Miniprep DNA isolation for automated sequencing of multiple samples.
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Base Sequence↗
Biomedical subjects
W A Frazier
Publications and source records attributed to W A Frazier.
Thrombspondin acts via integrin-associated protein to activate the platelet integrin alphaIIbbeta3.
Integrin-associated protein (IAP or CD47) is a receptor for the cell/platelet-binding domain (CBD) of thrombospondin-1 (TS1), the most abundant protein of platelet alpha granules. Although it associates with alphaIIbbeta3, IAP has no known function in platelets. TS1, the CBD, and an IAP agonist peptide (4N1K) from the CBD of TS1 activate the platelet integrin alphaIIbbeta3, resulting in platelet spreading on immobilized fibrinogen, stimulation of platelet aggregation, and enhanced tyrosine phosphorylation of focal adhesion kinase. Furthermore, 4N1K peptide selectively stimulates the phosphorylation of LYN and SYK and their association with FAK. The phosphorylation of SYK is blocked by pertussis toxin, implicating a Gi-like heterotrimeric G protein. IAP solublized from membranes of unstimulated platelets binds specifically to an affinity column of 4N1K peptide. Both alphaIIb and beta3 integrin subunits and c-Src bind along with IAP. This complex of proteins is also detected with immunoprecipitation. Activation of platelets with the agonist peptide 4N1K results in the association of FAK with the IAP-alphaIIbbeta3 complex. Thus an important function of TS1 in platelets is that of a secreted costimulator of alphaIIbbeta3 whose unique properties result in its localization to the platelet surface and the fibrin clot.
Amino Acid Sequence↗
Thrombospondin-1, a natural inhibitor of angiogenesis, regulates platelet-endothelial cell adhesion molecule-1 expression and endothelial cell morphogenesis.
Expression of thrombospondin-1 (TS1) in polyoma middle-sized T (tumor)-transformed mouse brain endothelial cells (bEND.3) restores a normal phenotype and suppresses their ability to form hemangiomas in mice. We show that TS1 expression results in complete suppression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) expression and altered cell-cell interactions in bEND.3 cells. To further investigate the role of PECAM-1 in regulation of endothelial cell-cell interactions and morphogenesis, we expressed human (full length) or murine (delta 15) PECAM-1 isoforms in TS1-transfected bEND.3 (bEND/TS) cells. Expression of either human or murine PECAM-1 resulted in an enhanced ability to organize and form networks of cords on Matrigel, an effect that was specifically blocked by antibodies to PECAM-1. Anti-PECAM-1 antibodies also inhibited tube formation in Matrigel by normal human umbilical vein endothelial cells. However, PECAM-1-transfected bEND/TS cells did not regain the ability to form hemangiomas in mice and the expressed PECAM-1, unlike the endogenous PECAM-1 expressed in bEND.3 cells, failed to localize to sites of cell-cell contact. This may be, in part, attributed to the different isoforms of PECAM-1 expressed in bEND.3 cells. Using reverse transcription-polymerase chain reaction, we determined that bEND.3 cells express mRNA encoding six different PECAM-1 isoforms, the isoform lacking both exons 14 and 15 (delta 14&15) being most abundant. Expression of the murine delta 14&15 PECAM-1 isoform in bEND/TS cells resulted in a similar phenotype to that described for the full-length human or murine delta 15 PECAM-1 isoform. The delta 14&15 isoform, despite the lack of exon 14, failed to localize to sites of cell-cell contact even in clones that expressed it at very high levels. Thus, contrary to recent reports, lack of exon 14 is not sufficient to result in junctional localization of PECAM-1 isoforms in bEND/TS cells.
Animals↗
Repression of thrombospondin-1 expression, a natural inhibitor of angiogenesis, in polyoma middle T transformed NIH3T3 cells.
Thrombospondin-1 (TSI) is a modular extracellular matrix glycoprotein and expressed by many cell types in culture. Thrombospondin-1 inhibits angiogenesis and its expression inversely correlates with the degree of invasiveness and metastasis in tumor cell lines. Here, we demonstrate that expression of Polyoma middle T oncogene in NIH3T3 cells results not only in transformation but also represses expression of three thrombospondin isoforms, TS1, TS2, and TS3. Similar results were observed in ras, and to a lesser extent in src transformed NIH3T3 cells. Middle T and ras transformed cells expressed higher levels of c-jun mRNA, while the src transformed cells expressed higher levels of junB mRNA when compared to control cells. Thus, repression of thrombospondin levels appears to play an important role in establishment and maintenance of a malignant phenotype. This is mediated, at least in part, by alteration in c-jun activity in middle T and ras transformed NIH3T3 cells.
