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(Received for publication, August 19,
1994; and in revised form, November 18, 1994) From the
CD36 is a multifunctional cell surface glycoprotein that acts as
a surface receptor for thrombospondin (TSP), and thereby may mediate
adhesive interactions between cells and substrata, platelets and other
cells, and macrophages and apoptotic neutrophils. The identity of the
TSP binding site on CD36 is controversial and may involve more than one
structural domain. We have constructed a series of recombinant
bacterial GST/CD36 fusion proteins that span nearly all of the CD36
molecule and have demonstrated that fusion proteins containing the
region extending from amino acid 93 to 120 formed specific, saturable,
and reversible complexes with TSP. As with intact CD36, binding was
calcium-dependent, was independent of which ligand was immobilized, and
was blocked by monoclonal antibodies to both CD36 and TSP.
Stoichiometry and affinity of the fusion proteins for TSP were
consistent with that of the intact protein. We also demonstrated that
these fusion proteins competitively inhibited binding of TSP to
purified platelet CD36 and to cell surface CD36 on peripheral blood
monocytes and CD36 cDNA-transfected melanoma cells. These data
demonstrate that the region between amino acids 93 and 120 has all of
the characteristics required of the TSP binding domain. Thrombospondin (TSP) ( Much of the function of TSP occurs on cellular surfaces where it
interacts with several unrelated receptors, including the We have shown previously that the
CD36-TSP interaction is involved in platelet-monocyte adhesion (32, 41) and platelet-tumor cell adhesion (42) , whereas Leung (43) has demonstrated a role in
platelet aggregation, Asch et al.(44) a role in tumor
substratum adhesion, and Savill and Hogg (37) a role in
macrophage uptake of apoptotic neutrophils. Regulation of TSP receptor
function of CD36 is complex. Although we have shown that certain cells (e.g. melanoma cells and 3T3 fibroblasts) transfected with the
CD36 cDNA acquired TSP binding capacity(42) , other cells (e.g. COS-7) did not(28) . In addition, resting
platelets express CD36 but do not bind TSP with appropriate
stoichiometry. The structure of the TSP binding domain in CD36 is
controversial. Leung et al.(45) have proposed a
complex model based on studies using small synthetic peptides. They
hypothesized that CD36 receptor function is controlled by two
independent structural domains, one of which, located in the region
from amino acids 139-155, binds TSP with low affinity and then
induces a conformational change in TSP, resulting in high affinity
binding to a region from amino acids 93-110 in CD36. Asch et
al.(46) have further proposed that phosphorylation and
dephosphorylation of CD36 at Thr
Figure 1:
Orientation of the GST/CD36 fusion
proteins within the CD36 sequence. Fusion proteins are identified by
their start and stop sites indicated by numbers to the left and right. Tm refers to the single
transmembrane domain at the carboxyl terminus of CD36(48) . The shaded fusion proteins are those that bind TSP. The predicted
molecular weights followed by the mean of those obtained from SDS-PAGE (n = 4) are as follows: FP93-298, 48,166/49,000;
FP67-157, 34,950/35,000; FP5-143, 40,253/41,000;
FP298-439, 40,876/43,400; FP390-471, 37,870/38,000;
FP118-182, 32,113/31,500; FP93-120, 30,233/30,000. The
cleaved peptides have the following experimentally derived mean
molecular weights (n = 4): 93-298, 20,666;
67-157, 9000; 5-143, 13,000; 298-439, 14,433;
390-471, 11,700; 118-182, 4500; 93-120,
2800.
Figure 2:
Time
course and reversibility of
Figure 3:
Binding of
Similar saturation binding isotherms to
those in Fig. 3were obtained when CD36 fusion proteins were
immobilized and labeled TSP was used as a ligand or when CD36 peptides
cleaved from the CD36/GST fusion proteins replaced the fusion proteins
in these assays (data not shown). All of the binding data were analyzed
using nonlinear curve fitting program ENZFITTER (by Robin J.
