From the Program in Cell Biology and Genetics,
Cornell University Graduate School of Medical Sciences, Cornell
University Medical College, New York, New York 10021 and the
§ Department of Medicine, Division of Hematology-Oncology,
The New York Hospital-Cornell Medical Center, New York, New
York 10021
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ABSTRACT |
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LIMPII (lysosomal integral membrane protein II) is one of a family of proteins structurally related to the cell surface glycoprotein CD36. We recently defined a single structural domain on CD36 that mediates binding to adhesive glycoprotein thrombospondin-1 (TSP1). The CD36-TSP1 interaction is known to play a role in platelet-tumor and platelet-monocyte adhesion, angiogenesis, and in monocyte uptake of apoptotic cells. To test whether LIMPII also binds TSP1, a LIMPII peptide corresponding to the TSP1 binding domain of CD36 was expressed as a recombinant glutathione S-transferase (GST) fusion protein. In solid phase binding assays, purified 125I-TSP1 bound to immobilized GST/LIMPII in a time-dependent and saturable manner. Inhibition by excess unlabeled TSP1 or EDTA demonstrated specificity. LIMPII·TSP1 complex formation was specifically blocked by soluble LIMPII fusion protein, by monospecific rabbit IgG directed against the LIMPII peptide and by CD36 fusion proteins containing the TSP1 binding domain. Transfection of Bowes melanoma cells with a chimeric LIMPII cDNA that targets expression to the plasma membrane conferred the ability to bind 125I-TSP1 and to adhere to TSP1-coated surfaces. This study defines a TSP1 binding site conserved between LIMPII and CD36 and suggests that cell surface LIMPII may function in some circumstances as an adhesion receptor for TSP1. Computer-assisted homology searches suggest that the TSP1 recognition motif identified from study of CD36 family members may be widely expressed in nature.
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INTRODUCTION |
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LIMPII1 (hLGP85) is a 74-85-kDa lysosomal integral membrane protein recently identified as a member of the CD36 gene family (1, 2). Other members of this family include the mammalian genes Cla-1 (CD36 and LIMPII analogous-1) (3), which is also known as SR-BI (scavenger receptor class B-I) (4), and invertebrate genes Drosophila croquemort (dCD36) (6)2 and emp (epithelial membrane protein) (7), and Caenorhabditis elegans cm08 h8 (8). Significant structural similarities include overall hydrophobicity, an N-terminal uncleaved signal peptide and C-terminal stop transfer signal, putative transmembrane domains yielding short cytoplasmic tails, one large extracellular/intraluminal domain, with highly conserved cysteine residues and potential asparagine-linked glycosylation sites (1). A novel leucine-isoleucine motif in the C-terminal cytoplasmic tail of LIMPII determines lysosomal localization via mannose 6-phosphate receptor-independent transport (9-11). CD36 (platelet glycoprotein GPIV), the prototype of this gene family, is a 78-88-kDa cell surface glycoprotein present on platelets, monocytes, erythroid precursors, mammary and retinal epithelium, adipocytes, activated keratinocytes, and certain microvascular endothelium (reviewed in Ref. 12). We and others have shown that CD36 serves as a receptor for TSP1 (13), collagen types I (14) and IV (15), and Plasmodium falciparum malaria-parasitized erythrocytes (16). CD36 participates in the uptake of apoptotic neutrophils (17, 18), long chain fatty acids (19), anionic phospholipids (20), oxidized low density lipoproteins (21, 22), and retinal photoreceptors (23). These latter functions have led Krieger and colleagues (4) to postulate that the CD36 gene family encodes a novel class of scavenger receptors. Supporting this are recent observations that SR-BI functions as a selective receptor for high density lipoproteins (24) and that croquemort functions in Drosophila embryogenesis as a monocyte receptor for apoptotic cells (6). CD36 also has been implicated in signal transduction (25), physically associated with cytoplasmic tyrosine kinases fyn, lyn, and yes (26).