3T3 Cells↗
Integrin-associated protein is a receptor for the C-terminal domain of thrombospondin.
The C-terminal "cell-binding domain" (CBD) of thrombospondin-1 (TS1) is a binding site for many cell types. Cell-binding peptides based on the sequence RFYVVM from the CBD of TS1 affinity label a 52-kDa cell surface glycoprotein, which we show is integrin-associated protein (IAP or CD47). IAP associates with alpha v beta 3 and thereby modulates the activity of several integrins. Cells that express IAP bind strongly to TS1, the CBD, and its active cell-binding peptides while IAP negative cells do not. The 52-kDa protein is affinity labeled on IAP-positive but not IAP-negative cells, and monoclonal antibodies against IAP specifically immunoprecipitate the affinity-labeled 52-kDa protein from lysates of IAP-positive cells. Consistent with the association of IAP with alpha v beta 3 integrin, the labeled 52-kDa protein is immunoprecipitated by an anti-alpha v beta 3 antibody. Endothelial cells exhibit chemotaxis toward TS1 (at concentrations above 10 nM) and RFYVVM peptides. Chemotaxis to both agents is specifically inhibited by a function blocking anti-IAP monoclonal antibody. These data establish IAP (CD47) as a receptor for the CBD of TS1 and suggest a mechanism for the well established effects of the CBD on cell motility.
Amino Acid Sequence↗
pH-Dependent self-association of the Src homology 2 (SH2) domain of the Src homologous and collagen-like (SHC) protein.
The Src homologous and collagen-like (SHC) protein plays an essential role in signal transduction pathways in that it participates in the chain of events that leads to the activation of the protein Ras. The crystal structure of the SH2 domain of SHC has been determined using the method of multiple isomorphous replacement at a resolution of 2.5 A. The SH2 domain of SHC is similar in fold to other SH2 domains. The peptide-binding surfaces resemble that of the SH2 domain of Src in that a deep pocket is formed where the third amino acid C-terminal to the phosphotyrosine can insert. A novel feature of this structure is the observation of a disulfide bond and an extensive dimer interface between two symmetry-related molecules. Solution studies under reducing conditions using analytical centrifugation and PAGE suggest that the SH2 domain of SHC dimerizes in a pH-dependent manner where low pH conditions (approximately 4.5) are conducive to dimer formation. Dimerization of SHC may have important biological implications in that it may promote the assembly of large heteromultimeric signaling complexes.
Amino Acid Sequence↗
Thrombospondin modulates alpha v beta 3 function through integrin-associated protein.
Integrin-associated protein (IAP) is a receptor for the carboxyl-terminal "cell-binding domain" (CBD) of thrombospondin 1 (TS1). IAP associates with alpha v beta 3 integrin and mAbs against IAP inhibit certain integrin functions. Here we examine the effects of the TS1 CBD and 4N1K (KRFYVVMWKK), a cell-binding peptide derived from it, on the adhesion and spreading on vitronectin (VN) of C32 human melanoma cells which express IAP, alpha v beta 3, and alpha v beta 5. Cells adhere to VN at low surface densities via alpha v beta 5 and spread very slowly while adhesion to higher density VN involves both alpha v beta 5 and alpha v beta 3 and results in rapid spreading. Spreading of the cells, but not adhesion, on sparse VN coatings is markedly enhanced by the presence of soluble TS1, the recombinant CBD and 4N1K, but not the "mutant" peptide 4NGG, KRFYGGMWKK, which fails to bind IAP. This enhanced spreading is completely blocked by mAb LM609 against alpha v beta 3 and the anti-IAP mAb B6H12. Correlated with this enhanced spreading is increased tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and a protein of ca. 90 kD. The enhanced spreading induced by TS1 and 4N1K and the constitutive spreading on higher density VN are both blocked by calphostin C (100 nM), wortmannin (10 nM), and tyrosine kinase inhibitors. In contrast, pertussis toxin specifically blocks only the TS1 stimulated spreading on low density VN, indicating that IAP exerts its effects on signal transduction via a heterotrimeric Gi protein acting upstream of a common cell spreading pathway which includes PI-3 kinase, PKC, and tyrosine kinases.
Amino Acid Sequence↗
Thrombospondin 1 expression in transformed endothelial cells restores a normal phenotype and suppresses their tumorigenesis.
