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• W2078698377 abstract "Thrombospondin 1 (TSP1) and thrombospondin 2 (TSP2) are members of the thrombospondin family that have a similar structural organization but somewhat different functional activities. Iodinated recombinant mouse TSP2 bound to NIH 3T3 cells and was internalized and degraded through a chloroquine-inhibitable pathway. TSP2 degradation was saturable, specific, and similar to the kinetics of degradation of TSP1. Human platelet TSP1, recombinant mouse TSP1, and recombinant mouse TSP2 cross-competed with one another for degradation by 3T3 cells. Degradation of TSP2 was less sensitive to inhibition by heparin than degradation of TSP1. This is in agreement with differences in heparin-binding affinity of the two TSPs. Degradation of TSP2 was slower in cultures of Chinese hamster ovary (CHO) cells lacking heparan sulfate proteoglycans than in wild type CHO cells or in cultures of 3T3 cells treated with heparitinase than in untreated 3T3 cells. Degradation of TSP2 was inhibited by antibodies against the low density lipoprotein receptor-related protein (LRP) or by the 39-kDa receptor-associated protein, a known antagonist of LRP. This study indicates that TSP2 and TSP1 are metabolized by a common internalization and degradation pathway involving heparan sulfate proteoglycan and LRP. Competition for this pathway is a possible mechanism whereby cells can control the levels and ratio of TSP1 and TSP2 in the extracellular milieu. Thrombospondin 1 (TSP1) and thrombospondin 2 (TSP2) are members of the thrombospondin family that have a similar structural organization but somewhat different functional activities. Iodinated recombinant mouse TSP2 bound to NIH 3T3 cells and was internalized and degraded through a chloroquine-inhibitable pathway. TSP2 degradation was saturable, specific, and similar to the kinetics of degradation of TSP1. Human platelet TSP1, recombinant mouse TSP1, and recombinant mouse TSP2 cross-competed with one another for degradation by 3T3 cells. Degradation of TSP2 was less sensitive to inhibition by heparin than degradation of TSP1. This is in agreement with differences in heparin-binding affinity of the two TSPs. Degradation of TSP2 was slower in cultures of Chinese hamster ovary (CHO) cells lacking heparan sulfate proteoglycans than in wild type CHO cells or in cultures of 3T3 cells treated with heparitinase than in untreated 3T3 cells. Degradation of TSP2 was inhibited by antibodies against the low density lipoprotein receptor-related protein (LRP) or by the 39-kDa receptor-associated protein, a known antagonist of LRP. This study indicates that TSP2 and TSP1 are metabolized by a common internalization and degradation pathway involving heparan sulfate proteoglycan and LRP. Competition for this pathway is a possible mechanism whereby cells can control the levels and ratio of TSP1 and TSP2 in the extracellular milieu. Thrombospondins (TSPs) 1The abbreviations used are: TSPthrombospondinmTSPmouse TSPhTSPhuman TSPCHOChinese hamster ovaryDMEhigh glucose Dulbecco's modified Eagle mediumELISAenzyme-linked immunosorbent assayLRPlow density lipoprotein receptor-related proteinPARPproline- and arginine-rich proteinPF-4platelet factor 4RAPreceptor-associated proteinTGF-β1transforming growth factor-β1. are a family of structurally related homologous glycoproteins. To date, five TSPs have been identified (1Lawler J. Hynes R.O. J. Cell Biol. 1986; 103: 1635-1648Crossref PubMed Scopus (390) Google Scholar, 2Lawler J. Duquette M. Ferro P. Copeland N.G. Gilbert D.J. Jenkins N.A. Genomics. 