TSP1, a 450-kDa trimeric glycoprotein component of extracellular
matrix, is a well characterized ligand of CD36 (reviewed in Refs. 27
and 35). It is a major component of platelet -granules secreted upon
platelet activation (36). TSP1 functions in various aspects of vascular
biology, including platelet aggregation (28), angiogenesis (29, 30),
transforming growth factor
activation (31), smooth muscle
proliferation (32), and plasmin generation (33, 34). It also modulates
adhesion and migration during embryonic development (37, 38) and has
been implicated in such disease processes as thrombosis, atherogenesis,
and tumor metastasis (39). While CD36 is only one of multiple TSP1
receptors, we and others have demonstrated that the CD36-TSP1
interaction is involved in platelet-monocyte adhesion (40, 41),
platelet-tumor cell adhesion (42), macrophage uptake of apoptotic cells
(17, 18), and tumor cell-substratum adhesion (43). CD36 recently has
been shown to mediate the anti-angiogenic effects of TSP1 (79)3 and to play a role in
progression and metastatic potential of breast cancer (44-46).
We recently defined a single TSP1 binding domain in CD36 (47). Given the extent of conserved structure between LIMPII and CD36, we were interested in learning whether LIMPII shares CD36 TSP1 binding activity. LIMPII has been identified as a lysosomal membrane component in all cell types examined to date (9, 48). It is not known whether LIMPII may function at the cell surface, nor has a ligand been identified to indicate that LIMPII could function as an authentic adhesion receptor. However, under certain circumstances, soluble lysosomal enzymes and membrane proteins are expressed at the plasma membrane. We reported previously that LAMP-1 and LAMP-2 (members of a subclass of lysosomal membrane glycoproteins not related to LIMPII) are expressed on activated platelets (49, 50), while others have shown LAMP expression on tumor cells and activated leukocytes (51-53). These observations support the possibility that LIMPII might act at the cell surface and thus may share TSP1 receptor function with CD36.
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EXPERIMENTAL PROCEDURES |
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LIMPII Fusion Proteins--
Sau3A fragments from base pairs
464-704 of the LIMPII cDNA (kindly provided by Dr. I. Sandoval,
Centro de Biología Molecular, Madrid, Spain (9)) encoding aa
75-155, and from base pairs 705-983 encoding aa 156-243, were
subcloned into prokaryotic expression vector pGEX-3X (Pharmacia Biotech
Inc.), directly adjacent to a fragment of the recombinant glutathione
S-transferase (GST) gene, downstream of an inducible LacZ
promoter. Fig. 1 shows the orientation of
LIMPII fusion proteins, and CD36 fusion proteins (CFP93-120,
CFP67-157, and CFP298-439 (47)), relative to the native LIMPII and
CD36 sequences. A truncation mutant terminating after codon four of
LFP75-155 was used as a control (GST1, LFP75-78). All plasmid
constructs were restriction-mapped and sequenced across fragment
junctions to confirm correct orientation and intact reading frame.
Large scale production of soluble fusion protein by the method of
Frangioni and Neel (54) included single step affinity purification on
glutathione-Sepharose 4B (Pharmacia). Glutathione eluates were dialyzed
extensively prior to storage at 70 °C in 10% glycerol. For some
experiments, LIMPII peptides were cleaved from the GST moiety with
coagulation Factor Xa (Boehringer Mannheim) and purified by size
exclusion chromatography (Centricon 10; Amicon, Beverly, MA). Fusion
proteins were analyzed by 8% SDS-polyacrylamide gel electrophoresis or
gel filtration (Superose-12, Pharmacia) to determine apparent molecular
weights (listed in Fig. 1) and by Western blot and enzyme-linked
immunosorbent assay to confirm size and document immunoreactivity.
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Purified Proteins, Peptides, and Antibodies-- Human platelet-rich plasma and outdated platelet concentrates were obtained from the New York Blood Center. Purified human calcium-replete TSP1 was prepared from releasate of thrombin-activated washed platelets by heparin affinity and anion exchange chromatography on Mono Q-Sepharose (Pharmacia) (34). TSP1 peptides were from Chiron Mimotopes, Victoria, Australia. Human thrombin was from Boehringer Mannheim, and epinephrine was from Biodata Corp., Horsham, PA. CD36 was prepared from human platelet Triton X-114 extracts as reported previously (40, 55, 56). Murine monoclonal anti-CD51 (69.6.1) was from Immunotech, Inc., Westbrook, ME. Monoclonal anti-LAMP-1 and -2 and anti-TSP1 IgGs are described elsewhere (40, 49, 50, 57). Anti-CD36 monoclonal antibody 8A6 was a gift of Dr. J. Barnwell, New York University Medical Center, New York. Domain-specific rabbit anti-LIMPII was generated by subcutaneous inoculation of the 10-kDa L75-155 thrombin-cleaved peptide. IgG was purified from serum by protein A chromatography (Pierce). Following depletion of GST-specific antibody on immobilized GST1 (HiTrap columns; Pharmacia), anti-LIMPII peptide IgG was purified by affinity capture on immobilized LFP75-155. These were determined to be monospecific by enzyme-linked immunosorbent assay and Western blot analyses.