Murine endothelial cells are readily transformed in a single step by the polyomavirus oncogene encoding middle-sized tumor antigen. These cells (bEND.3) form tumors (hemangiomas) in mice which are lethal in newborn animals. The bEND.3 cells rapidly proliferate in culture and express little or no thrombospondin 1 (TS1). To determine the role of TS1 in regulation of endothelial cell phenotype, we stably transfected bEND.3 cells with a human TS1 expression vector. The cells expressing human TS1 were readily identified by their altered morphology and exhibited a slower growth rate and lower saturation density than the parental bEND.3 cells. The TS1-expressing cells also formed aligned cords of cells instead of clumps or cysts in Matrigel. Moreover, while the bEND.3 cells formed large tumors in nude mice within 48 hr, the TS1-expressing cells failed to form tumors even after 1 month. The TS1-transfected cells expressed transforming growth factor beta mRNA and bioactivity at levels similar to those of the parental or vector-transfected bEND.3 cells, indicating that the effects of TS1 expression are not due to the activation of transforming growth factor beta by TS1. TS1 expression resulted in a > 100-fold decrease in net fibrinolytic (urokinase-type plasminogen activator, uPA) activity due to more plasminogen-activator inhibitor 1 and less uPA secretion. TS1 thus appears to be an important regulator of endothelial cell phenotype required for maintaining the quiescent, differentiated state.
Animals↗
Identification of integrin alpha 3 beta 1 as a neuronal thrombospondin receptor mediating neurite outgrowth.
Thrombospondins are a family of extracellular matrix proteins expressed throughout the developing nervous system that promote neurite outgrowth in vitro and help mediate the migration of granule cells across the molecular layer in explants of neonatal cerebellum. The receptors mediating these interactions have not previously been identified. In this study, monoclonal antibodies raised to the integrin alpha 3 beta 1 heterodimer are shown to inhibit neurite outgrowth by rat sympathetic neurons on thrombospondin-1. Alpha 3 beta 1 is found to be expressed on the cell body, neurites, and growth cones of sympathetic neurons in vitro and on sympathetic axons passing through the thrombospondin-rich outer sheath of the superior cervical ganglion in vivo, consistent with its role in mediating axon outgrowth. A receptor-ligand binding assay is used to demonstrate the direct binding of immunopurified alpha 3 beta 1 to thrombospondin-1. These results demonstrate a direct interaction between the integrin alpha 3 beta 1 and thrombospondin-1, which mediates neurite outgrowth in vitro and is likely to mediate the same interactions in vivo.
Adrenergic Fibers↗
Transfection of thrombospondin 1 complementary DNA into a human breast carcinoma cell line reduces primary tumor growth, metastatic potential, and angiogenesis.
Previous studies demonstrated that metastatic MDA-MB-435 breast carcinoma cells synthesized and secreted less of the extracellular matrix protein thrombospondin 1 (TSP1) than nonmetastatic breast carcinoma cell lines, a trend also observed for melanoma and lung carcinoma cell lines. To directly examine the effect of tumor cell TSP1 expression on tumor growth and metastasis. MDA-MB-435 cells were transfected with full length THBS-1 cDNA linked to a constitutive cytomegalovirus promoter, or with the cytomegalovirus vector alone. Injection of transfected clones that overexpressed TSP1 into the mammary fat pad of nude mice resulted in a dose-dependent inhibition of primary tumor size and an inhibition of spontaneous pulmonary metastases, which occurred in 21-30% of THBS-1 transfectants compared to 44-49% of controls (P = 0.007). An additional clone was identified that overexpressed a COOH-terminally truncated TSP1. This clone produced larger primary tumors and an increase in the occurrence of metastases relative to control transfectants, suggesting the participation of a previously understudied region of TSP1 in the regulation of tumor progression. The THBS-1 and control transfectants did not exhibit significant differences in growth, colonization, or motility in vitro. However, a relative reduction in capillary densities in primary tumors formed by the wild-type THBS-1 transfectants was observed, suggestive of an angiostatic effect. The data indicate that tumor cell production of TSP1 can exert a significant inhibitory effect on tumor progression in the MDA-MB-435 breast carcinoma cell line, which may be attributable in part to a reduction in angiogenesis.
Amino Acid Sequence↗
Identification of a receptor candidate for the carboxyl-terminal cell binding domain of thrombospondins.