1991; 11: 587-600Crossref PubMed Scopus (27) Google Scholar, 3Lawler J. Duquette M. Ferro P. J. Biol. 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These TSPs are divided into two subgroups, with TSP1 and TSP2 in one group and TSP3, TSP4, and TSP5 (also known as cartilage oligomeric matrix protein, COMP) in the other (17Bornstein P. Sage E.H. Methods Enzymol. 1994; 245: 62-85Crossref PubMed Scopus (155) Google Scholar). thrombospondin mouse TSP human TSP Chinese hamster ovary high glucose Dulbecco's modified Eagle medium enzyme-linked immunosorbent assay low density lipoprotein receptor-related protein proline- and arginine-rich protein platelet factor 4 receptor-associated protein transforming growth factor-β1. TSP1 is the best studied of the TSPs. It is a trimeric, secreted modular glycoprotein. Each subunit contains an NH2-terminal globular heparin-binding module (also called a PARP module), a domain that mediates disulfide-stabilized trimerization, a procollagen module, three type 1 (properdin) modules, three type 2 (epidermal growth factor) modules, a series of type 3 (Ca2+-binding) repeats, and a COOH-terminal globular domain (1Lawler J. Hynes R.O. J. Cell Biol. 1986; 103: 1635-1648Crossref PubMed Scopus (390) Google Scholar, 17Bornstein P. Sage E.H. Methods Enzymol. 1994; 245: 62-85Crossref PubMed Scopus (155) Google Scholar, 18Lawler J. Hynes R.O. Semin. Thromb. Hemostasis. 1987; 13: 245-254Crossref PubMed Scopus (14) Google Scholar, 19Mosher D.F. Annu. Rev. Med. 1990; 41: 85-97Crossref PubMed Scopus (146) Google Scholar, 20Frazier W.A. Curr. Opin. Cell Biol. 1991; 3: 792-799Crossref PubMed Scopus (162) Google Scholar). Human platelet TSP1 has been shown to bind to cells, platelets, Ca2+, and many matrix and plasma proteins including fibronectin, collagens, laminin, heparan sulfate, fibrinogen, plasminogen, osteonectin, histidine-rich glycoprotein, and transforming growth factor-β1 (19Mosher D.F. Annu. Rev. Med. 1990; 41: 85-97Crossref PubMed Scopus (146) Google Scholar, 20Frazier W.A. Curr. Opin. Cell Biol. 1991; 3: 792-799Crossref PubMed Scopus (162) Google Scholar, 21Murphy-Ullrich J.E. Schultz-Cherry S. Höök M. Mol. Biol. Cell. 1992; 3: 181-188Crossref PubMed Scopus (222) Google Scholar). There are several possible cell surface receptors for TSP1, including GPIIb/IIIa (22Tuszynski G.P. Karczewski J. Smith L. Murphy A. Rothman V.L. Knudsen K.A. Exp. Cell Res. 1989; 182: 473-481Crossref PubMed Scopus (28) Google Scholar, 23Lawler J. Hynes R.O. Blood. 1989; 74: 2022-2027Crossref PubMed Google Scholar), the vitronectin receptor integrin αvβ3 (23Lawler J. Hynes R.O. Blood. 1989; 74: 2022-2027Crossref PubMed Google Scholar, 24Lawler J. Weinstein R. Hynes R.O. J. Cell Biol. 1988; 107: 2351-2361Crossref PubMed Scopus (337) Google Scholar, 25Sun X. Skorstengaard K. Mosher D.F. J. Cell Biol. 1992; 118: 693-701Crossref PubMed Scopus (93) Google Scholar, 26Adams J.C. Lawler J. J. Cell Sci. 1993; 104: 1061-1071Crossref PubMed Google Scholar), heparan sulfate proteoglycans and sulfatides (27Roberts D.D. Cancer Res. 1988; 48: 6785-6793PubMed Google Scholar, 28Kaesberg P.R. Ershler W.B. Esko J.D. Mosher D.F. J. Clin. Invest. 1989; 83: 994-1001Crossref PubMed Scopus (60) Google Scholar, 29Sun X. Mosher D.F. Rapraeger A. J. Biol. Chem. 1989; 264: 2885-2889Abstract Full Text PDF PubMed Google Scholar, 30Sun X. Kaesberg P.R. Choay J. Harenberg J. Ershler W.B. Mosher D.F. Semin. Thromb. Hemostasis. 