Leatherbarrow, Elsevier Biosoft). Apparent dissociation constants are
listed in the figure legends. Analysis of these binding data using the
program LIGAND (Elsevier Biosoft) gave best fits for a single site
model. To estimate stoichiometry, the amount of immobilized fusion
protein was determined by measuring radiolabeled protein adsorbed to
the wells at saturating (10 µg/ml) input concentrations. Bound TSP
was then determined as the B Several approaches were used to demonstrate specificity of the
binding interactions between GST/CD36 fusion proteins and TSP. We
found, as shown in Fig. 4, that fluid phase CD36 blocked the
interaction of
Figure 4:
Fluid phase CD36 blocks
Figure 5:
Specificity of TSP interactions with
GST/CD36 fusion proteins. Binding of
Figure 6:
GST/CD36 fusion proteins block
Inhibition of binding of TSP to cell
surface CD36 was examined using Bowes melanoma cells stably transfected
with the CD36 cDNA and purified peripheral blood monocytes. We have
shown previously that Bowes CD36 transfectants acquired the capacity to
bind TSP in a specific, calcium-dependent manner(42) . We now
show, as seen in Fig. 7, that the GST/CD36 fusion proteins
93-298, 67-157, 93-120 and 118-182 at
concentrations
Figure 7:
GST/CD36 fusion proteins block
Figure 8:
GST/CD36 fusion proteins block binding to
Figure 9:
Calcium-independent binding of
FP118-182 to
Recombinant GST/CD36 fusion proteins produced by bacteria
were used to map the TSP binding domain of CD36. Bacterial fusion
proteins may have certain advantages compared with synthetic peptides
in domain analysis, because larger regions can be examined and
radiolabeling can be accomplished without affecting the sequence. Our
results showed specific, saturable, and reversible complex formation
between TSP and four fusion proteins that share a 27-amino acid region
of overlap between positions 93 and 120. As with intact CD36, we also
found that binding was calcium-dependent. Numerous approaches were
undertaken to demonstrate that these binding interactions were specific
and not related to artifactual influences of protein immobilization on
polystyrene. We found in all cases that binding of radiolabeled ligands
was inhibited by addition of excess unlabeled protein. In addition,
binding was independent of which ligand was immobilized, and was
blocked by monoclonal antibodies to both CD36 and TSP. Similar binding
of TSP to CD36 peptides cleaved by thrombin or Factor Xa from the GST
moiety and the lack of binding to fusion proteins not containing the
27-amino acid region demonstrated that complex formation was not
related to the GST portion of the fusion proteins. We also demonstrated
that these fusion proteins competitively inhibited binding of TSP to
purified platelet CD36 and to cell surface CD36 on peripheral blood
monocytes and CD36 cDNA transfected melanoma cells. From analysis of
the equilibrium binding isotherms we found that the four fusion
proteins had somewhat higher apparent affinities than CD36 for TSP.
This can be accounted for by either the size of the peptides which may
allow them to form conformations more advantageous for binding to TSP
or by the lack of glycosylation on the recombinant bacterial proteins
which might improve exposure of the amino acids required for binding.
That the smallest peptide, FP93-120, had the highest affinity
favors the first explanation. In sum, these data demonstrate that
peptides containing the region between amino acids 93 and 120 have all
of the characteristics required of the TSP binding domain: i.e. calcium-dependent complex formation with TSP, calcium-dependent
inhibition of TSP complex formation with intact CD36, inhibition of
calcium-dependent TSP binding to cell surface CD36, and stoichiometry
and affinity consistent with that of the intact protein. Asch et
al.(46) have shown that extracellular phosphorylation and
dephosphorylation of a Thr residue at position 92 of CD36 regulates TSP
binding, i.e. that phosphorylation blocked TSP binding and
that dephosphorylation led to ``activation'' of CD36 as a
functional TSP receptor. FP93-120 begins at position 93 and
therefore does not contain Thr Leung et al.(45) have recently shown that a synthetic peptide
corresponding to the region between amino acids 93 and 110 also
inhibited CD36-TSP complex formation. Unlike the slightly larger
FP93-120, however, this peptide did not bind directly to TSP.