The carboxyl-terminal cell binding domain (CBD) of thrombospondin-1 (TS1) contains two cell attachment peptides, 4N1s (RFYVVMWK) and 7N3 (FIRVVMY-EGKK), which share the sequence VVM. These peptides, and more soluble derivatives have been radiolabeled with 125I and used in conjunction with a variety of membrane impermeant cross-linking reagents to identify and characterize receptor candidates on several cell types. All of the VVM containing peptides tested with five different cross-linking reagents specifically labeled a 52-kDa protein, which was also affinity labeled by the recombinant TS1 CBD. After cross-linking peptide to K562 cells to block the 52-kDa protein, both cell adhesion to and affinity labeling by VVM peptides were inhibited in a concentration-dependent manner. Peptide labeling, like cell adhesion, was partially inhibited by heparin and stimulated by EDTA. The 52-kDa protein did not appear to contain sulfated glycan chains and was trypsin sensitive. It was recovered in a membrane fraction and was readily solubilized with Triton X-100 and X-114. Upon phase separation of the Triton X-114, the 52-kDa protein partitioned into the hydrophobic detergent phase. The detergent-solubilized receptor candidate bound selectively to wheat germ agglutinin-Sepharose, and after cell surface labeling with a membrane impermeant biotinylating reagent, bound to streptavidin-Sepharose. Furthermore, fluorescent beads covalently derivatized with peptide specifically decorated intact K562 cells. Thus the properties of the 52-kDa protein are consistent with those of a receptor for the CBD of TS1 and other TS isoforms.
Amino Acid Sequence↗
Comparison of normal and tumorigenic endothelial cells: differences in thrombospondin production and responses to transforming growth factor-beta.
Cultured endothelial cells constitutively synthesize significant levels of thrombospondin, an extracellular matrix-associated protein with reported anti-anti-angiogenic properties. However, two murine endothelial cell lines, bEND.3 and Py-4-1, which have been immortalized with polyoma T oncogenes and which generate vascular malformations in vivo, produce little or no thrombospondin though bEND.3 (but not Py-4-1) growth is inhibited by the addition of exogenous thrombospondin. In addition, Py-4-1 cells are not growth-inhibited by transforming growth factor-beta, a potent endothelial inhibitor. These results indicate that these two cell lines may be useful tools in understanding the role and mechanism of action of thrombospondin and transforming growth factor-beta in endothelial cell biology. A role for thrombospondin in vascular development is further suggested by the observation of significant differences in the levels of thrombospondin mRNA and protein between capillary and aortic endothelial cells. Transforming growth factor-beta-1 treatment of normal endothelial cells increases steady-state levels of thrombospondin mRNA and protein and results in extensive deposition of thrombospondin into the extracellular matrix. In contrast, transforming growth factor-beta-1 has little effect on thrombospondin levels in the tumorigenic endothelial cell lines. In view of our earlier finding that contact between endothelial cells and mural cells generates activated transforming growth factor-beta-1, and the fact that thrombospondin is present in a fibrillar network around vascular structures in vitro, we speculate that modulation of thrombospondin production and distribution by transforming growth factor-beta may be a physiological process to enjoin stabilization of vessels and cessation of vessel growth.
Animals↗
Heparin-binding peptides from thrombospondins 1 and 2 contain focal adhesion-labilizing activity.
The cell adhesion regulating extracellular matrix glycoprotein, thrombospondin (TSP), causes a loss of focal adhesion plaques from spread endothelial cells and fibroblasts. To localize the site on TSP that has focal adhesion-labilizing activity, we initially tested proteolytic fragments of TSP for activity. The heparin-binding fragment has significant focal adhesion-labilizing activity, whereas the nonheparin-binding 140-kDa fragment had no significant activity. These results were consistent with previous data that showed that both a monoclonal antibody to the heparin-binding domain of TSP (A2.5) and heparin neutralized TSP activity. Peptides from putative heparin binding sequences of the amino-terminal heparin-binding domain of TSP were synthesized and tested for their ability to cause loss of focal adhesions. The hep I peptide (amino acids 17-35) caused maximal loss of focal adhesions and was active at 0.1 microM, whereas peptide hep II (74-95) and peptide hep III (170-189) were inactive. The activity of the hep I peptide was neutralized by the addition of heparin and heparan sulfate but not by chondroitin sulfate. The basic amino acids in the hep I sequence appear to be required for focal adhesion-labilizing activity, because modification of the lysine residues at amino acids 24 and 32 rendered the peptide completely inactive. In addition, a peptide from the analogous sequence of mouse TSP 2, in which basic residues are conserved, was nearly as active as hep I from TSP1. These data show that the anti-adhesive activity of TSP is conserved in both TSP1 and TSP2 and that the active site is located in a 19-amino acid sequence in the heparin-binding domain of TSPs.
