conformation-sensitive TSP1 antibody that binds weakly to soluble
TSP1 in the presence of divalent cations. However, binding of the
antibody to melanoma cells was strongly stimulated by adding exogenous
TSP1 in the presence of calcium, suggesting that TSP1 undergoes a
conformational change following its binding to the cell surface. This
conformation was not induced by known cell surface TSP1 receptors,
whereas binding of F18 was stimulated when TSP1 bound to fibronectin
but not to heparin or fibrinogen. Conversely, binding of F18 to TSP1 enhanced TSP1 binding to fibronectin. Exogenous fibronectin also stimulated TSP1-dependent binding of F18 to melanoma cells.
Binding of the fibronectin-TSP1 complex to melanoma cells was
mediated by Thrombospondin-1 (TSP1)1
is an extracellular matrix protein that displays a complex variety of
biological activities (reviewed in Refs. 1 and 2). TSP1 can both
promote and inhibit cell adhesion, motility, and proliferation. TSP1
also contains sequences that can both activate latent transforming
growth factor- The latter mechanism for regulating TSP1 activity is consistent with
several previous reports that soluble TSP1 has activities distinct from
immobilized TSP1. Soluble TSP1 generally inhibits angiogenic responses
(14, 15), but immobilized TSP1 can stimulate angiogenic responses in
several contexts (13, 16, 17). Such distinct responses could arise from
differences in TSP1 receptor signaling because of the increased valency
of immobilized versus soluble TSP1 or from alterations in
signal transduction resulting from physical immobilization of a
specific TSP1 receptor. Both of these models have ample precedent from
investigation of other cell-matrix interactions (reviewed in Refs. 18
and 19). However, a third possibility is suggested by conformational
studies of TSP1. Based on electron microscopic and spectroscopic
analyses, the conformation of TSP1 is modulated by calcium binding
(20-22). Characterization of the epitopes recognized by several
monoclonal antibodies to TSP1 has identified
calcium-dependent epitopes on the molecule (21). Thus, TSP1
exists in at least two major conformational states, one of which is
induced by calcium binding. Remarkably, some antibodies that recognize
only the calcium-free state in vitro readily bind to TSP1 in
a calcium-rich environment in some tissues (23, 24). Therefore, binding
to some TSP1 receptors may induce conformational changes that resemble
those induced by removing calcium, and both states may exist in
vivo. Other extracellular matrix components may regulate the
conformation of TSP1. A recent study of protease sensitivity suggested
that binding of TSP1 to fibronectin reversibly alters its conformation to resemble the calcium-depleted state (25).
In addition to the reversible conformational changes induced by binding
of calcium or specific TSP1 ligands, TSP1 is a substrate for
protein-disulfide isomerase (26, 27). Several disulfide isomer forms of
TSP1 have been described and can also be differentiated by specific
TSP1 antibodies (26, 28). These may also have different functional
activities relating to inactivation of specific proteases or binding to
the integrin While characterizing a new conformation-specific antibody for TSP1, we
found that the TSP1 conformer recognized by this antibody is
specifically induced following binding of TSP1 to the surface of some
cells. We subsequently found that this conformation is induced by
binding to fibronectin in vitro or by association with cell
surface-associated fibronectin on cells. We further report that the
conformation induced by binding of this antibody and fibronectin
enhance the recognition of TSP1 by one of its cell surface receptors,
Proteins and Peptides--
TSP1 and plasma fibronectin were
purified from human platelets or plasma, respectively, obtained from
the National Institutes of Health Blood Bank (30, 31). Fibrinogen and
heparin-BSA were obtained from Sigma. Human fibroblast and lung
fibronectins were provided by Dr. Ralph Silverman, Fibrogenex, Inc.,
Chicago, IL. Purified Antibodies--
F18 1G8 is an IgG1 secreted by a
hybridoma derived from a BALB/c mouse immunized with
formalin-fixed, thrombin-activated human platelets. Fab fragments were
prepared from intact immunoglobulin using immobilized papain (Pierce)
and radiolabeled using IODO-Beads according to the manufacturer's
instructions (Pierce). TSP1 antibodies A6.1, D4.6, and C6.7 were
provided by Dr. William Frazier (Washington University, St. Louis, MO).
TSP1 antibody ah-TSP1 (HB8432) and the Cell Culture--
OH-1 small cell lung carcinoma (40), A2058
melanoma (41), and MDA-MB-231 and MDA-MB-435 breast carcinoma
cells (American Type Culture Collection, Manassas, VA) were
maintained in RPMI medium supplemented with 15% (for OH-1 cells) or
10% fetal calf serum. Human umbilical vein endothelial cells were
maintained in medium 199E supplemented with 20% fetal calf serum, 10 µg/ml heparin, 50 µg/ml endothelial mitogen (Biomedical
Technologies, Inc., Stoughton, MA), glutamine, penicillin, and
streptomycin sulfate.
Adhesion Assays--
TSP1 and TSP1 peptides in Dulbecco's PBS
or the indicated buffers were adsorbed on bacteriological polystyrene
dishes by incubation overnight at 4 °C. After blocking with 1% BSA
in Dulbecco's PBS with or without divalent cations, adhesion assays
were performed by adding cells suspended in the appropriate medium
containing 1 mg/ml BSA. Cell attachment and spreading were quantified
microscopically after fixing the adherent cells with 1% glutaraldehyde
and staining with Diff-Quik stain. For breast carcinoma and endothelial
cells, a spreading response was defined as a more than 3-fold increase in cell diameter. Neurite-like process formation was assessed as
described previously (11).
F18 Binding Assays--
Immulon-2 Removawell strips (Dynatech
Laboratories) were coated with 50 µl of plasma fibronectin or the
indicated proteins in Dulbecco's PBS by incubating overnight at
4 °C. The wells were aspirated and blocked by incubating with 1%
BSA in Tris-buffered saline, pH 7.6 (BSA/Tris), for 30 min. The wells
were then incubated with TSP1 (25 µg/ml) in 1% BSA/Tris with 10 mM EDTA for 1 h at 37 °C, washed three times with
PBS, and then incubated with 50 µl of 125I-F18 for 1 h at room temperature. The wells were washed three times, and the bound
radioactivity was quantified with a gamma counter (Packard Instruments).
