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• W1972163715 abstract "Thymic shared antigen-1 (TSA-1)/stem cell Ag-2 (Sca-2) is a glycosylphosphatidylinositol (GPI)-anchored antigen expressed on lymphocytes. We have previously demonstrated that a signal via TSA-1/Sca-2 inhibits T cell receptor (TCR)-mediated T cell activation and apoptosis. To elucidate a molecular mechanism for TSA-1-mediated modulation of the TCR-signaling pathway, we examined whether TSA-1 is physically coupled to the TCR in the present study. TSA-1 was clearly associated with CD3ζ chains in T cell hybridomas, activated T cells, and COS-7 cells transfected with TSA-1 and CD3ζ cDNA. The physical association was confirmed on the surface of T cells in immunoprecipitation and confocal microscopy. The analysis using stable and transient transfectants expressing a transmembrane form of TSA-1 revealed that the association of CD3ζ did not require the GPI anchor of TSA-1. Finally, tyrosine phosphorylation of CD3ζ chains was induced after stimulation with anti-TSA-1, suggesting that a functional association between these two molecules also exists. These results imply that the physical association to CD3ζ underlies a regulatory role of TSA-1/Sca-2 in the TCR-signaling pathway. Thymic shared antigen-1 (TSA-1)/stem cell Ag-2 (Sca-2) is a glycosylphosphatidylinositol (GPI)-anchored antigen expressed on lymphocytes. We have previously demonstrated that a signal via TSA-1/Sca-2 inhibits T cell receptor (TCR)-mediated T cell activation and apoptosis. To elucidate a molecular mechanism for TSA-1-mediated modulation of the TCR-signaling pathway, we examined whether TSA-1 is physically coupled to the TCR in the present study. TSA-1 was clearly associated with CD3ζ chains in T cell hybridomas, activated T cells, and COS-7 cells transfected with TSA-1 and CD3ζ cDNA. The physical association was confirmed on the surface of T cells in immunoprecipitation and confocal microscopy. The analysis using stable and transient transfectants expressing a transmembrane form of TSA-1 revealed that the association of CD3ζ did not require the GPI anchor of TSA-1. Finally, tyrosine phosphorylation of CD3ζ chains was induced after stimulation with anti-TSA-1, suggesting that a functional association between these two molecules also exists. These results imply that the physical association to CD3ζ underlies a regulatory role of TSA-1/Sca-2 in the TCR-signaling pathway. Thymic shared antigen-1 (TSA-1) 1The abbreviations used are: TSA-1, thymic shared Ag-1; Sca-2, stem cell Ag-2; TCR, T cell receptor; GPI, glycosylphosphatidylinositol; LFA-1, leukocyte function-associated antigen-1; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; NRIgG, normal rat immunoglobulin G.1The abbreviations used are: TSA-1, thymic shared Ag-1; Sca-2, stem cell Ag-2; TCR, T cell receptor; GPI, glycosylphosphatidylinositol; LFA-1, leukocyte function-associated antigen-1; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; NRIgG, normal rat immunoglobulin G./stem cell antigen-2 (Sca-2) is a Ly-6-related differentiation antigen expressed on immature thymocytes and thymic epithelial cells (1Godfrey D.I. Masciantonio M. Tucek C.L. Malin M.A. Boyd R.L. Hugo P. J. Immunol. 1992; 148: 2006-2011PubMed Google Scholar, 2MacNeil I. Kennedy J. Godfrey D.I. Jenkins N.A. Masciantonio M. Mineo C. Gilbert D.J. Copeland N.G. Boyd R.L. Zlotnik A. J. Immunol. 1993; 151: 6913-6923PubMed Google Scholar, 3Spangrude G.J. Aihara Y. Weissman I.L. Klein J. J. Immunol. 