Amino Acid Sequence↗
Identification of a new cell adhesion motif in two homologous peptides from the COOH-terminal cell binding domain of human thrombospondin.
Thrombospondin-1 (TS1) contains at least four domains that support cell attachment. The COOH-terminal cell binding domain (CBD) was first identified with a monoclonal antibody against TS1 that blocked secretion-dependent platelet aggregation. Subsequently, this domain of TS1 has been found to bind a number of normal and transformed cells. We have localized attachment sites for human melanoma cells (G361) within the CBD to two noncontiguous 30-residue peptides designated C4 and C7 (Kosfeld, M. D., and Frazier, W. A. (1991) J. Biol. Chem. 267, 16230-16236). Here we report studies to define the active sequences within C4 and C7. An octapeptide, RFYVVMWK (4N1-1), from C4 and a pentapeptide, IRVVM (7N3-1), from C7 were found to support attachment of G361 melanomas, K562 erythroleukemia cells, HT1080 fibrosarcomas, C32 amelanotic melanomas, and endothelial cells. These peptides also inhibit the adhesion of cells to the recombinant CBD of TS1. The hexapeptide RFYVVM (4N1-2) also inhibits cell attachment. The inhibitory effect of combinations of C4- and C7-derived peptides is synergistic. The sequences 4N1-1 and 7N3-1 of TS1 share homology with two cell adhesive peptides from laminin (LM), LMF9 and LMPA22-2, respectively. These TS1 and LM peptides are interchangeable in inhibiting the adhesion of G361 cells to LM or TS1, suggesting a possible sharing of receptors by LM and TS1. K562 cells, however, bound only to TS1, and this binding was inhibited preferentially by the TS1 CBD peptides, indicating a receptor specific for TS1 which does not recognize LM. The active TS1 peptides are highly conserved among five species and four isoforms of TS1. Homologs of the TS1 peptides are found in tenascin, a matrix protein that shares several properties with TS1 and in factor VIII, alpha 2-macroglobulin, and von Willebrand factor.
Amino Acid Sequence↗
Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity.
Thrombospondin-1 (TSP1) is a large modular matrix protein containing three identical disulfide-linked 180-kD chains that inhibits neovascularization in vivo (Good et al., 1990). To determine which of the structural motifs present in the 180-kD TSP1 polypeptide mediate the anti-angiogenic activity, a series of protease-generated fragments were tested using several in vitro and in vivo assays that reflect angiogenic activity. The majority of the anti-angiogenic activity of TSP1 resides in the central 70-kD stalk region which alone could block neovascularization induced by bFGF in the rat cornea in vivo and inhibit both migration in a modified Boyden chamber and [3H]thymidine incorporation stimulated by bFGF in cultured capillary endothelial cells. Although TSP1 has been shown to bind active TGF beta 1, this cytokine could not account for the inhibitory effects of the stalk region of TSP1 on cultured endothelial cells. Peptides and truncated molecules were used to further localize inhibitory activity to two domains of the central stalk, the procollagen homology region and the properdin-like type 1 repeats. Trimeric recombinant TSP1 containing NH2-terminal sequences truncated after the procollagen-like module inhibited endothelial cell migration in vitro and corneal neovascularization in vivo whereas trimeric molecules truncated before this domain were inactive as was the NH2-terminal heparin-binding domain that is present in both recombinant molecules. A series of peptides from the procollagen-like region, the smallest of which consisted of residues 303-309 of TSP1, inhibited angiogenesis in vivo in the rat cornea and the migration of endothelial cells in vitro. A 19-residue peptide containing these sequences blocked vessel formation in the granulation tissue invading a polyvinyl sponge implanted into the mouse. Nineteen residue peptides derived from two of the three type 1 repeats present in the intact TSP1 molecule blocked neovascularization in vivo in the rat cornea and inhibited the migration of cultured endothelial cells with ED50's of 0.6-7 microM. One of these peptides, containing residues 481-499 of TSP1, also inhibited vessel formation in granulation tissue invading sponges in vivo. These results suggest that the large TSP1 molecule employs at least two different structural domains and perhaps two different mechanisms to accomplish a single physiological function, the inhibition of neovascularization. The definition of short peptides from each of these domains that are able to block the angiogenic process may be of use in designing targeted inhibitors of the pathological neovascularization that underlies many diseases.