They reported, however, that the 93-110 peptide bound TSP if a
second peptide corresponding to the region from amino acids
139-155 was included in the reaction mixture. This latter peptide
was shown to bind to TSP in a calcium-independent, low affinity manner,
but not to inhibit TSP-CD36 complex formation. In fact, complex
formation was enhanced in the presence of this second peptide. From
these data the authors put forward a two step mechanism for TSP
binding; one region (amino acids 139-155) attaches to TSP and
induces a conformational change in the TSP molecule exposing a second
site for the second CD36 region(93-110) to bind. Our kinetic
data, however, do not show a two step binding process, a change in rate
in the binding interaction, or two sites in the form of a high affinity
site and a low affinity site in the calculation of the apparent
dissociation constant. In addition, two of the fusion proteins,
93-120 and 118-182, each contain one of the putative
domains, whereas two, 67-157 and 93-298, contain both. No
improvement in binding of the two-domain peptides to TSP was seen
compared with FP93-120 alone nor did addition of FP118-182
enhance TSP binding to intact CD36 or any of the fusion proteins. An
alternative explanation of the role of peptide 139-155 may be in
stabilizing peptide 93-110 spatially so that binding to TSP
becomes possible. Our results would then suggest additional amino acids
from position 110-119 could serve the same function. FP118-182, the protein containing only the putative regulatory
domain, does, however, present an interesting anomaly. Rather than
augmenting TSP binding it inhibited binding between CD36 and TSP.
FP118-182 also bound TSP in a specific, although
calcium-independent, manner. The physiological significance of this
calcium-independent interaction between FP118-182 and TSP is
unclear, since TSP did not bind to intact CD36 in the absence of
calcium, and since neither intact CD36 nor fusion proteins containing
either one or both regions were able to block the calcium-independent
interaction between FP118-182 and TSP. However, that the
inhibitory monoclonal antibody 8A6 partially blocked binding of TSP to
both FP93-120 and FP118-182 suggests that the region of
CD36 containing the 118-182 sequence may be structurally close to
the TSP binding domain. The simplest conclusion from our studies is
that the TSP binding domain in CD36 resides in the 27-amino acid region
between positions 93 and 120. Although an important regulatory role of
Thr
Volume 270,
Number 7,
Issue of February 17, 1995 pp. 2981-2986
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
EVIDENCE FOR A SINGLE CALCIUM-DEPENDENT BINDING SITE ON CD36 (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)is a large molecular weight
adhesive glycoprotein functionally implicated in numerous processes
relevant to vascular biology, development, and tumor
biology(1) . These include platelet aggregation(2) ,
angiogenesis(3, 4) , cell-substratum
adhesion(5) , transforming growth factor 
activation(6) , smooth muscle cell proliferation(7) ,
and plasmin generation(8, 9) . TSP is a major
component of platelet 
-granules from which it is secreted upon
platelet activation (10) and is synthesized and incorporated
into extracellular matrix by a variety of cell types (5, 11, 12, 13) in response to
cytokines. TSP is also produced by monocytes, by certain epithelial
cells, such as breast epithelia, and by neural
tissues(14, 15, 16, 17, 18) . 
integrin 
/(19) ,
sulfated glycolipids(20) , heparan sulfate
glycosaminoglycans(21) , and CD36(22) . Specific and
independent binding domains in TSP for these receptors have been
described. These include an RGD domain that interacts with the integrin
receptor(23) , an NH
-terminal region that interacts
with heparin and sulfated glycolipids(1, 20) , and a
properdin-like repeat containing SVTCG sequences that interacts with
CD36 (24, 25) and/or an incompletely characterized M
50,000 glycoprotein(26) . CD36, which is
also known as platelet glycoprotein IV(27) , is an M 88,000 transmembrane glycoprotein expressed on
platelets(28, 29) , erythroid precursors(30) ,
monocytes and macrophages(31, 32) , and certain
endothelial and specialized epithelial cells(33) . It is a
member of a small gene family (34) and is a multifunctional
receptor involved in binding and/or uptake of fatty acids(35) ,
oxidized low density lipoprotein(36) , apoptotic
cells(37) , photoreceptor outer segments(38) ,
malaria-infected erythrocytes(39) , and collagen(40) .