In some experiments, binding of TSP1 antibodies to TSP1 bound to
immobilized proteins as described above was detected by an enzyme-linked immunoassay. After incubating with the indicated antibodies for 1 h, the wells were washed three times and
incubated for 1 h with horseradish peroxidase-conjugated goat
anti-mouse IgG (Kirkegaard and Perry). The wells were washed again, and
binding was detected using o-phenylenediamine substrate (Sigma).
Fibronectin Binding Assays--
Immulon-2 Removawell strips
(Dynatech) were coated with 50 µl of TSP1 or F18 in Dulbecco's PBS
(± calcium) alone or complexed with the indicated antibodies by
incubating overnight at 4 °C. The wells were aspirated and then
blocked with 1% BSA/Tris for 30 min. The wells were washed three times
with Dulbecco's PBS and then incubated with 50 µl of
125I-fibronectin for 1 h at room temperature. Wells
were then washed three times, and the bound radioactivity was
quantified using a gamma counter.
Ligand Binding to Purified Cell Binding Assays--
Platelets were obtained by venipuncture
using a two-syringe technique and purified where indicated using a
discontinuous arabinogalactan gradient as described previously (42).
Platelets were diluted to 200,000/µl in 0.145 M NaCl, 2.7 mM KCl, 3.8 mM Hepes, 0.1% glucose, 0.35%
bovine serum albumin, pH 7.4. Divalent cations were added before use as
indicated for each experiment. 125I-Labeled antibody
binding was measured as previously described (42).
A2058 melanoma cells were harvested using PBS containing 2.5 mM EDTA, centrifuged for 3 min at 2000 rpm, and resuspended
at 2 × 105 cells/50 µl in complete cell medium
(RPMI 1640 with 10% fetal calf serum) with or without fibronectin or
fibrinogen. The cell suspensions were incubated at room temperature for
30 min with rocking and then re-centrifuged and resuspended in the
appropriate cell binding buffer (0.1% BSA in PBS, pH 6, with the
indicated divalent cations). A 50-µl volume of the cell suspension
was then incubated with 125I-F18 alone or with 5 µg/ml of
TSP1 and the indicated inhibitors, where indicated, in a final volume
of 200 µl for 3 h with orbital mixing. The suspension was then
transferred to a tube containing 100 µl of Nyosil oil (William F. Nye, Inc., New Bedford, MA), centrifuged, and washed with cell binding
buffer. The pellet was collected, and the bound radioactivity was
quantified with a gamma counter.
To examine the role of cytoskeleton in induction of the F18 epitope,
melanoma cells were harvested with 2.5 mM EDTA in PBS and
resuspended at 1×107 cells/ml in complete medium (RPMI
1640 with 10% fetal calf serum). Cell suspensions (200 µl) were
combined with 50 µl of 0.5 mg/ml fibronectin or 50 µl of
Dulbecco's PBS, plus 1.25 µl of Me2SO, cytochalasin D (final 1 µM), or nocodazole (10 µM) dissolved in the same volume of Me2SO and
incubated for 30 min at room temperature. The cells were centrifuged
and resuspended in 0.5 ml of binding buffer. A 50-µl volume of each
cell suspension was incubated with 125I-F18 (6.2 µCi/µg) alone or with 5 µg/ml TSP1 and 10 µg/ml fibronectin in
a final volume of 250 µl for 1 h with orbital mixing at
4 °C.
F18 Recognizes a Calcium-dependent Epitope on Platelets
and TSP1--
F18 is secreted by a hybridoma derived from a mouse
immunized with activated human platelets. The binding sites on
platelets for the antibody were increased 10-fold following
activation with thrombin and induction of binding-required
divalent cations (Table I). Fab fragments
were used for these studies to preclude interactions with Fc receptors
on the platelets, but a similar induction of binding was observed using
the intact antibody (data not shown). Calcium was more active than
magnesium for inducing the F18 epitope, and a combination of both
cations was somewhat less effective than calcium alone. The antibody
bound to purified platelet TSP1 (Fig. 1)
formed a stable complex with TSP1 by gel filtration and specifically
immunoprecipitated TSP1 from surface-labeled activated platelets (data
not shown).
The divalent cation dependence for binding to platelets could indicate
either that the antibody recognizes an epitope specific to a
calcium-replete conformation of TSP1 or that the TSP1 receptor mediating its binding to platelets is calcium-dependent.
Based on solid phase binding assays, however, F18 bound to TSP1
immobilized on plastic independent of divalent cations (Fig.
1A). In a reverse assay, soluble TSP1 bound preferentially
to immobilized F18 when calcium was absent (Fig. 1B). This
divalent cation dependence is similar to that of the previously
reported TSP1 antibody A6.1 (21), but the two antibodies did not
cross-compete for binding to TSP1 (data not shown). Therefore, this
antibody defines a distinct calcium-dependent epitope on
TSP1 that is preferentially exposed when calcium is removed, but this
divalent cation dependence could not account for the inverse
calcium-dependence for F18 binding to activated platelets.
Induction of the F18 Epitope by TSP1 Binding to
Fibronectin--
The above data suggested that binding of TSP1, either
to a cell surface TSP1 receptor or to another TSP1 ligand bound to its respective cell surface receptor, induces a TSP1 conformational epitope
that is recognized by F18. Preliminary studies using the blocking
antibody OKM5 excluded a role for the platelet TSP1 receptor CD36
(11 ± 15% inhibition of 125I-F18 Fab binding for six
experiments at 20 µg/ml antibody). Of the platelet receptor
antibodies examined, only the
To examine the ability of other known TSP1 ligands to induce this
epitope, we immobilized several TSP1 ligands on plastic and examined
the ability of labeled F18 to recognize TSP1 bound to each (Fig.
2A). Of those tested, only
immobilized fibronectin enhanced F18 binding to TSP1 (Fig.
2A). Fibrinogen also binds to TSP1 (45), but binding to a
fibrinogen substrate did not induce the F18 epitope (Fig.
2A). Type I collagen was also inactive (results not shown).