1988; 141: 3697-3707PubMed Google Scholar, 4Classon B.J. Coverdale L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5296-5300Crossref PubMed Scopus (48) Google Scholar). Recently, cDNA encoding human TSA-1 has been isolated, and it was shown that TSA-1 mRNA is expressed in human lymphoid tissues as well as various nonlymphoid tissues (5Capone M.C. Gorman D.M. Ching E.P. Zlotnik A. J. Immunol. 1996; 157: 969-973PubMed Google Scholar). Although TSA-1/Sca-2 is a useful marker in early T cell development and T cell activation and seems to play a regulatory role in thymocyte differentiation (6Kosugi A. Saitoh S.-I. Narumiya S. Miyake K. Hamaoka T. Int. Immunol. 1994; 6: 1967-1976Crossref PubMed Scopus (32) Google Scholar, 7Wu L. Antica M. Johnson G.R. Scollay R. Shortman K. J. Exp. Med. 1991; 174: 1617-1627Crossref PubMed Scopus (268) Google Scholar, 8Randle E.S. Waanders G.A. Masciantonio M. Godfrey D.I. Boyd R.L. J. Immunol. 1993; 151: 6027-6035PubMed Google Scholar), functions of TSA-1/Sca-2 remain largely obscure.In a previous study, we have analyzed a role of TSA-1 in mature T cells and demonstrated that it functions as a modulator of T cell receptor (TCR)-signaling pathway (6Kosugi A. Saitoh S.-I. Narumiya S. Miyake K. Hamaoka T. Int. Immunol. 1994; 6: 1967-1976Crossref PubMed Scopus (32) Google Scholar, 9Saitoh S.-I. Kosugi A. Noda S. Yamamoto N. Ogata M. Minami Y. Miyake K. Hamaoka T. J. Immunol. 1995; 155: 5574-5581PubMed Google Scholar, 10Noda S. Kosugi A. Saitoh S.-I. Narumiya S. Hamaoka T. J. Exp. Med. 1996; 183: 2355-2360Crossref PubMed Scopus (46) Google Scholar). Anti-TSA-1 mAb inhibited tyrosine phosphorylation of CD3ζ chains and IL-2 production induced by anti-CD3 stimulation in T cell hybridomas (9Saitoh S.-I. Kosugi A. Noda S. Yamamoto N. Ogata M. Minami Y. Miyake K. Hamaoka T. J. Immunol. 1995; 155: 5574-5581PubMed Google Scholar), suggesting that a signal via TSA-1 regulates early and late events in TCR signaling. The findings observed in this in vitro study were further strengthened by the fact that in vivo injection of anti-TSA-1 mAb completely blocked anti-TCR/CD3-mediated apoptosis of thymocytes (10Noda S. Kosugi A. Saitoh S.-I. Narumiya S. Hamaoka T. J. Exp. Med. 1996; 183: 2355-2360Crossref PubMed Scopus (46) Google Scholar). Thus, TSA-1/Sca-2 seems to be an important cell surface molecule regulating T cell differentiation and activation by virtue of its ability for modulating TCR-mediated signal transduction. However, since TSA-1 is a glycosylphosphatidylinositol (GPI)-anchored membrane protein and thus does not have its transmembrane and cytoplasmic regions, it is not known how TSA-1 transmit signals into the cytoplasm of the cell.In the present study, we addressed the above question by analyzing the molecular interaction between TSA-1 and the TCR. The data clearly demonstrated that TSA-1 is physically and functionally associated with CD3ζ chains of the TCR complex, and strongly suggested that the regulatory role of TSA-1 on TCR signaling is based on this intermolecular association.DISCUSSIONMany rodent and human GPI-anchored proteins have been implicated in regulation of T cell activation, since mAbs against these GPI-anchored proteins induce T cell activation as monitored by interleukin-2 production and proliferation. T cell activation induced by a signal through GPI-anchored proteins is dependent upon expression of the TCR; anti-Thy-1 and anti-Ly-6 mAbs fail to stimulate a TCR− variant cell line, and the defect was able to be restored by TCR expression in these variant cell lines (22Gunter K.C. Germain R.N. Kroczek R.A. Saito T. Yokoyama W.M. Chan C. Weiss A. Shevach E.M. Nature. 1987; 326: 505-507Crossref PubMed Scopus (132) Google Scholar, 23Sussman J.J. Saito T. Shevach E.M. Germain R.N. Ashwell J.D. J. Immunol. 