Dissecting the functional domains of this receptor is thus critical to
understanding its complex biology.
might control TSP
binding. They also showed, using small synthetic peptides that a domain
encompassing residues 87-99 bound TSP, although its affinity,
stoichiometry, and effect on TSP binding to cells were not measured. To
address these issues we have constructed a series of recombinant
bacterial GST/CD36 fusion proteins that span nearly all of the CD36
molecule and that include all of these putative domains, either alone
or in combination. We have found that the CD36 region extending from
amino acid 93 to 120 has all of the properties required of a TSP
binding domain.
Materials
Glutathione-agarose was obtained from
Sigma. Percoll, Ficoll-Paque, and all other chromatography media were
purchased from Pharmacia Biotech Inc. Na
I was obtained
from Amersham Corp., and 96-well removable strips (Immulon-4
Removawell) from Dynatech Laboratories, Inc. Platelet TSP and CD36 were
purified as described
previously(40, 41, 48, 49) . Murine
monoclonal anti-TSP IgG (46.4 and 11.4) were prepared as described
previously (41) . These antibodies recognize a
conformational-sensitive epitope that has not been mapped. They have
been shown previously to block CD36-TSP
binding(32, 42) . Anti-CD36 IgG (8A6) was a kind gift
from Dr. J. Barnwell, New York University Medical Center (New York).
This antibody has also been shown to inhibit CD36-TSP
interactions(24, 32, 42, 44, 48) .
Rabbit antisera were raised against purified platelet CD36 and were
specific as assayed by enzyme-linked immunosorbent assay and Western
blot(48) .Cells and Cell Lines
Platelet-rich plasma and
outdated platelet concentrates were obtained from the New York Blood
Center. Bowes melanoma cells stably transfected with human CD36 cDNA
(or control plasmid) were prepared and maintained as described
previously (42) . CD36 expression was confirmed prior to all
studies by immunofluorescence flow cytometry. Peripheral blood
monocytes were isolated from buffy coats obtained from the Puget Sound
Blood Center (Seattle, WA) by sequential centrifugation on Ficoll and
Percoll gradients(47) . Purified monocytes were washed in
phosphate-buffered saline and resuspended in RPMI 1640 containing
gentamicin and supplemented with 5% heat-inactivated human AB serum
(Sigma). Monocytes were >98% viable as determined by trypan blue
exclusion. More than 90% of the purified cells were monocytes as
determined by immunofluorescence flow cytometry using a panel of
anti-human monocyte monoclonal antibodies.Preparation of GST/CD36 Fusion Proteins
CD36 cDNA
was digested with restriction enzymes to yield seven fragments spanning
98% of the coding region. These include a 270-bp Sau3A
fragment from bp 408-676 that encodes from amino acid 67 to 157
in intact CD36, a 620-bp SnaBI-HaeIII fragment from
bp 483-1103 that encodes from amino acid 93 to 298, a 433-bp HaeIII fragment from bp 1103-1536 that encodes from
amino acid 298 to 439, a 351-bp HincII fragment from bp
1376-1737 that encodes from amino acid 390 to 471 (the carboxyl
terminus), a 416-bp HpaII-PvuII fragment from bp
222-638 that encodes from amino acid 5 to 143, a 194-bp NlaIV fragment from bp 565-759 that encodes from amino
acid 118 to 182, and an 82-bp SnaBI-NlaIV fragment
from bp 483-565 that encodes from amino acid 93 to 120. These
fragments were gel-purified and subcloned into the prokaryotic
expression plasmids pGEX-2T or pGEX-3X (Pharmacia) so as to maintain
reading frames downstream to the inducible LacZ promotor, and adjacent
to a fragment of the recombinant glutathione S-transferase
gene. GST/CD36 fusion proteins were prepared from large scale bacterial
cultures by chromatography of bacterial lysates on glutathione-agarose
beads. Proteins were extensively dialyzed against phosphate-buffered
saline after purification to remove soluble glutathione and were stored
at -20 °C. Their orientation within native CD36 is shown in Fig. 1. All plasmid constructs were mapped and insertion sites
sequenced by the dideoxynucleotide method (U. S. Biochemical Corp.) to
confirm that the fusion protein sequences were correct and in frame.