1992; 18: 243-251Crossref PubMed Scopus (15) Google Scholar, 31Guo N.H. Krutzsch H.C. Negre E. Vogel T. Blake D.A. Roberts D.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3040-3044Crossref PubMed Scopus (142) Google Scholar, 32Guo N. Krutzsch H.C. Negre E. Zabrenetzky V.S. Roberts D.D. J. Biol. Chem. 1992; 267: 19349-19355Abstract Full Text PDF PubMed Google Scholar), GPIV (CD36) (33Asch A.S. Silbiger S. Heimer E. Nachman R.L. Biochem. Biophys. Res. Commun. 1992; 182: 1208-1217Crossref PubMed Scopus (164) Google Scholar), integrin-associated protein (34Gao A.G. Lindberg F.P. Finn M.B. Blystone S.D. Brown E.J. Frazier W.A. J. Biol. Chem. 1996; 271: 21-24Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar), and possibly other receptors (35Yabkowitz R. Dixit V.M. Cancer Res. 1991; 51: 3648-3656PubMed Google Scholar, 36Adams J.C. Lawler J. Mol. Biol. Cell. 1994; 5: 423-437Crossref PubMed Scopus (68) Google Scholar). TSP1 displays interesting biological activities. It modulates substratum adhesion of normal and tumor cells (25Sun X. Skorstengaard K. Mosher D.F. J. Cell Biol. 1992; 118: 693-701Crossref PubMed Scopus (93) Google Scholar, 26Adams J.C. Lawler J. J. Cell Sci. 1993; 104: 1061-1071Crossref PubMed Google Scholar, 31Guo N.H. Krutzsch H.C. Negre E. Vogel T. Blake D.A. Roberts D.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3040-3044Crossref PubMed Scopus (142) Google Scholar, 36Adams J.C. Lawler J. Mol. Biol. Cell. 1994; 5: 423-437Crossref PubMed Scopus (68) Google Scholar, 37Tuszynski G.P. Rothman V. Murphy A. Siegler K. Smith L. Smith S. Karczewski J. Knudsen K.A. Science. 1987; 236: 1570-1573Crossref PubMed Scopus (110) Google Scholar, 38Roberts D.D. Sherwood J.A. Ginsburg V. J. Cell Biol. 1987; 104: 131-139Crossref PubMed Scopus (137) Google Scholar, 39Kosfeld M.D. Frazier W.A. J. Biol. Chem. 1993; 268: 8808-8814Abstract Full Text PDF PubMed Google Scholar, 40Vogel T. Guo N.H. Krutzsch H.C. Blake D.A. Hartman J. Mendelovitz S. Panet A. Roberts D.D. J. Cell. Biochem. 1993; 53: 74-84Crossref PubMed Scopus (139) Google Scholar) and has antiangiogenic activity in that it can inhibit endothelial cell proliferation, tube formation in vitro and neovascularization in vivo (40Vogel T. Guo N.H. Krutzsch H.C. Blake D.A. Hartman J. Mendelovitz S. Panet A. Roberts D.D. J. Cell. Biochem. 1993; 53: 74-84Crossref PubMed Scopus (139) Google Scholar, 41Bagavandoss P. Wilks J.W. Biochem. Biophys. Res. Commun. 1990; 170: 867-872Crossref PubMed Scopus (150) Google Scholar, 42Taraboletti G. Roberts D. Liotta L.A. Giavazzi R. J. Cell Biol. 1990; 111: 765-772Crossref PubMed Scopus (350) Google Scholar, 43Tolsma S.S. Volpert O.V. Good D.J. Frazier W.A. Polverini P.J. Bouck N. J. Cell Biol. 1993; 122: 497-511Crossref PubMed Scopus (511) Google Scholar). In contrast to the effects on endothelial cells, TSP1 promotes growth and migration of smooth muscle cells and fibroblasts (44Majack R.A. Goodman L.V. Dixit V.M. J. Cell Biol. 1988; 106: 415-422Crossref PubMed Scopus (190) Google Scholar, 45Phan S.H. Dillon R.G. McGarry B.M. Dixit V.M. Biochem. Biophys. Res. Commun. 1989; 163: 56-63Crossref PubMed Scopus (45) Google Scholar, 46Yabkowitz R. Mansfield P.J. Ryan U.S. Suchard S.J. J. Cell. Physiol. 1993; 157: 24-32Crossref PubMed Scopus (81) Google Scholar, 47Nicosia R.F. Tuszynski G.P. J. Cell Biol. 1994; 124: 183-193Crossref PubMed Scopus (155) Google Scholar). TSP1 has also been shown to stimulate migration of keratinocytes (48Nickoloff B.J. Mitra R.S. Riser B.L. Dixit V.M. Varani J. Am. J. Pathol. 