1988; 140: 2520-2526PubMed Google Scholar). In addition to the positive regulation by GPI-anchored proteins in T cell activation, some GPI-anchored proteins transduce a negative signal that inhibits anti-CD3-mediated TCR signaling (24Codias E.K. Rutter J.E. Fleming T.J. Malek T.R. J. Immunol. 1990; 145: 1407-1414PubMed Google Scholar). We have previously demonstrated that a signal via TSA-1/Sca-2 inhibits TCR/CD3-mediated activation and apoptosis both in vitro and in vivo (6Kosugi A. Saitoh S.-I. Narumiya S. Miyake K. Hamaoka T. Int. Immunol. 1994; 6: 1967-1976Crossref PubMed Scopus (32) Google Scholar, 9Saitoh S.-I. Kosugi A. Noda S. Yamamoto N. Ogata M. Minami Y. Miyake K. Hamaoka T. J. Immunol. 1995; 155: 5574-5581PubMed Google Scholar, 10Noda S. Kosugi A. Saitoh S.-I. Narumiya S. Hamaoka T. J. Exp. Med. 1996; 183: 2355-2360Crossref PubMed Scopus (46) Google Scholar). Thus, the TCR seems to be an essential molecule in signaling pathway of GPI-anchored proteins at least in T cells.A number of studies have indirectly suggested that there is a physical and/or functional association between some GPI-anchored proteins and the TCR. By using chemical cross-linkers, it was reported that CD45 is mutually associated with Thy-1 and the TCR, indicating that Thy-1 can physically interact with the TCR through CD45 (25Volarevic S. Burns C.M. Sussman J.J. Ashwell J.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7085-7089Crossref PubMed Scopus (119) Google Scholar). In another study, a T cell clone was stably transfected with antisense Ly-6A RNA (26Lee S.-K. Su B. Maher S.E. Bothwell A.L.M. EMBO J. 1994; 13: 2167-2176Crossref PubMed Scopus (53) Google Scholar). Cell surface expression of Ly-6A was markedly suppressed in this transfectant, but surprisingly surface expression of the TCR was greatly inhibited as well because of the reduction of TCR-β mRNA. The Ly-6A antisense transfectant was then transfected with TCR-β cDNA, and surface TCR expression was reconstituted without the expression of Ly-6A. However, TCR signaling was still impaired in this transfectant due to the absence of Ly-6A.Despite these observations, it seems to be very difficult to demonstrate a direct association of the TCR to Thy-1, Ly-6, or other GPI-anchored proteins in immunoprecipitation analysis. Nonetheless, we are able to provide evidence that TSA-1/Sca-2 is physically associated with TCR in the present study. When TSA-1/Sca-2 expressed on the cell surface was stimulated with anti-TSA-1 mAbs, CD3ζ in the TCR complex was induced to be phosphorylated in its tyrosine residues (Fig. 9). This result indicates that a functional association also exists between these two molecules, and argues against the possibility that the interaction between TSA-1 and CD3ζ occurs merely during the process of solubilization and immunoprecipitation.We do not know why we can successfully detect the physical association of the TCR to TSA-1 among many GPI-anchored proteins. Since none of mAbs against Thy-1, Ly-6A/E, and Ly-6C co-precipitated CD3ζ in an experiment in which both mAbs, PRST1 and GR12, against TSA-1 clearly co-precipitated CD3ζ (Fig. 2), the association between TSA-1 and CD3ζ is considered to be specific. Given that TSA-1 is a GPI-anchored protein and CD3ζ has a very short extracellular portion, the interaction between TSA-1 and CD3ζ could be mediated by an as yet undefined membrane protein, which could serve as a linker between these two proteins (27Brown D. Curr. Opin. Immunol. 