Enhancement by fibronectin was specific for the F18 antibody in that
the TSP1 antibody A6.1 exhibited similar reactivities for TSP1 bound to
immobilized fibronectin, fibrinogen, or type I collagen substrates
(Fig. 2B). The latter result confirmed that similar amounts
of TSP1 bound to each immobilized protein and that the increased
binding of F18 to TSP1 bound to fibronectin was not an artifact of an
increased TSP1 binding capacity on fibronectin compared with
fibrinogen. The response for immobilized plasma fibronectin was
dose-dependent, and cellular fibronectins from two sources
produced similar enhancements of F18 binding to the TSP1-fibronectin
complex (Fig. 2C).
Although some binding of F18 could be detected to TSP1 complexed
with high concentrations of immobilized heparin-BSA (results not
shown), when heparin and fibronectin concentrations that supported comparable levels of TSP1 binding were compared, as verified by quantifying direct 125I-TSP1 binding and binding of two
other TSP1 antibodies, antibody F18 demonstrated enhanced binding to
TSP1 complexed with fibronectin but much weaker binding to TSP1
complexed with heparin-BSA (Fig. 2D). Therefore, binding to
fibronectin specifically induces the F18 epitope on TSP1.
Binding of F18 to TSP1 Enhances Fibronectin Binding--
If the
enhancement of antibody F18 binding induced by fibronectin resulted
from a conformational change in TSP1, then binding of these two ligands
to TSP1 may be thermodynamically linked. To test this hypothesis, we
compared the binding of radiolabeled fibronectin to TSP1 and to TSP1
complexed with F18 (Fig. 3). Fibronectin binding was ~3-fold higher to the immobilized
TSP1-F18 complex than to immobilized TSP1 itself. This enhancement was
specific, because immobilized F18 alone had no binding activity for
fibronectin and complexing TSP1 with a control TSP1 antibody, ah-TSP1,
did not enhance fibronectin binding relative to immobilized TSP1
without antibodies (Fig. 3).
Based on the dose-dependence for binding of
125I-fibronectin to immobilized TSP1 alone or complexed to
F18, we determined that F18 increased the number of binding sites for
fibronectin (10 versus 22 pmol/well, respectively) but did
not significantly alter the apparent affinity of fibronectin for
immobilized TSP1.
F18 Binding to Melanoma Cells Is Mediated by a TSP1-Fibronectin
Complex--
F18 bound at low levels to a suspension of A2058 melanoma
cells, but its binding was reproducibly and
dose-dependently enhanced by adding exogenous TSP1 (Fig.
4A). In contrast, binding of
labeled TSP1 to the same cells was not significantly induced by the
addition of F18 (Fig. 4B). This result indicates either that
these two TSP1 binding interactions are not thermodynamically linked or that TSP1 bound to its major binding site on these cells is not recognized by the F18 antibody. Because high affinity binding of TSP1
to A2058 cells was previously shown to be mediated by the
heparin-binding domain of TSP1 (37), this result also suggests that
cell surface heparan sulfate proteoglycans are not the TSP1 receptor that induces the epitope recognized by F18 and is consistent with the failure of heparin binding to induce the F18 epitope on TSP1
in vitro.
TSP1-dependent binding of 125I-F18 to A2058
cells was further enhanced by the addition of plasma fibronectin with
the TSP1 (Fig. 4C). As observed for F18 binding to
platelets, the enhancement of F18 binding was at least partially
calcium-dependent, because addition of EDTA significantly
inhibited both basal binding of F18 and the TSP1-fibronectin-stimulated
binding of F18 (Fig. 4C).
The
Inhibition of TSP1/fibronectin-dependent F18 binding to
melanoma cells by several integrin
The preceding data show that fibronectin adsorbed on plastic (Fig. 2)
or bound to Binding of TSP1 to F18 Augments
Integrin antagonists were used to verify that the enhanced adhesion
response of breast carcinoma cells to immobilized TSP1 was mediated by
Complexing TSP1 with F18 similarly enhanced
Complex Formation with Fibronectin Enhances
Fibronectin Enhances TSP1 Binding to Purified
Much progress has been made toward defining both ligand- and
receptor-binding specificities of extracellular matrix proteins. However, to understand the diverse biological activities that have been
reported for these proteins we must also consider the possibility that
some of their binding activities are differentially regulated.
Previously, the regulation of cellular responses to integrin ligands
been studied primarily in terms of the activation state of integrin
receptors ("inside out signaling," reviewed in Ref. 18) and
intracellular cross-talk between the signals from integrins and other
cell surface receptors (reviewed in Ref. 49). Activation of the
F18 is a TSP1 antibody that recognizes a conformation that is not
favored when soluble platelet TSP1 is in physiological media containing
divalent cations but is induced when TSP1 is bound on the surface of
platelets and melanoma cells or adsorbed on plastic. Fibronectin
specifically induces this conformational change and can serve as a
bridging ligand to present TSP1 in this conformation on the surface of
cells (see Fig. 10A).
Exposure of this TSP1 epitope on melanoma cells requires the
In addition to mediating the binding of TSP1 to the melanoma cell
surface, fibronectin binding alters the biological activity of TSP1 for
processes mediated by one of its receptors,
Although both The binding of TSP1 to fibronectin is mediated by at least two domains
in each protein (45, 53-56), an interaction that was recently shown to
modify the sensitivity of TSP1 to limited proteolysis (25). Although
the proteolysis data implied that binding to fibronectin alters the
conformation of TSP1, its functional relevance to the biological
activities of TSP1 were unknown. Cell-adhesive activities of
TSP1/fibronectin mixtures have been examined using myoblasts,
and specific morphological responses to both proteins could be
identified in myoblasts attaching on TSP1/fibronectin mixtures (57).
Although alterations in stress fiber assembly were noted using the
mixed matrices, this could not be attributed to alterations in specific
integrin-ligand binding activities of fibronectin or TSP1
versus cross-talk among the intercellular signals induced by
each protein. Additional reports of cross-talk between fibronectin and
TSP1 to regulate cell behavior include the suppression of TSP1-induced
endothelial cell apoptosis by fibronectin (58) and disruption by TSP1
of focal adhesions in endothelial cells attached on fibronectin (59).