Cell Lines-- An EcoRI fragment encoding a chimeric LIMPII cDNA was subcloned into eukaryotic expression vector pMV7 (58) (gift of Dr. Moses Chao, Cornell Medical College). This cDNA contains the luminal and transmembrane domains of rat LIMPII coupled to the C-terminal cytoplasmic tail of human CD36 and is targeted to the plasma membrane (9). Bowes melanoma cells were transfected by liposome-mediated fusion (LipofectinTM; Life Technologies, Inc.) and stable lines selected and maintained as described for CD36-transfected Bowes cells (42). Cells with high LIMPII surface expression were enriched by adsorption with polyclonal anti-L75-155 IgG and subsequent selection with sheep anti-rabbit IgG-conjugated magnetic beads (Dynabeads M-450; Dynal Inc. Great Neck, NY). Surface expression was confirmed prior to all studies by immunofluorescence flow cytometry as described previously (42).
Solid Phase Binding Assays--
TSP1 (5-10 µg/ml) or fusion
proteins and peptides (10-20 µg/ml) were immobilized on detachable
96-well strips (Immulon-4 Remove-a-well, Dynatech Laboratories, Inc.)
by overnight incubation at 4 °C in carbonate buffer (100 mM Na2CO3, 1 mM
MgCl2, 0.02% NaN3, pH 9.8). Saturable coating
determined using radiolabeled proteins ranged from 200 to 280 ng/well.
Washed wells were blocked with 1% BSA, then incubated in triplicate
with soluble radiolabeled ligand as described previously (47). After
extensive washing, bound radioactivity was quantified by a counter.
Radiolabeling was performed with Na125I (Amersham Life
Science Inc.) using immobilized chloramine T (IODO-BEADS; Pierce) (40,
56). Specific activity determined for each experiment ranged from 0.06 to 1.0 µCi/µg. Nonspecific binding was determined by carrying out
binding in the presence of 10 mM EDTA or excess unlabeled
ligand and was generally less than 10% of total.
Cell Binding Assays-- Binding of 125I-labeled TSP1 to suspensions of LIMPII-transfected Bowes cells was measured as described previously (40, 42). Inhibition studies used 5-10-fold molar excess competitor protein, 80 µg/ml IgG, or 1 mM EDTA. All assays were performed in triplicate. Binding of 125I-TSP1 to activated platelets was measured using platelet monolayers prepared as described below.
Melanoma Cell Adhesion Assays-- Platelet-tumor cell adhesion assays were performed as described (42). Briefly, washed rat or human platelet suspensions (4 × 107/ml) were aliquoted into 96-well tissue culture plates, and adherent "monolayers" formed by centrifuging plates at 1100 × g for 10 min at 22 °C. Platelets then were activated with 0.5 units/ml thrombin for 2 min at 37 °C. Triplicate wells were washed, blocked with 20 mM HEPES, 150 mM NaCl, 0.75 mM Na2HPO4, pH 7.04, 1% BSA, then incubated with 2 × 104 melanoma cells/well for 1 h at 37 °C in 5% CO2. Wells were gently washed, fixed in 1% glutaraldehyde, and adherent cells scored by counting identical 1-mm2 sectors under phase microscopy. Nonspecific adhesion was assessed as binding to BSA-coated wells. TSP1 adhesion assays were performed with identical conditions, except that instead of platelet monolayers, wells were coated with 5 µg/ml TSP1 in carbonate buffer overnight at 4 °C.
Immunofluorescence Flow Cytometry of Rat Platelets-- Platelets were obtained by exsanguination of 1-year-old Sprague-Dawley rats. Platelet-rich plasma was harvested after low speed centrifugation of anticoagulated blood and washed platelet suspensions prepared as described (42). Surface expression of LIMPII and LAMP-1 and -2 on resting platelets, or on platelets treated with either 0.5 unit/ml human thrombin or 0.1 unit/ml epinephrine, was assessed by indirect immunofluorescence flow cytometry as described (49, 50).