1993; 5: 349-354Crossref PubMed Scopus (189) Google Scholar). This “linker” protein presumably functions not only in T cells but in COS-7 cells (Fig. 4). Moreover, the association between TSA-1 and the “linker” protein could not be dependent on the GPI anchor, but on primary sequence motifs of TSA-1. An effort should be made to identify the “linker” protein in biochemical analysis.Alternatively, another possibility may account for the mechanism underlying the physical association between TSA-1 and CD3ζ. GPI-anchored proteins are known to be localized to caveolae, glycosphingolipid-rich areas in the cell membrane (28Bamezai A. Goldmacher V. Reiser H. Rock K.L. J. Immunol. 1989; 143: 3107-3116PubMed Google Scholar, 29Anderson R.G.W. Kamen B.A. Rothberg K.G. Lacey S.W. Science. 1992; 255: 410-411Crossref PubMed Scopus (657) Google Scholar, 30Parton R.G. Simons K. Science. 1995; 269: 1398-1399Crossref PubMed Scopus (295) Google Scholar). Caveolae are also enriched in signal-transducing molecules, such as GTP-binding proteins, small G proteins, and nonreceptor-type tyrosine kinases (31Anderson R.G.W. Curr. Opin. Cell Biol. 1993; 5: 647-652Crossref PubMed Scopus (171) Google Scholar). It has been proposed that caveolae could represent a specialized signaling compartment at the cell surface (32Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8019) Google Scholar). Although lymphocytes do not have caveolae due to the lack of caveolin, there is the same membrane microdomain that are enriched in glycosphingolipids in lymphocytes (33Fra A.M. Williamson E. Simons K. Parton R.G. J. Biol. Chem. 1994; 269: 30745-30748Abstract Full Text PDF PubMed Google Scholar). Thus, if the TCR complex or the CD3ζ may reside in this microdomain, GPI-anchored proteins could be associated with CD3ζ by lipid-protein interactions, thereby forming a signaling compartment at the surface of T cells. Stimulation of GPI-anchored proteins with mAbs results in the delivery of a signal through this signaling compartment. If this possibility is correct, TSA-1 is not special among other GPI-anchored proteins, but our mAbs against TSA-1 could be special among other mAbs against GPI-anchored proteins. Although we can not thus far explain whether and how a transmembrane TSA-1 could be localized in this signaling compartment, the above hypothesis is very attractive, given that most of GPI-anchored proteins have a signal-transducing ability when cross-linked with mAbs.Although the mechanism is not fully understood, our previous finding that a signal through TSA-1 down-modulates the TCR signaling pathway could be explained by the physical association between TSA-1 and CD3ζ. Cross-linking of TSA-1 with the mAb induces the phosphorylation of tyrosine residues in CD3ζ chains (Fig. 9) through the activation of the Src family tyrosine kinases, which may subsequently cause recruitment of another protein tyrosine kinase, ZAP-70 (34Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1944) Google Scholar). Thus, intracellular signal-transducing molecules could be sequestered from TCR-signaling pathways to the TSA-1/CD3ζ complex, resulting in down-modulation of TCR signaling. Studies are in progress to elucidate a molecular mechanism for the TSA-1-signaling pathway. Thymic shared antigen-1 (TSA-1) 1The abbreviations used are: TSA-1, thymic shared Ag-1; Sca-2, stem cell Ag-2; TCR, T cell receptor; GPI, glycosylphosphatidylinositol; LFA-1, leukocyte function-associated antigen-1; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; NRIgG, normal rat immunoglobulin G.1The abbreviations used are: TSA-1, thymic shared Ag-1; Sca-2, stem cell Ag-2; TCR, T cell receptor; GPI, glycosylphosphatidylinositol; LFA-1, leukocyte function-associated antigen-1; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; NRIgG, normal rat immunoglobulin G./stem cell antigen-2 (Sca-2) is a Ly-6-related differentiation antigen expressed on immature thymocytes and thymic epithelial cells (1Godfrey D.I. Masciantonio M. Tucek C.L. Malin M.A. Boyd R.L. Hugo P. J. Immunol. 