Using a cell line that lacks functional fibronectin-binding integrins,
we have now demonstrated that fibronectin binding can alter the ability of TSP1 to be recognized by The interaction of TSP1 with fibronectin on the surface of activated
platelets was previously demonstrated by cross-linking (60). Because
TSP1 was not required for fibronectin binding to platelets (61), TSP1
may also bind to the surface of activated platelets through
integrin-associated fibronectin, as demonstrated here for its binding
to melanoma cells. Both Although conformational regulation of integrin function has been widely
observed, only a few examples of conformational regulation of
extracellular matrix protein function have been reported. Binding of
fibronectin to We thank Drs. Steven Akiyama, William
Frazier, Ralph Isberg, William Miller, Ralph Silverman, and Kenneth
Yamada for providing reagents.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, May 17, 2001, DOI 10.1074/jbc.M009518200
2
R. G. Rodrigues, N.-h. Guo, and D. D. Roberts, our unpublished results.
The abbreviations used are:
TSP1, human platelet
thrombospondin-1;
BSA, bovine serum albumin;
EGF, epidermal growth
factor;
IGF1, insulin-like growth factor-1;
MBP, maltose-binding
protein;
PBS, phosphate-buffered saline.
Conformational Regulation of the Fibronectin Binding and
3
1 Integrin-mediated Adhesive
Activities of Thrombospondin-1*
,,,,
Laboratory of Pathology, NCI, National
Institutes of Health and § Hematology Service, Clinical
Center, National Institutes of Health, Bethesda, Maryland 20892 and the
¶ Center for Cell and Gene Therapy and Department of Molecular and
Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4
1 and
51 integrins. Furthermore, binding to F18
or fibronectin strongly enhanced the adhesive activity of immobilized
TSP1 for some cell types. This enhancement of adhesion was mediated by 31 integrin and required that the
31 integrin be in an active state.
Fibronectin also enhanced TSP1 binding to purified
31 integrin. Therefore, both fibronectin
and the F18 antibody induce conformational changes in TSP1 that enhance
the ability of TSP1 to be recognized by
31 integrin. The conformational and
functional regulation of TSP1 activity by fibronectin represents a
novel mechanism for extracellular signal transduction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and inhibit its activation (3). In various
animal models, TSP1 expression has been both positively and negatively
correlated with tumor growth and angiogenesis (4-10). Some of these
disparities in the cellular responses to TSP1 may arise from the
presence of several functional sites on TSP1, which, when coupled with differences in the expression or activation state of the corresponding cell surface receptors between cell types, could result in opposing responses to this protein in different cell types. Yet, a growing list
of examples are known in which a single cell type can respond to TSP1
as both an activator and an inhibitor of a specific response (11-13).
Therefore, additional mechanisms must exist either to regulate cellular
responsiveness to TSP1 or specific ligand binding activities of TSP1 itself.
v3 (29). Therefore, TSP1 can
exist in multiple conformational states, some of which can interconvert
reversibly, whereas others are restricted by covalent disulfide bonds.
31 integrin. This reversible
conformational change therefore modulates the ability of TSP1 to
differentially engage specific cell surface TSP1 receptors and to
thereby trigger specific biological responses.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
31 integrin was
obtained from Chemicon. MBP-invasin 497 fusion protein was purified as
described (32). Synthetic peptides derived from TSP1 that are
recognized by the 31 integrin or other
TSP1 receptors were synthesized as previously described (33). Integrin
antagonists GRGDNP (51 (34)) and
(4-((2-methylphenyl)aminocarbonyl)aminophenyl)acetyl-LDVP (41 (35)) were obtained from Bachem
(Torrance, CA). A non-peptide antagonist of v-containing
integrins (SB223245) was provided by Dr. William H. Miller (SmithKline
Beecham Pharmaceuticals, King of Prussia, PA) (36). Proteins were
labeled with 125I using Iodogen (Pierce) as described
previously (37).
1
integrin-activating antibody TS2/16 (38) were prepared from the
respective hybridomas obtained from the American Type Culture
Collection. A 1 integrin function-blocking antibody
(mAb13) was provided by Dr. Ken Yamada (NIDCR, National Institutes of Health) (39). The 31 integrin blocking
antibody P1B5 and 41 blocking antibody
P4C2 were obtained from Life Technologies, Inc. Purified function
blocking 51 integrin antibody P1D6 was
obtained from Chemicon. The CD36 blocking antibody OKM5 was from Ortho Diagnostics.
31
Integrin--
Immulon-2 Removawell strips (Dynatech) were coated
directly with 50 µl of 5 µg/ml 31
integrin by incubating overnight at 4 °C in Dulbecco's PBS or were
coated with 5 µg/ml of antibody TS2/16 and then incubated with the
integrin. Immulon-2 Removawell strips (Dynatech) were pre-coated with
50 µl of 10 µg/ml TS2/16 anti-1 integrin in 0.1 M sodium carbonate (pH 9.0) overnight at 4 °C. The wells
were aspirated and washed three times with PBS and then coated with 50 µl of 3 µg/ml 31 integrin for 3 h at 4 °C. The wells were aspirated and blocked with 1% BSA in 0.1 mM MnCl2, 20 mM Tris, 150 mM NaCl, pH 7.6, for 30 min. The wells were then incubated
with 125I-labeled MBP-invasin-497 (32) (7.75 µCi/µg)
alone or in the presence of the indicated ligands at room temperature
for 3 h. The wells were then washed three times, and the bound
radioactivity was quantified using a gamma counter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Binding of TSP1 antibody F18 to platelets
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Fig. 1.
Antibody F18 1G8 recognizes TSP1.