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RESULTS |
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TSP1 Binds LIMPII
Interaction of TSP1 with LIMPII/GST Fusion Proteins-- In solid phase binding assays, soluble 125I-labeled TSP1 bound to immobilized LIMPII fusion protein LFP75-155, spanning aa 75-155 (Fig. 2A), but not to immobilized control fusion protein GST1 (LFP75-78), nor to a downstream LIMPII fusion protein (LFP156-243). Binding was time-dependent (Fig. 2B), reaching equilibrium within 3 h, and upon addition of excess unlabeled TSP1 at equilibrium, was reversed by ~50% within 30 min and ~75% at 2 h. In complementary studies in which TSP1 was immobilized, soluble 125I-LFP75-155 bound to TSP1 in a time-dependent reversible manner and reached equilibrium by 4 h (not shown), suggesting that binding was not related to artifactual influences of protein immobilization.
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Specificity of TSP1-LIMPII Interactions-- To characterize further the LIMPII-TSP1 interaction, competitive inhibition assays were performed. As summarized in Fig. 3, 125I-TSP1 binding to solid phase LFP75-155 was inhibited by excess TSP1 (75 ± 5%) and excess LFP75-155 (86 ± 8%), but not by GST1 (17 ± 8%), demonstrating specificity. Inhibition isotherms over a range of concentrations (1 nM to 1 µM, not shown) revealed IC50 values for TSP1 and LFP75-155 similar to the apparent affinities determined from Fig. 2 (listed in Table I). As further demonstration of specificity, TSP1·LFP75-155 complex formation was blocked by anti-TSP1 or domain-specific anti-LIMPII IgG directed against L75-155 peptide (98 and 96 ± 1%, respectively), whereas nonimmune IgG (4 ± 7%) or IgG directed against a downstream peptide of LIMPII (LFP156-243) did not inhibit.
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CD36 Competes with LIMPII for TSP1 Binding-- Because identity between the TSP1 binding domain of CD36 and the corresponding region of LIMPII is higher than overall homology (see below), we tested whether they might compete for a common binding site on TSP1. Intact purified platelet CD36 inhibited binding of 125I-TSP1 to immobilized LFP75-155 by 92 ± 2% (Fig. 3). This result was reproducible using CD36 fusion proteins containing the TSP1 binding domain. CFP67-157 inhibited by 81 ± 3%, while the 27-aa minimal binding site of CFP93-120 showed 95 ± 1% inhibition. In contrast, neither CFP298-439 nor LFP156-243 fusion proteins derived from regions downstream of the TSP1 binding domain were able to block this interaction (not shown). Results over a range of competitor concentrations (10 nM to 1 µM, not shown) indicated apparent IC50 values for CFP67-157, CFP93-120, and native CD36 of ~80, 55, and 46 nM, respectively (Table I). These results strongly suggest that the corresponding region of LIMPII shares overlapping function with the CD36 TSP1 binding domain.
CD36 is known to bind to the sequence CSVTCG within the type I properdin repeat of TSP1 (43). Therefore, we tested the ability of this TSP1-derived peptide to inhibit 125I-TSP1 binding to immobilized LIMPII fusion protein. Excess soluble CSVTCG peptide blocked 66 ± 3% of specific TSP1 binding (Fig. 3), compared with a scrambled control peptide VGSCCT (20 ± 4%). An RGD-containing peptide, also a cell recognition motif found in the C-terminal type 3 calcium-binding repeat of TSP1, showed less efficient inhibition (32 ± 6%). We conclude that the TSP1 binding domain of LIMPII is functionally identical to that of CD36 and interacts directly with the CD36-binding sequence on TSP1.Surface Expression of LIMPII Confers TSP1 Binding Ability to Transfected Melanoma Cells
LIMPII-transfected Cells Acquire the Capacity to Bind TSP1-- To investigate whether LIMPII could serve as a cell surface receptor for TSP1, we employed a transfected cell system, which serves as a paradigm for studying CD36 function (42). Bowes melanoma cells were stably transfected with a chimeric LIMPII cDNA containing the luminal and transmembrane domains of LIMPII coupled to the C-terminal cytoplasmic tail (6 residues) of CD36 (9), that targets expression to the plasma membrane, such that the intraluminal domain of LIMPII is oriented extracellularly. Surface expression of LIMPII on transfected Bowes cells was confirmed by flow cytometry using affinity-purified anti-LFP75-155 IgG (Fig. 4). As shown in Fig. 5, LIMPII-transfected Bowes cells bound soluble 125I-TSP1, in contrast to control cells transfected with pMV7 vector alone and to a similar extent as CD36-transfected cells (not shown). Binding was inhibited in the presence of excess unlabeled TSP1 (93 ± 0.5%) and was completely abolished by 1 mM EDTA (not shown), demonstrating specificity. In addition, fusion proteins LFP75-155 (95 ± 0.3%) and CFP93-120 (93 ± 2%), but not GST-1, were able to compete significantly for TSP1 binding, providing further evidence of a common TSP1 binding domain.