1992; 148: 2006-2011PubMed Google Scholar, 2MacNeil I. Kennedy J. Godfrey D.I. Jenkins N.A. Masciantonio M. Mineo C. Gilbert D.J. Copeland N.G. Boyd R.L. Zlotnik A. J. Immunol. 1993; 151: 6913-6923PubMed Google Scholar, 3Spangrude G.J. Aihara Y. Weissman I.L. Klein J. J. Immunol. 1988; 141: 3697-3707PubMed Google Scholar, 4Classon B.J. Coverdale L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5296-5300Crossref PubMed Scopus (48) Google Scholar). Recently, cDNA encoding human TSA-1 has been isolated, and it was shown that TSA-1 mRNA is expressed in human lymphoid tissues as well as various nonlymphoid tissues (5Capone M.C. Gorman D.M. Ching E.P. Zlotnik A. J. Immunol. 1996; 157: 969-973PubMed Google Scholar). Although TSA-1/Sca-2 is a useful marker in early T cell development and T cell activation and seems to play a regulatory role in thymocyte differentiation (6Kosugi A. Saitoh S.-I. Narumiya S. Miyake K. Hamaoka T. Int. Immunol. 1994; 6: 1967-1976Crossref PubMed Scopus (32) Google Scholar, 7Wu L. Antica M. Johnson G.R. Scollay R. Shortman K. J. Exp. Med. 1991; 174: 1617-1627Crossref PubMed Scopus (268) Google Scholar, 8Randle E.S. Waanders G.A. Masciantonio M. Godfrey D.I. Boyd R.L. J. Immunol. 1993; 151: 6027-6035PubMed Google Scholar), functions of TSA-1/Sca-2 remain largely obscure. In a previous study, we have analyzed a role of TSA-1 in mature T cells and demonstrated that it functions as a modulator of T cell receptor (TCR)-signaling pathway (6Kosugi A. Saitoh S.-I. Narumiya S. Miyake K. Hamaoka T. Int. Immunol. 1994; 6: 1967-1976Crossref PubMed Scopus (32) Google Scholar, 9Saitoh S.-I. Kosugi A. Noda S. Yamamoto N. Ogata M. Minami Y. Miyake K. Hamaoka T. J. Immunol. 1995; 155: 5574-5581PubMed Google Scholar, 10Noda S. Kosugi A. Saitoh S.-I. Narumiya S. Hamaoka T. J. Exp. Med. 1996; 183: 2355-2360Crossref PubMed Scopus (46) Google Scholar). Anti-TSA-1 mAb inhibited tyrosine phosphorylation of CD3ζ chains and IL-2 production induced by anti-CD3 stimulation in T cell hybridomas (9Saitoh S.-I. Kosugi A. Noda S. Yamamoto N. Ogata M. Minami Y. Miyake K. Hamaoka T. J. Immunol. 1995; 155: 5574-5581PubMed Google Scholar), suggesting that a signal via TSA-1 regulates early and late events in TCR signaling. The findings observed in this in vitro study were further strengthened by the fact that in vivo injection of anti-TSA-1 mAb completely blocked anti-TCR/CD3-mediated apoptosis of thymocytes (10Noda S. Kosugi A. Saitoh S.-I. Narumiya S. Hamaoka T. J. Exp. Med. 1996; 183: 2355-2360Crossref PubMed Scopus (46) Google Scholar). Thus, TSA-1/Sca-2 seems to be an important cell surface molecule regulating T cell differentiation and activation by virtue of its ability for modulating TCR-mediated signal transduction. However, since TSA-1 is a glycosylphosphatidylinositol (GPI)-anchored membrane protein and thus does not have its transmembrane and cytoplasmic regions, it is not known how TSA-1 transmit signals into the cytoplasm of the cell. In the present study, we addressed the above question by analyzing the molecular interaction between TSA-1 and the TCR. The data clearly demonstrated that TSA-1 is physically and functionally associated with CD3ζ chains of the TCR complex, and strongly suggested that the regulatory role of TSA-1 on TCR signaling is based on this intermolecular association. DISCUSSIONMany rodent and human GPI-anchored proteins have been implicated in regulation of T cell activation, since mAbs against these GPI-anchored proteins induce T cell activation as monitored by interleukin-2 production and proliferation. T cell activation induced by a signal through GPI-anchored proteins is dependent upon expression of the TCR; anti-Thy-1 and anti-Ly-6 mAbs fail to stimulate a TCR− variant cell line, and the defect was able to be restored by TCR expression in these variant cell lines (22Gunter K.C. Germain R.N. Kroczek R.A. Saito T. Yokoyama W.M. Chan C. Weiss A. Shevach E.M. Nature. 