A, wells in a 96-well plate were coated by incubating with
TSP1 (1 µg/well) diluted in PBS with 1 mM calcium
or 2.5 mM EDTA. After removing unbound TSP1 and blocking
nonspecific sites with 1% BSA, binding of F18 to the respective wells
was measured in buffer containing 1 mM CaCl2 or
5 mM EDTA as indicated. Wells were incubated with 15 µg/ml F18 for 2 h followed by washing and incubation with
125I-anti-mouse IgG (84,000 cpm/well) for 1 h. Bound
radioactivity was corrected for nonspecific binding to wells without
TSP1 (248 cpm for EDTA and 174 cpm for calcium) and is presented as
mean ± S.D., n = 2. B, wells were
coated with F18 (2 µg/well) diluted in 0.1 M sodium
carbonate, pH 9. Binding of 125I-TSP1 (10 µCi/µg,
105,000 cpm/well) was determined in the presence of calcium or EDTA as
indicated. Nonspecific binding of 125I-TSP1 to wells
without antibody (576 cpm in EDTA and 311 cpm in Ca) was subtracted,
and net binding is presented.
IIb3
integrin antibody 10E5 reproducibly reduced
activation-dependent binding of F18 to platelets (35 ± 7% inhibition for six independent experiments). The TSP1 antibody
C6.7, which blocks platelet aggregation and TSP1 binding to the
platelet TSP1 receptor CD47 (44), also did not significantly inhibit
binding of F18 Fab to activated platelets (22% at 20 µg/ml).
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Fig. 2.
Binding to fibronectin specifically induces
the TSP1 epitope recognized by F18. A, binding of
125 I-F18 to TSP1 immobilized on various matrix proteins.
The indicated proteins (2.5 µg/well in PBS) were coated on Immulon 2 polystyrene wells. After blocking with PBS containing 1% BSA, TSP1 at
the indicated concentrations was added and incubated for 1 h at
37 °C. The wells were washed three times and incubated with
125I-F18 (15 µCi/µg) for 1 h at 37o.
The wells were washed, and bound radioactivity was quantified and
presented as mean ± S.D., n = 2. F18 binding to
TSP1 was significantly enhanced in a dose-dependent fashion
when TSP1 was coated on fibronectin (FN) ( 
), but no significant effect on binding occurred when
TSP1 was bound to fibrinogen (FG) (
) or the BSA control
(
). B, binding of 125 I-A6.1 (14 µCi/µg)
to TSP1 immobilized on various matrix proteins. A6.1 binding was
determined to TSP1 incubated with immobilized fibronectin (),
fibrinogen (
), type I collagen (Coll) (), or BSA
control (). C, both plasma and cellular fibronectins
induce the F18 epitope on TSP1. Immulon 2 wells were coated using the
indicated concentrations of human plasma fibronectin (), foreskin
fibroblast fibronectin (
), or lung fibronectin (). The wells were
incubated with 25 µg/ml TSP1 followed by 125I-F18 (3 µCi/µg). Net binding, corrected for nonspecific binding to wells
without fibronectin and preincubated with TSP1 (198 cpm), is presented
as mean ± S.D., n = 2. D, fibronectin
but not heparin binding induces the F18 epitope. Wells coated with 1 µg/well plasma fibronectin, 1 ng of heparin-BSA, or BSA alone were
incubated with 0.5 µg/ml TSP1 followed by the indicated TSP1
antibodies, horseradish peroxidase-conjugated anti-mouse antibody, and
o-phenylenediamine. A duplicate plate with removable wells
was incubated with 0.5 µg/ml 125I-TSP1 to directly
quantify the bound TSP1 under each condition. The immobilized ligand
concentrations were chosen based on this assay to obtain equivalent
levels of bound TSP1. Antibody binding quantified by the enzyme-linked
immunoassay was normalized to the actual TSP1 bound and is presented as
mean ± S.D., n = 2.
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Fig. 3.
Effects of TSP1 antibodies on
125I-fibronectin binding to TSP1. TSP1 (20 µg/ml)
was coated on Immulon-2 wells alone or after mixing with the indicated
TSP1 antibody (4 µg/ml). Control wells were coated with BSA. The
wells were incubated with 125I-fibronectin (FN)
for 1 h, washed, and counted. Fibronectin binding is presented as
mean ± S.D. F18 (4 µg/ml) complexed with TSP1 significantly
increased fibronectin binding (p = 0.04), but the
control antibody ah-TSP1 did not (p > 0.6).
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Fig. 4.
TSP1 complexed with fibronectin induces the
F18 epitope on melanoma cells. A, exogenous TSP1 induces
binding of 125 I-F18 to melanoma cells. A2058 cells were
incubated for 1 h at 4 °C with 125I-F18 (15 µCi/µg) in the presence of the indicated concentrations of TSP1.
Bound radioactivity was determined after centrifugation of the cells
through oil; mean ± S.D., n = 3. B,
exogenous F18 does not induce binding of 125 I-TSP1 to
melanoma cells. A2058 cells were incubated with 125I-TSP1
(10 µCi/µg) in the presence of the indicated concentrations of F18.
C, fibronectin enhances TSP1-dependent binding
of F18 to melanoma cells. 125I-F18 (3 µCi/µg) binding
to A2058 melanoma cells at 25 °C was measured in binding medium
containing divalent cations alone (Basal) or in the presence
of 5 µg/ml TSP1. In cells pre-incubated with fibronectin (10 µg/ml), F18 binding was further enhanced. In buffer without cations
and containing 5 mM EDTA (basal+EDTA),
fibronectin/TSP1-stimulated binding of F18 was inhibited with
p = 0.0002. The addition of mAb13 (10 µg/ml), a
1-specific blocking antibody, completely inhibited
TSP1-fibronectin-stimulated F18 binding in the presence of calcium
(p < 0.0001). D, TSP1 does not enhance
binding of 125I-F18 to MDA-MB-435 breast carcinoma cells.
125I-F18 (3 µCi/µg) binding in MDA-MB-435 cells was
measured in the presence of 5 µg/ml TSP1 alone or with the addition
of the 1-specific blocking antibody mAb13 (10 µg/ml).
1 Integrin-dependent Binding of TSP1
Antibody F18 to Melanoma Cells--
Of the known cell surface TSP1
receptors, only integrins require divalent cations for binding to TSP1,
suggesting that binding of TSP1 to an integrin induced the epitope
recognized by F18 on platelets and melanoma cells. We considered both
integrins that have been reported to bind directly to TSP1
(31, 41,
51, IIb3,
and v3) and integrins that could bind
fibronectin to serve as a bridging ligand for TSP1
(41, v1,
and 51). Using A2058 melanoma cells, we
could not inhibit TSP1-enhanced F18 binding using an v
integrin antagonist (Fig. 5), but a
1 integrin-blocking antibody inhibited the
fibronectin-enhanced binding of F18 to melanoma cells (Fig.
