©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Recombinant GST/CD36 Fusion Proteins Define a Thrombospondin Binding Domain
EVIDENCE FOR A SINGLE CALCIUM-DEPENDENT BINDING SITE ON CD36 (*)

(Received for publication, August 19, 1994; and in revised form, November 18, 1994)

S. Frieda A. Pearce (§) Jun Wu Roy L. Silverstein

From the Department of Medicine (Hematology/Oncology), Cornell University Medical College, New York, New York 10021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Thrombospondin (TSP) (^1)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 beta activation(6) , smooth muscle cell proliferation(7) , and plasmin generation(8, 9) . TSP is a major component of platelet alpha-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 beta(3) integrin alpha(v)/beta(3)(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(2)-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(r) 50,000 glycoprotein(26) . CD36, which is also known as platelet glycoprotein IV(27) , is an M(r) 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.


EXPERIMENTAL PROCEDURES

Materials

Glutathione-agarose was obtained from Sigma. Percoll, Ficoll-Paque, and all other chromatography media were purchased from Pharmacia Biotech Inc. NaI 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 approx98% 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.


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.



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(3), 1 mM MgCl(2), 0.02% NaN(3), 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(2) 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.


RESULTS

TSP Binds to GST/CD36 Fusion Proteins

Binding of radiolabeled TSP to recombinant immobilized GST/CD36 fusion proteins was time-dependent, reaching equilibrium at approx2 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.


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 (up triangle).




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 (circle), FP93-298 (up triangle), FP67-157 (), FP298-439 (down triangle), FP390-471 (), and FP5-143 (). The binding isotherms in the lower panel represent FP93-120 (circle) and FP118-182 (up triangle). 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(max) (maximal velocity) from the binding isotherm (Fig. 3). From these data we determined that approx 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(d) 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(d) 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 (up triangle), FP67-157 (), FP298-439 (bullet), FP390-471 (), FP118-182 (circle), 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 (box), 5 mM EDTA (), 0.45 µM unlabeled TSP (open column with timess), 1 µg/ml murine anti-CD36 IgG 8A6 (&cjs2112;), and 10 µgbulletml 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(d) values derived from the radioligand binding studies.


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 (up triangle), 60.3 ± 7.7 nM; FP67-157 (), 57.4 ± 6.8 nM; FP5-143 (), 32.4 ± 7.8 nM; FP118-182 (bullet), 70.2 ± 10.5 nM; and FP93-120 (), 6.1 ± 4.2 nM and FP298-439 (bullet bullet bullet bullet bullet bullet bullet) 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 leq 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 (10^5 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(2) (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 (10^6 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(2) (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.


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(2), column 2 is in the presence of purified platelet CD36 (1 µM) in 1 mM CaCl(2), 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.).




DISCUSSION

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.


FOOTNOTES

*
This work was supported by Research Grants RO1-HL42540 (to R. L. S.) and P5O-HL46403 (to R. L. S.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of National Research Service Award T32 HL-07029. To whom all correspondence should be addressed: Division of Hematology-Oncology, Rm. C606, Cornell University Medical College, 1300 York Ave., New York, NY 10021. Tel.: 212-746-2068; Fax: 212-746-8866.

(^1)
The abbreviations used are: TSP, thrombospondin 1; bp, base pair; FP, fusion proteins; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Qinghu Zhang for his assistance with the preparations of peripheral blood monocytes and for maintenance of the Bowes melanoma transfected cells.


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