1987; 326: 505-507Crossref PubMed Scopus (132) Google Scholar, 23Sussman J.J. Saito T. Shevach E.M. Germain R.N. Ashwell J.D. J. Immunol. 1988; 140: 2520-2526PubMed Google Scholar). In addition to the positive regulation by GPI-anchored proteins in T cell activation, some GPI-anchored proteins transduce a negative signal that inhibits anti-CD3-mediated TCR signaling (24Codias E.K. Rutter J.E. Fleming T.J. Malek T.R. J. Immunol. 1990; 145: 1407-1414PubMed Google Scholar). We have previously demonstrated that a signal via TSA-1/Sca-2 inhibits TCR/CD3-mediated activation and apoptosis both in vitro and in vivo (6Kosugi A. Saitoh S.-I. Narumiya S. Miyake K. Hamaoka T. Int. Immunol. 1994; 6: 1967-1976Crossref PubMed Scopus (32) Google Scholar, 9Saitoh S.-I. Kosugi A. Noda S. Yamamoto N. Ogata M. Minami Y. Miyake K. Hamaoka T. J. Immunol. 1995; 155: 5574-5581PubMed Google Scholar, 10Noda S. Kosugi A. Saitoh S.-I. Narumiya S. Hamaoka T. J. Exp. Med. 1996; 183: 2355-2360Crossref PubMed Scopus (46) Google Scholar). Thus, the TCR seems to be an essential molecule in signaling pathway of GPI-anchored proteins at least in T cells.A number of studies have indirectly suggested that there is a physical and/or functional association between some GPI-anchored proteins and the TCR. By using chemical cross-linkers, it was reported that CD45 is mutually associated with Thy-1 and the TCR, indicating that Thy-1 can physically interact with the TCR through CD45 (25Volarevic S. Burns C.M. Sussman J.J. Ashwell J.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7085-7089Crossref PubMed Scopus (119) Google Scholar). In another study, a T cell clone was stably transfected with antisense Ly-6A RNA (26Lee S.-K. Su B. Maher S.E. Bothwell A.L.M. EMBO J. 1994; 13: 2167-2176Crossref PubMed Scopus (53) Google Scholar). Cell surface expression of Ly-6A was markedly suppressed in this transfectant, but surprisingly surface expression of the TCR was greatly inhibited as well because of the reduction of TCR-β mRNA. The Ly-6A antisense transfectant was then transfected with TCR-β cDNA, and surface TCR expression was reconstituted without the expression of Ly-6A. However, TCR signaling was still impaired in this transfectant due to the absence of Ly-6A.Despite these observations, it seems to be very difficult to demonstrate a direct association of the TCR to Thy-1, Ly-6, or other GPI-anchored proteins in immunoprecipitation analysis. Nonetheless, we are able to provide evidence that TSA-1/Sca-2 is physically associated with TCR in the present study. When TSA-1/Sca-2 expressed on the cell surface was stimulated with anti-TSA-1 mAbs, CD3ζ in the TCR complex was induced to be phosphorylated in its tyrosine residues (Fig. 9). This result indicates that a functional association also exists between these two molecules, and argues against the possibility that the interaction between TSA-1 and CD3ζ occurs merely during the process of solubilization and immunoprecipitation.We do not know why we can successfully detect the physical association of the TCR to TSA-1 among many GPI-anchored proteins. Since none of mAbs against Thy-1, Ly-6A/E, and Ly-6C co-precipitated CD3ζ in an experiment in which both mAbs, PRST1 and GR12, against TSA-1 clearly co-precipitated CD3ζ (Fig. 2), the association between TSA-1 and CD3ζ is considered to be specific. Given that TSA-1 is a GPI-anchored protein and CD3ζ has a very short extracellular portion, the interaction between TSA-1 and CD3ζ could be mediated by an as yet undefined membrane protein, which could serve as a linker between these two proteins (27Brown D. Curr. Opin. Immunol. 