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LIMPII-transfected Melanoma Cells Adhere to TSP1-coated Surfaces-- Fig. 6A shows that LIMPII transfectants adhered to TSP1-coated wells (235 ± 37 cells/mm2) to a similar extent as CD36 transfectants (92 ± 17% of maximum LIMPII adhesion), while control cells transfected with vector alone showed only minimal adhesion (25 ± 9%) above background to BSA-coated wells (7 ± 4%, not shown). Adhesion was inhibited upon addition of 200 nM TSP1 (22 ± 7%) and abolished in the presence of 1 mM EDTA (not shown), demonstrating TSP1-specific LIMPII-dependent adhesion.
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LIMPII-transfected Melanoma Cells Adhere to Platelet Surface
TSP1--
We have shown previously that CD36-transfected Bowes cells
adhered specifically to TSP1-expressing platelets immobilized on microtiter wells (42). To determine if LIMPII also could serve to
mediate tumor cell-platelet adhesion, we measured the ability of the
chimeric LIMPII-transfected Bowes cells to adhere to activated platelets. As shown in Fig. 6A, these cells adhered to
thrombin-stimulated rat platelet monolayers (246 ± 37 cells/mm2) better than CD36 transfectants (58 ± 12%
of LIMPII maximum) and significantly greater than control cells
(33 ± 7%). Adhesion was completely abolished by addition of 1 mM EDTA (not shown) and was abrogated in the presence of
soluble TSP1 (26 ± 6% of LIMPII maximum). In addition,
LFP75-155 and CFP93-120 inhibited adhesion of LIMPII transfectants by
79 ± 6% and 67 ± 6%, respectively (Fig. 6B),
compared with GST1 (21 ± 20%). Affinity-purified rabbit anti-L75-155 IgG or anti-TSP1 IgG also inhibited adhesion (25 and
19 ± 5%, respectively), while control IgG had no effect
(~2%). An inhibitory monoclonal antibody to another TSP1 receptor,
v
3 integrin, blocked only ~32%,
suggesting that adhesion of these cells was predominantly attributable
to the LIMPII-TSP1 interaction. These data suggest that LIMPII, like
CD36, could mediate TSP1-dependent tumor cell-platelet
interactions and thus may contribute to the metastatic potential of
these cells.
LIMPII Is Expressed on the Surface of Activated Platelets and May Participate in TSP1 Binding-- We previously established that lysosome-associated membrane proteins are expressed preferentially on the surface of activated platelets (49, 50). Evidence of lysosomal membrane flow and fusion predicts that LIMPII also would appear as a surface membrane component upon stimulation of platelet secretion. To test this hypothesis, we used immunofluorescence flow cytometric analysis to assess the presence of native LIMPII. As shown in Fig. 7, anti-L75-155 IgG bound to the surface of thrombin-activated platelets (mean channel fluorescence (mcf) = 0.256), but not to resting platelets (mcf = 0.165). The level of anti-LIMPII IgG fluorescence appeared similar to that of anti-LAMP-1 IgG (mcf = 0.242), while nonimmune IgG reactivity with activated or resting platelets was indistinguishable from that of anti-LIMPII IgG reactivity to resting platelets (mcf = 0.155). LIMPII expression did not follow stimulation with epinephrine (mcf = 0.174), consistent with previous data showing that weak agonists did not induce surface appearance of platelet lysosomal proteins (49, 50). By inference from anti-LAMP-1 relative fluorescence intensity, LIMPII expression probably also is in the range of 1500 molecules/platelet (50). This is in contrast to 20,000-60,000 TSP1 binding sites on activated platelets (27), suggesting that LIMPII accounts for a minor fraction of potential TSP1 binding sites.