Amino Acid Sequence↗
Identification of active peptide sequences in the carboxyl-terminal cell binding domain of human thrombospondin-1.
Thrombospondin (TS) mediates attachment, spreading, and motility of several cell types through at least four cell binding domains: the amino-terminal heparin binding domain, the type I repeats containing the CSVTCG sequence, the RGDA sequence in the last of the type III calcium binding repeats and the carboxyl-terminal cell or platelet binding domain (CBD). The attachment of human melanoma cells (G361) to the COOH-terminal domain is independent of the RGDA sequence and is inhibited by the monoclonal antibody C6.7. To define the cell binding site(s) within this 212-residue COOH-terminal domain, we have synthesized eight overlapping peptides (seven 30-mers and a final 37-mer) representing the entire sequence of the CBD. Several of these peptides are insoluble in aqueous buffers at high concentration. Cell adhesion assays have been devised which employ covalent coupling of peptides in chaotropic solvents to chemically derivatized plastic 96-well plates. Three synthetic peptides, two of which are nonadjacent in the linear sequence, are potent attachment factors for G361 cells. C6.7 blocks adhesion to one of these peptides, whereas sulfated glycoconjugates inhibit adhesion of cells to all three. Polyclonal antibodies raised against the peptides inhibit cell adhesion to the peptides, the recombinant CBD, and to intact TS. The peptides GRGDSP and VTCG are not inhibitory. These sites are thus independent from the type I repeats and the RGDA sequence of TS. Each of the active peptides inhibits cell attachment to the other active peptides as well as to the CBD and to intact TS. This mutual inhibition suggests that the peptides share a common cellular receptor which may contain an associated glycoconjugate chain. These data indicate that the COOH-terminal cell binding domain of TS contains at least two peptide sequences which contribute to the attachment of a wide variety of cells.
Amino Acid Sequence↗
Thrombospondin promotes process outgrowth in neurons from the peripheral and central nervous systems.
Thrombospondin (TSP) is a prominent constituent of the extracellular matrix of the developing nervous system. We have examined the effects of TSP on the morphological differentiation of neurons. In short-term cultures (less than or equal to 24 hr) of embryonic rat sympathetic neurons, TSP stimulated neurite outgrowth, causing significant increase in the number of processes and their length. Similar effects were observed in cultures of rat dorsal root ganglion, hippocampal, and cerebral cortical neurons. Moreover, in cultures of central neurons, TSP was more effective than laminin in enhancing process extension. Analysis of long-term (5-7 days) cultures of sympathetic neurons indicated that processes formed in the presence of TSP had the cytochemical characteristics of axons. Thus, TSP can influence neuronal development by selectively enhancing axonal growth. The neurite-promoting region of the molecule was identified using a panel of monoclonal antibodies targeted to different regions of the protein. Process outgrowth could be totally inhibited with antibody A4.1, which recognizes the stalk region of TSP. These data suggest that the neurite-promoting activity is localized to a single region of the TSP molecule.
Animals↗
Cell attachment activity of the carboxyl-terminal domain of human thrombospondin expressed in Escherichia coli.
Thrombospondin (TS) is a multidomain, adhesive glycoprotein that associates with cells through multiple cell attachment sites. One of these has been located in or near the globular COOH-terminal region of TS by the monoclonal antibody (mAb) C6.7, which inhibits the attachment of human melanoma cells (G361) to TS. The epitope for C6.7 lies within the last 122 residues of the COOH-terminal domain of TS. This domain is distant from two known cell attachment sites in TS, namely the NH2-terminal heparin-binding domain and the CSVTCG sequences in the type I repeats, but is close to the RGDA sequence, an integrin-dependent cell attachment site. In order to separate the adhesive activity of the TS COOH-terminal domain from that of the RGD sequence, we have expressed the COOH-terminal 212 amino acids (residues 941-1152) of TS in Escherichia coli using the expression vector pRIT2T. The resultant fusion protein is effective in supporting G361 cell attachment even though it lacks the RGD sequence. In addition, the expressed protein inhibits adhesion of G361 cells to intact TS. mAb C6.7 blocks adhesion to the expressed TS COOH-terminal domain whereas GRGDSP and VTCG peptides are not inhibitory. These results show that the TS COOH-terminal domain contains a separate cell adhesion site, defined by mAb C6.7, that is distinct from the other adhesion sites of TS.
Amino Acid Sequence↗