1988; 132: 543-551PubMed Google Scholar) and stimulate chemotaxis and haptotaxis of neutrophils (49Mansfield P.J. Boxer L.A. Suchard S.J. J. Cell Biol. 1990; 111: 3077-3086Crossref PubMed Scopus (69) Google Scholar), smooth muscle cells (46Yabkowitz R. Mansfield P.J. Ryan U.S. Suchard S.J. J. Cell. Physiol. 1993; 157: 24-32Crossref PubMed Scopus (81) Google Scholar), and several carcinoma and melanoma cells (50Taraboletti G. Roberts D.D. Liotta L.A. J. Cell Biol. 1987; 105: 2409-2415Crossref PubMed Scopus (174) Google Scholar, 51Yabkowitz R. Mansfield P.J. Dixit V.M. Suchard S.J. Cancer Res. 1993; 53: 378-387PubMed Google Scholar). TSP1, when secreted from platelets, can complex with activated TGF-β1. TSP1 also binds and activates latent TGF-β1 (52Schultz-Cherry S. Murphy-Ullrich J.E. J. Cell Biol. 1993; 122: 923-932Crossref PubMed Scopus (401) Google Scholar, 53Schultz-Cherry S. Lawler J. Murphy-Ullrich J.E. J. Biol. Chem. 1994; 269: 26783-26788Abstract Full Text PDF PubMed Google Scholar, 54Schultz-Cherry S. Ribeiro S. Gentry L. Murphy-Ullrich J.E. J. Biol. Chem. 1994; 269: 26775-26782Abstract Full Text PDF PubMed Google Scholar, 55Schultz-Cherry S. Chen H. Mosher D.F. Misenheimer T.M. Krutzsch H.C. Roberts D.D. Murphy-Ullrich J.E. J. Biol. Chem. 1995; 270: 7304-7310Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). Since most cell types secrete latent TGF-β and express TGF-β receptors on their surfaces, it is postulated that TSP1 is a key regulator of TGF-β1 activity under physiological conditions (52Schultz-Cherry S. Murphy-Ullrich J.E. J. Cell Biol. 1993; 122: 923-932Crossref PubMed Scopus (401) Google Scholar, 53Schultz-Cherry S. Lawler J. Murphy-Ullrich J.E. J. Biol. Chem. 1994; 269: 26783-26788Abstract Full Text PDF PubMed Google Scholar, 54Schultz-Cherry S. Ribeiro S. Gentry L. Murphy-Ullrich J.E. J. Biol. Chem. 1994; 269: 26775-26782Abstract Full Text PDF PubMed Google Scholar, 55Schultz-Cherry S. Chen H. Mosher D.F. Misenheimer T.M. Krutzsch H.C. Roberts D.D. Murphy-Ullrich J.E. J. Biol. Chem. 1995; 270: 7304-7310Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). The structural modules of TSP2 are similar to those of TSP1, with an increasing gradient of sequence identity from the NH2-terminal module (38% identity) to the COOH-terminal domain (82% identity) (4Bornstein P. O'Rourke K. Wikstrom K. Wolf F.W. Katz R. Li P. Dixit V.M. J. Biol. Chem. 1991; 266: 12821-12824Abstract Full Text PDF PubMed Google Scholar, 6Laherty C.D. O'Rourke K. Wolf F.W. Katz R. Seldin M.F. Dixit V.M. J. Biol. Chem. 1992; 267: 3274-3281Abstract Full Text PDF PubMed Google Scholar). The patterns of expression of TSP1 and TSP2 mRNAs are distinct in tissues of embryonic and developed mice (56Iruela-Arispe M.L. Liska D.J. Sage E.H. Bornstein P. Dev. Dyn. 1993; 197: 40-56Crossref PubMed Scopus (184) Google Scholar, 57Liska D.J. Hawkins R. Wikstrom K. Bornstein P. J. Cell. Physiol. 1994; 158: 495-505Crossref PubMed Scopus (22) Google Scholar). To compare the structure and function of TSP1 and TSP2, we have expressed mouse TSP2 (mTSP2) in a baculovirus system as a disulfide-bonded homotrimer (58Chen H. Sottile J. O'Rourke K.M. Dixit V.M. Mosher D.F. J. Biol. Chem. 1994; 269: 32226-32232Abstract Full Text PDF PubMed Google Scholar). mTSP2 supports adhesion for endothelial cells, osteosarcoma cells, and colon carcinoma cells by mechanisms similar but not identical to those of TSP1. Adherence to both TSPs appears to utilize heparan sulfate proteoglycans and αvβ3 integrin, and is regulated