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Identification of an Evolutionarily Conserved Structural Domain from Analysis of the TSP1 Binding Motif in CD36 Gene Family Members
Sequence comparison within the CD36 gene family was performed to identify evolutionarily conserved amino acid residues likely important in interactions with TSP1. Using huCD36 aa 76-163 (corresponding to LIMPII aa 75-155) as query, a BLAST search (59) successfully identified additional C. elegans homologs. These included F11C1.3 and CO3F11 (531 and 562 residues, chromosome 3), R07B1.3 (536 residues, chromosome X), and F07A5.3 (590 residues, chromosome I), bringing the total number of CD36 gene family members to 14. Two Saccharomyces cerevisiae genes, Nes24p and HDF1, may represent candidate yeast homologs. Multiple sequence alignments (ClustalW (60), MAP (61), PIMA (62, 63)) of deduced amino acid sequences corresponding to LIMPII aa 75-155 (Fig. 9) revealed a highly homologous stretch from aa 89-123, with 59% identity and 68% similarity between LIMPII and CD36, suggesting the existence of a conserved TSP1 recognition motif. Using BlockMaker (combined Gibbs/MOTIF analysis (64)), which identifies short runs of ungapped homology within related sequences indicative of common motifs, three highly conserved segments were identified. Block A (Gibbs only) represents a protein kinase C consensus phosphorylation site (GPYTYR). Block B (MOTIF only) begins at the 5' boundary of CD36 Exon 5 (aa 95-143) (65). Block C appeared identical by either analysis. This provides strong evidence of actual motif structure, since each algorithm is based on different principles with complementary strengths and weaknesses. The average interblock distance of 10 residues between Blocks B and C predicts that this motif could appear in other proteins as a bipartite domain.
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DISCUSSION |
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The studies reported here demonstrate that a TSP1 binding domain originally identified in CD36 is conserved in the genetically related protein LIMPII. In vitro binding of TSP1 to a LIMPII peptide and to LIMPII cDNA-transfected cells was similar in apparent affinity to CD36 and CD36-derived peptides. Close structural similarity was confirmed further by the ability of the CD36 fusion protein CFP93-120 (minimal TSP1-binding peptide) and TSP1-derived peptide CSVTCG to block TSP1 binding to LFP75-155 and by the ability of LIMPII and CD36 peptides to cross-compete for TSP1 binding to LIMPII-transfected cells. These data indicate that both CD36 and LIMPII recognize the same binding site on TSP1.
The biological significance of the TSP1-LIMPII interaction is not known. Recently, Cuervo and Dice (66) described LAMP-2 as a receptor for selective uptake of intracellular substrates for lysosomal proteolysis in a reconstituted in vitro system. This raises the possibility that native LIMPII also may function in a specialized import pathway and might serve as a lysosomal receptor for TSP1. Rapid internalization and degradation of fluorescently-labeled TSP1 by LIMPII-transfected melanoma cells similar to CD36 transfectants further supports such a role.4 Lysosomal CD68 (LAMP-related macrosialin) also has been implicated as a surface receptor for oxidized low density lipoproteins on activated macrophages (67). The possibility that LIMPII shares scavenger receptor function with CD36 and SR-BI is supported by recent studies from our laboratory indicating that LFP75-155 specifically inhibited binding of oxidized low density lipoproteins to CD36.5
In addition to providing insight into the structural basis of TSP1 binding by CD36-related proteins, demonstration of activation-dependent expression of LIMPII on the surface of platelets presents the possibility of a direct role as a cell adhesion molecule. In certain physiological settings, surface expression of lysosomal membrane proteins is a common feature. For example, we have shown previously that LAMP-1 and -2 translocate to the cell surface during platelet activation (49, 50). Others have confirmed this phenomenon in neutrophil and monocyte activation (69, 70). LIMPII might participate in normal function of platelets and leukocytes by contributing to pro-adhesive changes characteristic during coagulation, inflammation, and immune responses. Another resident lysosomal protein recognized as a platelet activation marker, the four-transmembrane superfamily prototype CD63 (LIMPI, LAMP-3), recently was shown to interact directly with integrin cell adhesion receptors (71).