The fusion proteins were also examined by SDS-PAGE, Western blot, and
enzyme-linked immunosorbent assay to confirm size and document CD36
immunoreactivity. Molecular weights of the fusion proteins were also
determined by nondenaturing PAGE analysis as well as by gel filtration
(Superose 12, Pharmacia). The molecular weights are listed in the
legend of Fig. 1and were within the calculated range with less
than 5% difference between the calculated and experimentally determined
values. Sizing chromatography also indicated that the fusion proteins
did not form dimers or larger multimers. In addition, none of the
fusion proteins bound to purified intact CD36 in solid phase binding
assays. For some experiments the CD36 peptides were cleaved and eluted
from the fusion proteins bound to the agarose beads by treatment with
thrombin or factor Xa. The protease was then removed by incubation with
benzamidine-Sepharose.
Iodination of CD36, TSP, and Purified Fusion
Proteins
TSP, CD36, and GST/CD36 fusion proteins were labeled
with NaI using immobilized chloramine T (IODOBEAD;
Pierce) as described previously(41, 49) . Specific
activity was determined for each of the labeled proteins prior to each
experiment and ranged from 0.1 to 0.5 µCi/µg.Solid Phase Binding Assays
Solid phase binding
assays were used to quantify TSP interactions with CD36 and recombinant
GST/CD36 fusion proteins. One of the ligands (e.g. TSP, CD36,
or fusion protein) was immobilized on wells in a detachable 96
microwell plate by overnight incubation at 4 °C. Saturable coating
conditions were first determined using radiolabeled proteins. CD36 was
thus adsorbed at 4 µg/ml in phosphate-buffered saline, whereas TSP
and fusion proteins were adsorbed at 10 µg/ml in carbonate buffer
(100 mM NaHCO, 1 mM MgCl,
0.02% NaN, pH 9.8). The amount of protein coated on the
wells ranged from 200-280 ng. The wells were then washed three
times with 20 mM Tris, 150 mM NaCl, pH 7.4,
containing 0.05% Tween 20 (TBS-Tween) and then blocked with TBS-Tween
containing 0.5% bovine serum albumin. Radiolabeled ligands were then
added in TBS-Tween containing 1 mM CaCl and the
mixture incubated for 3 h at 22 °C. The wells were then washed
thoroughly three to four times with TBS-Tween, dried, and bound
radioactivity quantified by 
counter. Nonspecific binding was
determined by carrying out the binding in the presence of 5 mM EDTA or excess unlabeled ligand and was generally less than 10% of
total. For competition experiments the competing proteins were added
along with the labeled protein and incubated for 3 h.Cell Binding Assays
Binding of I-TSP
to suspensions of CD36-transfected Bowes melanoma cells or peripheral
blood monocytes was measured as described
previously(41, 42) . Inhibition studies were done
using 0.045 µM input concentrations of I-TSP
and CD36 or GST/CD36 fusion proteins at concentrations of 100-500
nM.