by Ca2+ and reduction. One major difference between adhesive activities of TSP1 and TSP2 is the differential sensitivity to inhibition of adhesion by heparin (58Chen H. Sottile J. O'Rourke K.M. Dixit V.M. Mosher D.F. J. Biol. Chem. 1994; 269: 32226-32232Abstract Full Text PDF PubMed Google Scholar). In another adhesion system where adrenocortical cells are used as a source, bovine TSP2 (also known as corticotropin-induced secreted protein, CISP) shows an antiadhesive activity (59Pellerin S. Lafeuillade B. Chambaz E.M. Feige J.J. Mol. Cell. Endocrinol. 1994; 106: 181-186Crossref PubMed Scopus (18) Google Scholar). TSP2, like TSP1, binds TGF-β1 (55Schultz-Cherry S. Chen H. Mosher D.F. Misenheimer T.M. Krutzsch H.C. Roberts D.D. Murphy-Ullrich J.E. J. Biol. Chem. 1995; 270: 7304-7310Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). TSP2 does not, however, activate latent TGF-β1 (55Schultz-Cherry S. Chen H. Mosher D.F. Misenheimer T.M. Krutzsch H.C. Roberts D.D. Murphy-Ullrich J.E. J. Biol. Chem. 1995; 270: 7304-7310Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). This lack of activity apparently is due to substitution of the activating RFK sequence in TSP1 (55Schultz-Cherry S. Chen H. Mosher D.F. Misenheimer T.M. Krutzsch H.C. Roberts D.D. Murphy-Ullrich J.E. J. Biol. Chem. 1995; 270: 7304-7310Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar) with the trypsin-susceptible, nonactivating RIR sequence in TSP2 (55Schultz-Cherry S. Chen H. Mosher D.F. Misenheimer T.M. Krutzsch H.C. Roberts D.D. Murphy-Ullrich J.E. J. Biol. Chem. 1995; 270: 7304-7310Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 58Chen H. Sottile J. O'Rourke K.M. Dixit V.M. Mosher D.F. J. Biol. Chem. 1994; 269: 32226-32232Abstract Full Text PDF PubMed Google Scholar). TSP2 inhibits the activation of latent TGF-β1 by TSP1, presumably through the common TGF-β1 binding sequence GGWSHW present in both TSP1 and TSP2 (55Schultz-Cherry S. Chen H. Mosher D.F. Misenheimer T.M. Krutzsch H.C. Roberts D.D. Murphy-Ullrich J.E. J. Biol. Chem. 1995; 270: 7304-7310Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). Therefore TSP2 may act as a buffer to the activation of latent TGF-β1 by TSP1. Because TSP1 has potent biological activities, and TSP2, besides its own functions, may regulate TSP1 function, one may postulate that the ratio of TSP1 and TSP2, as well as their amount, is regulated by their expression and half-life. TSP1 is secreted from platelet α-granules upon activation (19Mosher D.F. Annu. Rev. Med. 1990; 41: 85-97Crossref PubMed Scopus (146) Google Scholar, 60Lawler J.W. Slayter H.S. Coligan J.E. J. Biol. Chem. 1978; 253: 8609-8616Abstract Full Text PDF PubMed Google Scholar). It is also produced by a variety of normal and transformed cell lines (19Mosher D.F. Annu. Rev. Med. 1990; 41: 85-97Crossref PubMed Scopus (146) Google Scholar, 61Raugi G.J. Mumby S.M. Abbott-Brown D. Bornstein P. J. Cell Biol. 1982; 95: 351-354Crossref PubMed Scopus (167) Google Scholar, 62Jaffe E.A. Ruggiero J.T. Leung L.K. Doyle M.J. McKeown-Longo P.J. Mosher D.F. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 998-1002Crossref PubMed Scopus (194) Google Scholar). Whereas TSP1 production is up-regulated dramatically by serum or growth factors, TSP2 expression is constitutive (5Bornstein P. Devarayalu S. Li P. Disteche C.M. Framson P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8636-8640Crossref PubMed Scopus (51) Google Scholar, 6Laherty C.D. O'Rourke K. Wolf F.W. Katz R. Seldin M.F. Dixit V.M. J. Biol. Chem. 1992; 267: 3274-3281Abstract Full Text PDF PubMed Google Scholar). It has been shown that platelet TSP1 can bind to cells and incorporate into the extracellular matrix (63McKeown-Longo P.J. Hanning R. Mosher D.F. J. Cell Biol. 1984; 98: 22-28Crossref PubMed Scopus (71) Google Scholar, 64Murphy-Ullrich J.E. Mosher D.F. Semin. Thromb. Hemostasis. 