4C, p < 0.0001).
View larger version (20K):
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Fig. 5.
TSP1-fibronectin-dependent
binding of 125I-F18 to melanoma cells is dependent on
41
and
51
integrins. A, a suspension of A2058 melanoma cells was
incubated with 125I-F18 (3 µCi/µg) alone
(Control) or in the presence of 5 µg/ml TSP1
(+TSP1). For all other conditions, A2058 cells were
pre-incubated with fibronectin for 30 min, centrifuged, and resuspended
in binding buffer. Binding of 125I-F18 was measured to
these cells (+TSP1+FN) alone or in the presence of
function blocking antibodies for 41 (5 µg/ml) or 51 (5 µg/ml), 1 µM 4l integrin antagonist
(4-((2-methylphenyl)aminocarbonyl)aminophenyl)acetyl-LDVP
(
LDVP), 0.4 mM
51 peptide antagonist GRGDNP, or 1 µM v integrin antagonist SB223245.
Significant inhibition relative to the control containing both TSP1 and
fibronectin is indicated by an asterisk for:
41-blocking antibody, p = 0.0014; 51-blocking antibody,
p = 0.0016; 4l integrin
antagonist LDVP, p < 0.0001; and the
51 peptide antagonist GRGDNP,
p < 0.0001. B, TSP1-fibronectin-mediated
binding of F18 does not require an intact cytoskeleton. Melanoma cells
were resuspended in complete cell culture medium, treated with
cytochalasin D (1 µM), nocodazole (10 µM),
or an equivalent concentration of dimethyl sulfoxide (DMSO)
for 30 min at room temperature in the presence or absence of
fibronectin, and then centrifuged and resuspended with
125I-F18 (6.2 µCi/µg) alone (control) or with 5 µg/ml
TSP1 and 10 µg/ml fibronectin (TSP1+FN). Binding was
determined after 1 h with orbital mixing at 4 °C.
1 integrin dependence was specific to cells where
exogenous TSP1 enhanced F18 binding (Fig. 4D). Basal binding
of F18 to MDA-MB-435 breast carcinoma cells was only slightly
stimulated by exogenous TSP1, and the 1 integrin
antibody had no significant effect on F18 binding to MDA-MB-435 cells
(Fig. 4B). Both of these negative results suggested that
direct binding of TSP1 to the 31
integrin, which is the primary adhesive receptor for TSP1 in these
cells (46), does not induce the F18 epitope.
subunit antagonists further
suggested that the TSP1-fibronectin complex is bound through integrins
that recognize fibronectin (Fig. 5A). Function-blocking
antibodies specific for 41 and
51 integrins both inhibited F18 binding to or below basal levels (p < 0.002). Specific peptide
antagonists of 41 (35) and
51 integrins (34) yielded similar
inhibition, whereas an v-specific antagonist (36) was
inactive. Antibody and peptide antagonists of several other known TSP1
receptors, including CD36, CD47, and 31,
were tested but were also negative (results not shown).
1 integrins on the cell surface (Figs. 4 and
5) induces a conformation change in TSP1 detected by the F18 antibody.
Because binding of fibronectin to cell surface integrins induces
conformation changes that initiate fibronectin matrix assembly
(reviewed in Ref. 47), which may also be induced by adsorption of
fibronectin to plastic, the cytoskeleton could be required as a
scaffold for cell surface fibronectin to induce this epitope on TSP1.
However, neither disruption of the actin cytoskeleton using
cytochalasin D nor disruption of microtubules using nocodazole
significantly inhibited the TSP1/fibronectin-mediated binding of F18 to
melanoma cells (Fig. 5B). Therefore, the observed response
does not require an intact cytoskeleton.
31
Integrin-dependent Adhesion--
Because we noted
previously that spreading of breast carcinoma cells on a TSP1
substrate, which is mediated by 31
integrin, was enhanced when the protein was adsorbed in the absence of
divalent cations (46), we examined the effect of F18 binding on the
adhesive activity of immobilized TSP1 for this integrin. In preliminary experiments, we found that adding soluble F18 moderately stimulated spreading of MDA-MB-435 breast carcinoma cells on immobilized TSP1
(results not shown). Because TSP1 immobilized on a plastic substrate
may have limited ability to undergo conformational changes in response
to antibody binding, we repeated this experiment by pre-incubating TSP1
in solution with TSP1 antibodies prior to adsorbing the complex on
plastic (Fig. 6). Remarkably, F18
strongly enhanced the spreading of MDA-MB-435 and MDA-MB-231 breast
carcinoma cells on substrates coated with a low concentration of TSP1.
Formation of a complex of TSP1 with F18 dramatically increased the
outgrowth of both lamellar and filopodial processes on MDA-MB-231 cells attaching on TSP1 (Fig. 6A). This enhancement of cell
spreading was specific in that all of the other TSP1 antibodies
examined either had no effect or partially inhibited spreading of
MDA-MB-231 cells under the same conditions (Fig. 6B). The
spreading response in MDA-MB-231 cells, which have a largely inactive
31 integrin (46), was dependent on
activation of 1 integrins using antibody TS2/16 (Fig.
6C), indicating that 1 integrin activation
was required for the response to the F18-TSP1 complex. The
31 integrin in MDA-MB-435 cells is
normally partially active (46), and enhanced spreading on TSP1
complexed with F18 was stimulated only moderately by the
1 integrin-activating antibody (results not shown).
View larger version (26K):
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Fig. 6.
Binding of TSP1 to F18 enhances
31
integrin-mediated adhesive activity. A, phase contrast
micrographs of MDA-MB-231 breast carcinoma cells plated on substrates
coated with 15 µg/ml TSP1 alone in PBS with 2.5 mM EDTA
(left) or TSP1 in the same buffer complexed with 2.5 µg/ml
F18 (right). Cells were photographed after 60 min.