Our transfected melanoma cell model provides evidence that cell surface LIMPII has the capacity to act as an authentic TSP1 receptor on tumor cells. Of particular interest is the observation that surface expression of lysosomal membrane proteins correlates with increased metastatic potential (72-77). Expression of LAMP-1 and -2 and lysosomal CD63 on tumor cells is well documented (52, 53, 78), and monoclonal anti-LAMP-1 antibody was shown to inhibit tumor cell migration (76). It has been proposed that TSP1-mediated platelet-tumor cell thrombi facilitate extravasation by trapping circulating metastatic cells in the microvasculature (57). LIMPII-dependent modulation of tumor cell adhesion to matrix or to platelet TSP1 could promote tumor progression and metastasis.
Computer-assisted comparison of the TSP1 binding region in CD36-related genes revealed three blocks of homology (Fig. 9). The first is a protein kinase C consensus sequence (GPYTYR). Asch et al. (13) have shown that protein kinase C-dependent phosphorylation of CD36 Thr92 blocked TSP1 binding to CD36, and may regulate CD36 function in vivo. Consistent with this model, we have shown that while Thr92 is not necessary for TSP1 binding activity, phosphorylation of peptides containing Thr92 partially inhibited TSP1 binding (15). Thr93 of some family members, including Cla-1/SR-BI, is replaced by Val; therefore, one might predict constitutive rather than regulated binding. Definition of the protein kinase C phosphorylation consensus site as an optional regulatory domain, separate from the TSP1 binding motif, would be consistent with CD36 genomic organization. Exon 5 encodes aa 95-143 of human CD36, corresponding to aa 93-143 of LIMPII. The 48-50 residue peptide contains the other two TSP1 binding motif blocks and thus is an excellent candidate to fit a model of evolutionary exon shuffling of a functional "module." As the first structural domain delineated within the CD36 gene family, we propose to designate this motif CLESH-1 (CD36, LIMPII, emp, SR-BI Homology sequence 1).
Finally, using a pattern-based BEAUTY homology search (68), we have identified CLESH-1 related sequences in several classes of proteins outside the CD36 family. These include several proteins known to bind TSP1. Some of the matched sequences conform to a bipartite organization, as predicted by separate motif blocks (Fig. 9). Our studies provide direct evidence that this motif can function as a site for protein-protein interaction, showing TSP1 binding by either the CD36 or LIMPII domain and blockade of TSP1 binding by fusion proteins containing the CD36/LIMPII TSP1 binding motif. While the role of CLESH-1 sequences in other proteins remains to be determined, we have shown that a protein not previously known to interact with TSP1 (human immunodeficiency virus type 1 envelope gp120) contains a split CLESH-1 motif and complexes with TSP1 (5). Thus, the CLESH-1 motif clearly is indicative of a potential TSP1 recognition module. Further identification and functional characterization of CLESH-1-related domains may facilitate elucidation of novel TSP1 interactions.
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ACKNOWLEDGEMENTS |
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We thank Dr. Frieda Pearce for generously providing purified platelet CD36 and CD36 fusion proteins, Qinghu Zhang for excellent technical support, Dr. Leif Bergsagel for assisting with sequence analysis, Dr. Tim McCaffrey for rat blood, and Dr. Siu Lo for helpful discussion.
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FOOTNOTES |
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* This work was supported by Grants HL46403, HL18828 (Thrombosis SCOR), and DE11348 from the National Institutes of Health and by the Charles Fogarty Trust.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.
¶ To whom correspondence should be addressed: Division of Hematology-Oncology, Rm C606, Dept. of Medicine, Cornell University Medical College, 1300 York Ave., New York, NY 10021. Tel.: 212-746-2060; Fax: 212-746-8866.
1
The abbreviations used are: LIMPII, lysosomal
integral membrane protein II; aa, amino acid(s); CD51,
v
3 integrin; Cla-1, CD36/LIMPII analogous
protein 1; GST, glutathione S-transferase; LAMP,
lysosome-associated membrane protein; SR-BI, scavenger receptor class
B-I; TSP1, thrombospondin-1; BSA, bovine serum albumin; mcf, mean
channel fluorescence.
2 N. Franc, J. Dimarcq, J. Hoffmann, and M. Lagueux, GenBankTM accession number Z31582.
3 M. Febbraio, O. V. Volpert, S. E. Crawford, N. P. Bouck, and R. L. Silverstein, personal communication.
4 R. Crombie and S. F. A. Pearce, unpublished data.
5 S. F. A. Pearce, M. Febbraio, A. C. Nicholson, and R. Silverstein, submitted for publication.
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REFERENCES |
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