1987; 13: 343-351Crossref PubMed Scopus (15) Google Scholar), or be cleared by cells via endocytosis and lysosomal degradation (30Sun X. Kaesberg P.R. Choay J. Harenberg J. Ershler W.B. Mosher D.F. Semin. Thromb. Hemostasis. 1992; 18: 243-251Crossref PubMed Scopus (15) Google Scholar, 63McKeown-Longo P.J. Hanning R. Mosher D.F. J. Cell Biol. 1984; 98: 22-28Crossref PubMed Scopus (71) Google Scholar, 64Murphy-Ullrich J.E. Mosher D.F. Semin. Thromb. Hemostasis. 1987; 13: 343-351Crossref PubMed Scopus (15) Google Scholar, 65Murphy-Ullrich J.E. Mosher D.F. J. Cell Biol. 1987; 105: 1603-1611Crossref PubMed Scopus (90) Google Scholar, 66Murphy-Ullrich J.E. Westrick L.G. Esko J.D. Mosher D.F. J. Biol. Chem. 1988; 263: 6400-6406Abstract Full Text PDF PubMed Google Scholar, 67Mikhailenko I. Kounnas M.Z. Strickland D.K. J. Biol. Chem. 1995; 270: 9543-9549Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 68Godyna S. Liau G. Popa I. Stefansson S. Argraves W.S. J. Cell Biol. 1995; 129: 1403-1410Crossref PubMed Scopus (128) Google Scholar). Cell surface heparan sulfate proteoglycans are required for binding and degradation of platelet TSP1 (30Sun X. Kaesberg P.R. Choay J. Harenberg J. Ershler W.B. Mosher D.F. Semin. Thromb. Hemostasis. 1992; 18: 243-251Crossref PubMed Scopus (15) Google Scholar, 63McKeown-Longo P.J. Hanning R. Mosher D.F. J. Cell Biol. 1984; 98: 22-28Crossref PubMed Scopus (71) Google Scholar, 64Murphy-Ullrich J.E. Mosher D.F. Semin. Thromb. Hemostasis. 1987; 13: 343-351Crossref PubMed Scopus (15) Google Scholar, 65Murphy-Ullrich J.E. Mosher D.F. J. Cell Biol. 1987; 105: 1603-1611Crossref PubMed Scopus (90) Google Scholar, 66Murphy-Ullrich J.E. Westrick L.G. Esko J.D. Mosher D.F. J. Biol. Chem. 1988; 263: 6400-6406Abstract Full Text PDF PubMed Google Scholar, 67Mikhailenko I. Kounnas M.Z. Strickland D.K. J. Biol. Chem. 1995; 270: 9543-9549Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 68Godyna S. Liau G. Popa I. Stefansson S. Argraves W.S. J. Cell Biol. 1995; 129: 1403-1410Crossref PubMed Scopus (128) Google Scholar). Low density lipoprotein receptor-related protein (LRP) has been shown recently to synergize with heparan sulfate proteoglycans in mediating internalization and degradation of platelet TSP1 (67Mikhailenko I. Kounnas M.Z. Strickland D.K. J. Biol. Chem. 1995; 270: 9543-9549Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 68Godyna S. Liau G. Popa I. Stefansson S. Argraves W.S. J. Cell Biol. 1995; 129: 1403-1410Crossref PubMed Scopus (128) Google Scholar). One may hypothesize, based on the homology between TSP1 and TSP2, that their metabolism is similar. However, many homologous proteins have different receptors, and the NH2-terminal region that mediates binding to heparin is the part with the lowest sequence identity between TSP1 and TSP2. Thus, one may also hypothesize that the metabolism of TSP1 and TSP2 is different. To evaluate these hypotheses, we carried out experiments to investigate whether recombinant mouse TSP2 is metabolized by a mechanism similar to that of platelet or recombinant TSP1 and whether TSP1 and TSP2 compete for the same degradation pathway. Human platelet TSP1 (hTSP1) and recombinant