1993; 5: 349-354Crossref PubMed Scopus (189) Google Scholar). This “linker” protein presumably functions not only in T cells but in COS-7 cells (Fig. 4). Moreover, the association between TSA-1 and the “linker” protein could not be dependent on the GPI anchor, but on primary sequence motifs of TSA-1. An effort should be made to identify the “linker” protein in biochemical analysis.Alternatively, another possibility may account for the mechanism underlying the physical association between TSA-1 and CD3ζ. GPI-anchored proteins are known to be localized to caveolae, glycosphingolipid-rich areas in the cell membrane (28Bamezai A. Goldmacher V. Reiser H. Rock K.L. J. Immunol. 1989; 143: 3107-3116PubMed Google Scholar, 29Anderson R.G.W. Kamen B.A. Rothberg K.G. Lacey S.W. Science. 1992; 255: 410-411Crossref PubMed Scopus (657) Google Scholar, 30Parton R.G. Simons K. Science. 1995; 269: 1398-1399Crossref PubMed Scopus (295) Google Scholar). Caveolae are also enriched in signal-transducing molecules, such as GTP-binding proteins, small G proteins, and nonreceptor-type tyrosine kinases (31Anderson R.G.W. Curr. Opin. Cell Biol. 1993; 5: 647-652Crossref PubMed Scopus (171) Google Scholar). It has been proposed that caveolae could represent a specialized signaling compartment at the cell surface (32Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8019) Google Scholar). Although lymphocytes do not have caveolae due to the lack of caveolin, there is the same membrane microdomain that are enriched in glycosphingolipids in lymphocytes (33Fra A.M. Williamson E. Simons K. Parton R.G. J. Biol. Chem. 1994; 269: 30745-30748Abstract Full Text PDF PubMed Google Scholar). Thus, if the TCR complex or the CD3ζ may reside in this microdomain, GPI-anchored proteins could be associated with CD3ζ by lipid-protein interactions, thereby forming a signaling compartment at the surface of T cells. Stimulation of GPI-anchored proteins with mAbs results in the delivery of a signal through this signaling compartment. If this possibility is correct, TSA-1 is not special among other GPI-anchored proteins, but our mAbs against TSA-1 could be special among other mAbs against GPI-anchored proteins. Although we can not thus far explain whether and how a transmembrane TSA-1 could be localized in this signaling compartment, the above hypothesis is very attractive, given that most of GPI-anchored proteins have a signal-transducing ability when cross-linked with mAbs.Although the mechanism is not fully understood, our previous finding that a signal through TSA-1 down-modulates the TCR signaling pathway could be explained by the physical association between TSA-1 and CD3ζ. Cross-linking of TSA-1 with the mAb induces the phosphorylation of tyrosine residues in CD3ζ chains (Fig. 9) through the activation of the Src family tyrosine kinases, which may subsequently cause recruitment of another protein tyrosine kinase, ZAP-70 (34Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1944) Google Scholar). Thus, intracellular signal-transducing molecules could be sequestered from TCR-signaling pathways to the TSA-1/CD3ζ complex, resulting in down-modulation of TCR signaling. Studies