TSP Binds to GST/CD36 Fusion Proteins
Binding of
radiolabeled TSP to recombinant immobilized GST/CD36 fusion proteins
was time-dependent, reaching equilibrium at 2 h. As shown in Fig. 2, binding of I-TSP (0.045 µM)
to a representative fusion protein, 93-298, was completely
reversed at equilibrium by the addition of a 10-fold excess (0.5
µM) of unlabeled TSP. Dissociation was rapid and complete
by 30 min. Equilibrium binding studies were carried out for all seven
fusion proteins both by immobilizing the fusion proteins and using
labeled TSP as the ligand or by immobilizing TSP and using labeled
fusion proteins as the ligand. As shown in Fig. 3, upper
panel, three of the fusion proteins (67-157, 93-298,
and 5-143) bound to immobilized TSP in a concentration-dependent
manner with similar affinity as purified intact platelet CD36. Two
proteins (390-471 and 298-439) did not bind TSP,
demonstrating specificity. These data suggest that the TSP binding
domain lies within the 50-amino acid overlap region between residues 93
and 143 shared by the three peptides. To define this domain more
precisely and to explore the potential role of the regulatory sequence
(residues 139-155) proposed by Leung et al.(45) we studied the binding of TSP to two additional
fusion proteins FP93-120 and FP118-182. As seen in Fig. 3, lower panel, both of these peptides bound to
TSP in a saturable manner.
I-TSP binding to
FP93-298. A fixed concentration of I-TSP (0.045
µM) was added to immobilized FP93-298 and incubated
for timed points of 1 min to 6 h. The amount of bound TSP was measured
after extensive washing (
). At equilibrium (
) a 10-fold
(0.5 µM) excess of unlabeled TSP was added and incubated
for timed points of 1 min to 6 h and the amount of remaining bound TSP
bound was measured (
).
I-GST/CD36 fusion
proteins to immobilized TSP. I-CD36 or I-fusion proteins were added in increasing concentrations
to immobilized TSP for 3 h at 22 °C. The binding isotherms in the upper panel represent CD36 (
), FP93-298 (),
FP67-157 (
), FP298-439 (
), FP390-471
(
), and FP5-143 (
). The binding isotherms in the lower panel represent FP93-120 () and
FP118-182 (). The figures are drawn from one data set
whereas the dissociation constants are calculated as a mean of all the
data sets (n = 6), and the errors are calculated as
standard deviation (S.D.). The apparent dissociation constants for each
curve were calculated by nonlinear curve fitting and are as follows:
CD36, 227 ± 19.89 nM; FP93-298, 305 ± 32
nM; FP67-157, 153 ± 16.6 nM;
FP5-143, 35 ± 3.7 nM; FP93-120, 8.6
± 3.6 nM; and FP118-182, 67.3 ± 11.5
nM.
(maximal velocity)
from the binding isotherm (Fig. 3). From these data we
determined that 2.4 ± 0.2 GST/CD36 fusion protein molecules
complexed with each TSP, consistent with the homotrimeric structure of
TSP.I-TSP with the immobilized CD36/GST fusion
proteins in a concentration-dependent manner with IC
values very similar to the calculated k
values. Similarly, displacement studies using increasing amounts
of unlabeled TSP along with a fixed concentration (3 nM) of
labeled TSP (data not shown) revealed IC values similar to
the calculated k values. As shown in Fig. 5, we found that TSP binding was blocked by >95% by a
10-fold molar excess of unlabeled TSP or by inhibitory murine
monoclonal antibodies to either CD36 (8A6; 1 µg/ml) or TSP (11.4 or
46.4; 10 µg/ml). Control antibodies had no effect. Murine
monoclonal anti-CD36 8A6 was only a partial inhibitor of TSP binding to
FP93-120 and FP118-182.
I-TSP binding to immobilized GST/CD36 fusion proteins.
Increasing concentrations (1 nM to 2 µM) of
platelet-derived CD36 were added with a fixed concentration of I-TSP to immobilized fusion proteins as in Fig. 2.