mTSP2 produced with baculovirus were purified as described previously (58Chen H. Sottile J. O'Rourke K.M. Dixit V.M. Mosher D.F. J. Biol. Chem. 1994; 269: 32226-32232Abstract Full Text PDF PubMed Google Scholar). Production and purification of recombinant mTSP1 were similar to those used for mTSP2. Briefly, mouse TSP1 cDNA including bases 51-3751 flanked by MluI linkers in the pJDM eukaryote expression vector (a generous gift from Dr. Vishva Dixit) (69O'Rourke K.M. Laherty C.D. Dixit V.M. J. Biol. Chem. 1992; 267: 24921-24924Abstract Full Text PDF PubMed Google Scholar) was used. NcoI and EcoRI digestion was used to generate a 5′ mouse TSP1 fragment containing bases 210-1404 which lacked excess 5′-untranslated region. A 3′ fragment was generated by EcoRI digestion and incomplete BamHI digestion which cut at the BamHI site in the pJDM multiple cloning region but not inside the mouse TSP1 3′ cDNA. These two fragments were subcloned into the baculovirus transfer vector pAcSG2 (Pharmingen, San Diego, CA) linearized by NcoI and BglII, utilizing the compatible cohesive ends of BamHI and BglII. Recombinant mTSP1 virus was generated using the BaculoGold transfection system (Pharmingen) with Lipofectin per the manufacturer's instructions. After transfection, recombinant viruses were plaque-purified once, and third passage virus in serum-free medium SF900 II (Life Technologies, Inc.) was used to infect Spodoptera frugiperda cells. Receptor-associated protein (RAP) and R777 antibodies against LRP were prepared as described previously (67Mikhailenko I. Kounnas M.Z. Strickland D.K. J. Biol. Chem. 1995; 270: 9543-9549Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Native and urea-treated vitronectin (70Bittorf S.V. Williams E.C. Mosher D.F. J. Biol. Chem. 1993; 268: 24838-24846Abstract Full Text PDF PubMed Google Scholar), platelet factor 4 (PF-4) (71Watson J.B. Getzler S.B. Mosher D.F. J. Clin. Invest. 1994; 94: 261-268Crossref PubMed Scopus (57) Google Scholar), fibronectin (72McKeown-Longo P.J. Mosher D.F. J. Cell Biol. 1983; 97: 466-472Crossref PubMed Scopus (201) Google Scholar), fibrinogen (73Mosher D.F. J. Biol. Chem. 1975; 250: 6614-6621Abstract Full Text PDF PubMed Google Scholar), and type I collagen (74Bornstein P. Piez K. J. Clin. Invest. 1964; 43: 1813-1823Crossref PubMed Scopus (73) Google Scholar) were purified as described elsewhere. Heparin was purchased from Sigma. Heparitinase, heparinase I, and chondroitinase ABC were from stocks in Dr. Alan Rapraeger's laboratory at the University of Wisconsin-Madison and purchased originally from ICN Biomedicals (Aurora, OH). Chondroitinase ABC from Seikagaku (Rockville, MD) was also tested. Purified mTSP2, 30 µg, was transferred to nitrocellulose paper after electrophoresis in SDS. The nitrocellulose paper containing the mTSP2 band was cut out, washed with H2O, air-dried, frozen in a dry ice/isopropanol bath, and crushed into a fine powder with a glass rod. The powder was emulsified with complete Freund's adjuvant and injected subcutaneously into two New Zealand White male rabbits (30 µg each). The rabbits were boosted three times at 1-month intervals with electrophoretically repurified mTSP2, 35 µg, on nitrocellulose in incomplete Freund's adjuvant, followed by boosts with purified soluble mTSP2 every 4–6 weeks. Antibody titers and antibody specificities were checked by enzyme-linked immunosorbent assay (ELISA) on 96-well plates coated with hTSP1, mTSP1, or mTSP2. Antibodies to hTSP1 were produced by a similar protocol. The