Bar = 50 µm. B, MDA-MB-435 cells were
plated on substrates coated with 15 µg/ml TSP1 alone (0)
or complexed with the indicated concentrations of F18 or 3 µg/ml TSP1
antibodies ah-TSP1, C6.7, A6.1, or A4.1. Cell spreading at 60 min was
quantified and is presented as mean ± S.D., n = 3. C, MDA-MB-231 cells were plated on substrates coated with
TSP1 alone or complexed with antibody F18. Adhesion of control cells
and cells activated with the 1 integrin antibody TS2/16
was assessed after 60 min. Cell attachment (solid bars) and
spreading (striped bars) are presented as mean ± S.D.,
n = 3. D, MDA-MB-231 cell spreading,
mean ± S.D., n = 3, was determined on substrates
coated with 30 µg/ml TSP1 alone (control), complexed with
7.5 µg/ml F18 (+F18) in the presence of 10 nM
IGF1 (+IGF), and complexed with F18 and in the presence of
IGF1 alone (IGF+F18) or in the presence of 5 µg/ml
indicated integrin function-blocking antibodies:
31, P1B5; 1, mAb13;
41, P4C2; or
51, P1D6.
31 integrin (Fig. 6D). In this
experiment, 31 integrin was activated
using IGF1, and complexing the TSP1 with F18 further stimulated the
IGF1-dependent spreading. Both
31- and 1-specific
function blocking antibodies reversed the F18-dependent spreading of IGF1-activated cells on TSP1, whereas the
41- and 51-blocking antibodies that prevented
TSP1-fibronectin-mediated F18 binding to melanoma cells had no effect
on F18-enhanced adhesion (Fig. 6D). Therefore, the
F18-enhanced spreading is not mediated by fibronectin on the surface of
breast carcinoma cells.
31 integrin-mediated responses in two
other cell types. F18 enhanced spreading of human umbilical vein
endothelial cells on TSP1 (Fig.
7A). As expected, based on the
known regulation of 31 integrin
activation by cell contact in endothelial cells (13), cells from sparse cultures showed greater stimulation of spreading on the immobilized F18-TSP1 complex than did cells from confluent cultures. Similarly, F18
stimulated outgrowth of neurite-like processes in OH-1 small cell lung carcinoma cells on a TSP1 substrate (Fig. 7B). A
2.3-fold stimulation of process formation was observed in cells without EGF, whereas cells treated with EGF did not show a significant response. In the presence of EGF, the outgrowth response to immobilized TSP1 is presumably maximal and could not be further stimulated.
View larger version (20K):
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Fig. 7.
Stimulation of
31-mediated
responses to TSP1 by F18 in endothelial and small cell lung carcinoma
cells. A, sparse or confluent human umbilical vein
endothelial cell (HUVEC) cultures were plated on substrates
coated with 25 µg/ml TSP1 alone or complexed with F18. Cell
spreading, mean ± S.D., was quantified microscopically.
B, OH-1 small cell lung carcinoma cells were dissociated
from floating aggregates and plated on substrates coated with 30 µg/ml TSP1 or TSP1 complexed with 10 µg/ml F18. Process outgrowth
was assessed after 100 min for untreated cells or cells in the presence
of 10 ng/ml EGF. Results are presented as mean ± S.D.,
n = 3.
31 Integrin-dependent
Adhesive Activity of Immobilized TSP1--
Most cell types that
interact with TSP1 through the 31
integrin also express integrins that recognize fibronectin, precluding an analysis of the effect of fibronectin binding on the interaction of
TSP1 with 31 integrin. However, some
small cell lung carcinoma cells lack any functional fibronectin
receptors but exhibit 31 integrin-dependent adhesion and outgrowth of neurite-like
processes on TSP1 (11). We therefore used these cells to examine the
effect of complexing TSP1 with fibronectin on its recognition by
31 integrin. Complexes of TSP1 with
fibronectin or with antibody F18 produced similar enhancements of
OH-1 process outgrowth relative to cells plated on TSP1 alone
(Fig. 8). Neither F18 nor
fibronectin-coated substrates alone induced neurite formation.
Therefore, conformational changes induced by binding of fibronectin to
TSP1 also enhance its ability to interact with the
31 integrin.
View larger version (8K):
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Fig. 8.
Binding to fibronectin enhances an
31
integrin-mediated cellular response to TSP1. OH-1 cells were
plated on substrates coated with 40 µg/ml TSP1 alone or complexed
with 10 µg/ml F18, 10 µg/ml F18, 40 µg/ml TSP1 complexed with 20 µg/ml fibronectin, or 20 µg/ml fibronectin alone. Neurite-like
process formation was assessed at 120 min; mean ± S.D.,
n = 3.
31 Integrin--
The above data
demonstrated that changing the conformation of TSP1 modulates
31 integrin-mediated responses to TSP1 in
three cell types but did not exclude the possibility that fibronectin modulated the function of this integrin indirectly. To directly examine
the effect of fibronectin on binding of TSP1 to
31 integrin, we established a competitive
assay to measure the affinity of TSP1 for
31 integrin (Fig.
9). We used an 125I-labeled
invasin fragment as a tracer, because preliminary experiments indicated
that TSP1 binding to 31 integrin was of
too low an affinity to quantify in a direct assay, consistent with a
previous report that recombinant 31
integrin binding to immobilized TSP1 could not be detected using a
direct binding assay (32). In contrast, TSP1 was an effective
competitive inhibitor of invasin binding to purified
31 integrin (Fig. 9A). Using
the LIGAND program (48), the apparent Ka for this
interaction was determined to be 2.6 ± 0.3 × 106 M
1. Inhibition by
TSP1 was specific, because fibronectin did not inhibit invasin binding
to the same integrin (Fig. 9B and results not shown).
Complexing TSP1 with fibronectin, however, increased the activity of
TSP1 to inhibit invasin binding (Fig. 9B, p = 0.012 compared with TSP1 alone using a 2-sided t test).
Based on the dose dependence for inhibition by the TSP1-fibronectin complex (Fig. 9A), complexing with fibronectin significantly
increased the affinity of TSP1 for 31
integrin (4.8 ± 1.5 × 106
M1, p = 0.004).