Based on the value of 0% inhibition in the absence of CD36, the percent
inhibition for each concentration of CD36 was calculated for
FP93-298 (), FP67-157 (), FP298-439
(
), FP390-471 (), FP118-182 (),
FP5-143 (), and FP93-120 (
). This figure
represents one data set, whereas the numbers below indicate a mean of
all the data sets (n = 6) and the error is calculated
as S.D. The IC values for the TSP displacement by CD36 for
the fusion proteins are as follows: FP93-298, 68.99 ± 8.5
nM; FP67-157, 63.14 ± 10.2 nM;
FP5-143, 25.4 ± 6.7 nM; FP93-120, 5.27
± 2.5 nM; and FP118-182, 66.15 ± 11.5
nM.
I-TSP to immobilized
GST/CD36 fusion proteins was determined in the presence of 1 mM calcium (
), 5 mM EDTA (), 0.45 µM unlabeled TSP (open column with 
s), 1 µg/ml
murine anti-CD36 IgG 8A6 (&cjs2112;), and 10 µgml murine
anti-TSP IgG 45.1 (&cjs2113;) (n = 6; error calculated
as S.D.). Bound TSP values for the GST/CD36 fusion proteins are
compared with the values obtained with immobilized intact
platelet-derived CD36.
GST/CD36 Fusion Proteins Are Effective Inhibitors of
CD36-TSP Complex Formation and of CD36-dependent Cell Surface TSP
Binding
As shown in Fig. 6, the five GST/CD36 peptides
that bound TSP in solid phase binding assays were also effective
inhibitors of TSP complex formation with intact platelet-derived CD36.
The control fusion proteins (298-439 and 390-471) had no
effect. The IC values are listed in the figure legends and
are comparable with the k values derived from the
radioligand binding studies.
I-TSP binding to immobilized CD36. Increasing amounts of
GST/CD36 fusion proteins were added along with a fixed concentration of I-TSP (20 µg/ml) to immobilized CD36 as in Fig. 2. Binding was normalized to that in the absence of fusion
proteins. This figure represents one data set for each line, whereas
the numbers below indicate a mean of all the data sets (n = 6) and the error is calculated as S.D. The IC values calculated for each peptide are as follows: FP93-298
(), 60.3 ± 7.7 nM; FP67-157 (), 57.4
± 6.8 nM; FP5-143 (), 32.4 ± 7.8
nM; FP118-182 (), 70.2 ± 10.5 nM;
and FP93-120 (), 6.1 ± 4.2 nM and
FP298-439 ( ) and
FP390-471 () show values out of range for this
graph.
500 nM blocked I-TSP binding
to these cells, whereas FP298-439 or GST alone had no effect.
Similarly, as shown in Fig. 8, these fusion proteins also
blocked I-TSP binding to peripheral blood monocytes.
Interestingly, FP93-120 which showed a 5-fold higher affinity
than the other peptides in the solid phase in vitro assays did
not show effective inhibition of cellular TSP binding at the lower
concentration. Effective inhibition was only seen at the same
concentration as that observed for the larger fusion proteins
(100-200 nM), suggesting that the solid phase assay
provided an overestimate of the true affinity.
I-TSP binding to Bowes melanoma cells transfected with
CD36 cDNA. CD36 or GST/CD36 fusion proteins (500 nM) were
added to a cell suspension of CD36 cDNA transfected Bowes melanoma
cells (10
cells) in the presence of a fixed concentration
of I-TSP. After incubation for 60 min at 4 °C, bound
and free radioactivity were separated by centrifugation through
silicone oil. Binding of I-TSP to Bowes melanoma cells in
the presence of 1 mM CaCl (column 1), 5
mM EDTA (column 2), 500 nM of purified
platelet CD36 (column 3), 500 nM of FP93-298 (column 4), 500 nM of FP67-157 (column
5), 500 nM of FP5-143 (column 6), 50
nM of FP93-120 (column 7), 500 nM of
FP118-182 (column 8), a mixture of 500 nM FP118-182 and 50 nM FP93-120 (column
9), and 1 µM FP298-439 (column 10) are
shown (n = 6; error calculated as
S.E.).