specificities and species cross-reactivities of rabbit antibodies to mTSP2 or hTSP1 were determined by direct ELISA. The anti-mTSP2 antisera had titers against mTSP2 of 1:20,000-1:40,000, against hTSP1 of 1:600 and against mTSP1 of 1:1,500. Anti-TSP1 antisera had a titer against mTSP1 of 1:15,000 and against mTSP2 of 1:1,200. Purified TSP, 100 µg, was iodinated with 0.5 mCi of Na125I in the presence of 0.5 mM chloramine-T as described previously (63McKeown-Longo P.J. Hanning R. Mosher D.F. J. Cell Biol. 1984; 98: 22-28Crossref PubMed Scopus (71) Google Scholar, 65Murphy-Ullrich J.E. Mosher D.F. J. Cell Biol. 1987; 105: 1603-1611Crossref PubMed Scopus (90) Google Scholar). After 1 min, phenylmethylsulfonyl fluoride-treated bovine albumin was added to a concentration of 10 mg/ml. 125I-TSP was repurified by affinity chromatography on heparin-agarose and eluted by 1 M NaCl in 0.3 mM Ca2+ and 10 mM Tris, pH 7.4. Albumin was added to a final concentration of 2 mg/ml, and 125I-TSPs were stored as small aliquots at −70°C until use. Iodinated TSPs had the expected mobilities in autoradiograms of polyacrylamide gels after electrophoretic separation in SDS without and with reduction. Specific activities were 1.2-5.2 mCi/mg TSP. Iodinated TSPs had 2–7% trichloroacetic acid-soluble radioactivity. NIH 3T3 cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained in high glucose Dulbecco's modified Eagle medium (DME) containing 10% fetal bovine serum at 37°C in an incubator containing 8% CO2. Chinese hamster ovary (CHO) cells were maintained as described previously (66Murphy-Ullrich J.E. Westrick L.G. Esko J.D. Mosher D.F. J. Biol. Chem. 1988; 263: 6400-6406Abstract Full Text PDF PubMed Google Scholar) in a 5% CO2 incubator. Cells were grown to confluence on 24-well tissue culture plates (Costar, Cambridge, MA) and washed three times with DME before assays. Binding and degradation assays were carried out in DME with 0.2% bovine albumin containing 100 units/ml penicillin G and 150 µg/ml streptomycin sulfate according to procedures described before (63McKeown-Longo P.J. Hanning R. Mosher D.F. J. Cell Biol. 1984; 98: 22-28Crossref PubMed Scopus (71) Google Scholar, 64Murphy-Ullrich J.E. Mosher D.F. Semin. Thromb. Hemostasis. 1987; 13: 343-351Crossref PubMed Scopus (15) Google Scholar, 65Murphy-Ullrich J.E. Mosher D.F. J. Cell Biol. 1987; 105: 1603-1611Crossref PubMed Scopus (90) Google Scholar, 66Murphy-Ullrich J.E. Westrick L.G. Esko J.D. Mosher D.F. J. Biol. Chem. 1988; 263: 6400-6406Abstract Full Text PDF PubMed Google Scholar). Binding medium containing 125I-TSP was incubated with cells in the CO2 incubator at 37°C for various times. After the incubation, binding medium was removed and mixed with trichloroacetic acid at a final concentration of 10%. After incubation on ice for 15 min, the precipitate was removed by centrifugation. The increase in trichloroacetic acid-soluble radioactivity above the baseline value during the incubation was taken as TSP that had been degraded by the cells. The negligible increase in trichloroacetic acid-soluble radioactivity during incubation in plates without cells was considered as the baseline. At the end of the incubation, cell layers were washed with cold Tris-buffered saline three times and" @default.
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• W2078698377 date "1996-07-01" @default.
• W2078698377 modified "2023-10-18" @default.
• W2078698377 title "Metabolism of Thrombospondin 2" @default.
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