Therefore, the stimulation of TSP1 binding to
31 integrin by fibronectin could be
verified using purified proteins.
View larger version (18K):
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Fig. 9.
Fibronectin directly modulates TSP1 binding
to
31
integrin. A, TSP1 inhibits MBP-invasin binding to purified
31 integrin.
31 integrin was adsorbed on Immunlon-2
Removawells (150 ng/well) pre-coated with the 1 integrin
antibody TS2/16. After blocking with BSA, the wells were incubated with
0.5 µg/ml 125I-MBP-invasin alone or in the presence of
the indicated concentrations of TSP1 () or TSP1 complexed with
fibronectin at a 4:1 molar ratio () and diluted in Tris-buffered
saline, pH 7.6, containing 0.1 mM MnCl2. The
bound fraction for a representative experiment is presented as
mean ± S.D., n = 3 and is corrected for
nonspecific binding of invasin to wells coated with BSA alone. Curves
were calculated by fitting the data to a single-site competitive
binding model using the LIGAND program. B, wells coated with
31 integrin were incubated with 0.5 µg/ml 125I-MBP-invasin alone (control) or in
the presence of 100 µg/ml TSP1, 100 µg/ml TSP1, and 10 µg/ml
fibronectin (TSP1+FN), or 10 µg/ml fibronectin. Net
binding is presented as mean ± S.D., n = 5.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
31 integrin to recognize TSP1 is
regulated by the former pathway in several cell types (11, 13, 46). However, we now present evidence for an additional mechanism to modulate the same integrin-ligand interaction based on allosteric regulation of integrin ligand conformation. The active conformation of
TSP1 is preferentially recognized and can be induced or stabilized by
the TSP1 antibody F18. We further identified fibronectin as an
extracellular matrix and cell surface-associated protein that specifically induces the same conformational and functional state of
TSP1.
41 and 51 integrins, presumably to bind fibronectin. Although most high affinity
binding of TSP1 to melanoma and other cell types is mediated by
sulfated glycoconjugates (37, 46), F18 does not recognize TSP1 bound to
this ligand but instead binds to a minor fraction of the TSP1 that
binds to the cell through fibronectin and its 1 integrin
receptors.
View larger version (34K):
[in a new window]
Fig. 10.
Models for the conformational modulation of
TSP1 interactions with cells by TSP1 antibody F18 and fibronectin.
A, antibody F18 recognizes an epitope on cell surface TSP1
specifically induced by complexing with fibronectin bound to cells via
the fibronectin-specific integrins 51 and
41. B, the same
conformation in immobilized TSP1, induced by either F18 or fibronectin,
enhances recognition of TSP1 by cells expressing
31 integrin.
31 integrin (Fig. 10B).
Because most cells also express fibronectin receptors, we could only
quantify this activity in small cell lung carcinoma cells that do not
recognize fibronectin. However, fibronectin presumably modulates
interactions of TSP1 with 31 integrin in other cell types as well. 31 integrin
participates in the interactions of several cell types with TSP1 (11,
13, 46, 50, 51), and therefore this regulation may be important for
understanding the effect of TSP1 on many cell types. In addition, the
apparently unfavorable equilibrium between the active conformation and
the less active calcium-replete form of TSP1 in physiological media may
explain the failure to detect direct binding of TSP1 to recombinant 31 integrin (32).
41 and
51 integrins have been reported to be
TSP1 receptors in several cell types (46, 51, 52), the present results
suggest that the role of these integrins in adhesion to TSP1 may be
indirect rather than direct. We have observed that medium containing
serum provides sufficient fibronectin to promote TSP1 binding via this
mechanism.2 Therefore,
fibronectin derived from the growth media or secreted by the cells
during processing may provide sufficient fibronectin to mediate
41- and
51-dependent interactions
with TSP1. We must therefore re-examine the roles of these two
integrins in directly mediating adhesion to TSP1. To date, we
have established a role for this pathway only in the binding of soluble
TSP1 to melanoma cells. However, this allosteric regulation of TSP1
probably affects the responses of other cell types to mixed
fibronectin/TSP1 matrices.
31 integrin.
This modulation of receptor binding appears to be specific in that
heparin and fibrinogen binding activities of TSP1 were not modulated by
the same conformational change. Conversely, fibronectin binding
specifically modulates signal transduction from TSP1 in the matrix by
regulating the ability of TSP1 to interact with a specific integrin
receptor (Fig. 9). Thus, both intracellular and extracellular signaling pathways may mediate the combined effects of these two proteins on cell function.
v3 and
IIb3 integrins have been implicated as
TSP1 receptors on platelets (62, 63). Although an
IIb3 antibody partially inhibited F18
binding to activated platelets, previous work has questioned the
relevance of this antibody inhibition to the proposed function of
IIb3 as a TSP1 receptor (64). Our results
in melanoma cells further suggest that the contribution of
IIb3 integrin in F18 binding to platelets
may also be to bind fibronectin as a bridging molecule rather than
acting as a direct receptor for TSP1. The role of 1
integrins that bind fibronectin as indirect receptors for TSP1 on
platelets should therefore be further examined.
51 integrin induces
exposure of a self-association site that mediates fibronectin fiber
assembly (reviewed in Ref. 47). The exposure of an anti-adhesive site
in fibronectin may also be conformationally regulated and is induced
following binding to glycosaminoglycans (65). The binding of fibrinogen
to the platelet surface also induces a change in its conformation and exposes a neoepitope recognized by a function-blocking antibody (42).
In TSP1, conformational changes are now known to regulate its binding
to two integrins, v3 (43) and
31. The regulation of
v3 integrin binding requires covalent
modification of disulfide bonds in TSP1 (43), whereas regulation of
31 integrin binding appears to be
reversible. It is likely that further examples of such functional
regulation will be found in other matrix proteins.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Bldg. 10, Rm.
2A33, 10 Center DR. MSC 1500, National Institutes of Health, Bethesda, MD 20892-1500. Tel.: 301-496-6264; Fax: 301-402-0043; E-mail: droberts@helix.nih.gov.
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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