I-TSP binding to peripheral blood monocytes. GST/CD36
fusion proteins were added to a cell suspension of purified peripheral
blood monocytes (10
cells) in the presence of a fixed
concentration of I-TSP. After incubation for 30 min at 4
°C, bound and free radioactivity were separated by centrifugation
through silicone oil. The binding of I-TSP to monocytes
in the presence of 1 mM CaCl (column 1),
5 mM EDTA (column 2), 1 µg/ml monoclonal
anti-CD36 IgG 8A6 (column 3), 500 nM of
FP93-298 (column 4), 500 nM of FP67-157 (column 5), 500 nM of FP93-120 (column
6), 500 nM of FP118-182 (column 7), 1
µM of FP298-439 (column 8), and 1
µM of GST (column 9) are shown (n = 4; error calculated as S.D.).
FP118-182 Binds TSP in a Calcium-independent
Manner
As shown in Fig. 5(closed bars) EDTA
completely inhibited the specific binding of TSP to immobilized CD36
and to all of the GST/CD36 fusion proteins except FP118-182. The
significance of this calcium-independent interaction between
FP118-182 and TSP is unclear, since TSP did not bind to intact
CD36 in the absence of calcium. Similarly, as shown in Fig. 9,
in the absence of calcium, neither intact CD36 nor the fusion proteins
containing the 50-amino acid overlap region (residues 93-143)
were able to block the interaction between FP118-182 and TSP.
FP93-298, which includes both the 93-143 overlap and the
118-182 domain, also did not block calcium independent binding to
FP118-182. Murine monoclonal anti-CD36 IgG 8A6, which blocked TSP
binding to purified platelet CD36, monocytes, CD36-transfected melanoma
cells, and GST/CD36 fusion proteins 67-157, 93-298, and
5-143 only partially blocked binding of TSP to 118-182. No
additive effect of FP118-182 on the calcium-dependent binding of
TSP to cells by 93-120 was seen (Fig. 7). In contrast to
the studies of Leung et al.(45) using a synthetic
peptide containing residues 139-154, we did not observe any
augmentation of TSP binding to cells or to purified CD36 in the
presence of FP118-182 and calcium. In fact, as seen in Fig. 6Fig. 7Fig. 8, FP118-182 inhibited
TSP-CD36 and TSP-cell interactions.
I-TSP. I-TSP (50
µg/ml) was added to immobilized FP118-182 in the presence of
various inhibitors. Column 1 shows the binding in the presence
of 1 mM CaCl, column 2 is in the presence
of purified platelet CD36 (1 µM) in 1 mM CaCl, column 3 is in 5 mM EDTA, and columns 4-7 are in 5 mM EDTA plus 1 µM CD36 and 500 nM FP93-298, FP67-157 or
FP5-143 respectively (n = 4; error calculated as
S.D.).
, whereas FP5-143 and
FP67-157 both contain Thr. All three of these
peptides bind TSP and inhibit TSP-CD36 complex formation with similar
kinetics, suggesting that Thr is not a necessary component
of the TSP binding domain. However, in vitro phosphorylation
of immobilized proteins by exposure to purified protein kinase C and
ATP (46) resulted in partial inhibition of TSP binding to
FP67-157 and intact CD36, but not to FP93-298 (data not
shown), suggesting that on the cell surface, phosphorylation at
position 92 could sterically hinder contact between TSP and the
immediately adjacent binding domain. is supported by its location immediately adjacent to
the TSP binding domain, no strong evidence supports a role for the
region between amino acids 118 and 182.
)
We thank Qinghu Zhang for his assistance with the
preparations of peripheral blood monocytes and for maintenance of the
Bowes melanoma transfected cells.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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