(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) ()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) .
Much of the function of TSP occurs on cellular surfaces where it
interacts with several unrelated receptors, including the 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.
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 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.
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 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 (
).
Figure 3:
Binding of 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.
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 (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.
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 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.
Figure 4:
Fluid phase CD36 blocks 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.
Figure 5:
Specificity of TSP interactions with
GST/CD36 fusion proteins. Binding of 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 µg
ml 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.
Figure 6:
GST/CD36 fusion proteins block 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.
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 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.
Figure 7:
GST/CD36 fusion proteins block 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.).
Figure 8:
GST/CD36 fusion proteins block binding to 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.).
Figure 9:
Calcium-independent binding of
FP118-182 to 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.).
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, 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.
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 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.