are in progress to elucidate a molecular mechanism for the TSA-1-signaling pathway. Many rodent and human GPI-anchored proteins have been implicated in regulation of T cell activation, since mAbs against these GPI-anchored proteins induce T cell activation as monitored by interleukin-2 production and proliferation. T cell activation induced by a signal through GPI-anchored proteins is dependent upon expression of the TCR; anti-Thy-1 and anti-Ly-6 mAbs fail to stimulate a TCR− variant cell line, and the defect was able to be restored by TCR expression in these variant cell lines (22Gunter K.C. Germain R.N. Kroczek R.A. Saito T. Yokoyama W.M. Chan C. Weiss A. Shevach E.M. Nature. 1987; 326: 505-507Crossref PubMed Scopus (132) Google Scholar, 23Sussman J.J. Saito T. Shevach E.M. Germain R.N. Ashwell J.D. J. Immunol. 1988; 140: 2520-2526PubMed Google Scholar). In addition to the positive regulation by GPI-anchored proteins in T cell activation, some GPI-anchored proteins transduce a negative signal that inhibits anti-CD3-mediated TCR signaling (24Codias E.K. Rutter J.E. Fleming T.J. Malek T.R. J. Immunol. 1990; 145: 1407-1414PubMed Google Scholar). We have previously demonstrated that a signal via TSA-1/Sca-2 inhibits TCR/CD3-mediated activation and apoptosis both in vitro and in vivo (6Kosugi A. Saitoh S.-I. Narumiya S. Miyake K. Hamaoka T. Int. Immunol. 1994; 6: 1967-1976Crossref PubMed Scopus (32) Google Scholar, 9Saitoh S.-I. Kosugi A. Noda S. Yamamoto N. Ogata M. Minami Y. Miyake K. Hamaoka T. J. Immunol. 1995; 155: 5574-5581PubMed Google Scholar, 10Noda S. Kosugi A. Saitoh S.-I. Narumiya S. Hamaoka T. J. Exp. Med. 1996; 183: 2355-2360Crossref PubMed Scopus (46) Google Scholar). Thus, the TCR seems to be an essential molecule in signaling pathway of GPI-anchored proteins at least in T cells. A number of studies have indirectly suggested that there is a physical and/or functional association between some GPI-anchored proteins and the TCR. By using chemical cross-linkers, it was reported that CD45 is mutually associated with Thy-1 and the TCR, indicating that Thy-1 can physically interact with the TCR through CD45 (25Volarevic S. Burns C.M. Sussman J.J. Ashwell J.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7085-7089Crossref PubMed Scopus (119) Google Scholar). In another study, a T cell clone was stably transfected with antisense Ly-6A RNA (26Lee S.-K. Su B. Maher S.E. Bothwell A.L.M. EMBO J. 1994; 13: 2167-2176Crossref PubMed Scopus (53) Google Scholar). Cell surface expression of Ly-6A was markedly suppressed in this transfectant, but surprisingly surface expression of the TCR was greatly inhibited as well because of the reduction of TCR-β mRNA. The Ly-6A antisense transfectant was then transfected with TCR-β cDNA, and surface TCR expression was reconstituted without the expression of Ly-6A. However, TCR signaling was still impaired in this transfectant due to the absence of Ly-6A. Despite these observations, it seems to be very difficult to demonstrate a direct association of the TCR to Thy-1, Ly-6, or other GPI-anchored proteins in immunoprecipitation analysis. Nonetheless, we are able to provide evidence that TSA-1/Sca-2 is physically associated with TCR in the present study. When TSA-1/Sca-2 expressed on the cell surface was stimulated with anti-TSA-1 mAbs, CD3ζ in the TCR complex was induced to be phosphorylated in its tyrosine residues (Fig. 9). This result indicates that a functional association also exists between these two molecules, and argues against the possibility that the interaction between TSA-1 and CD3ζ occurs merely during the process of solubilization and immunoprecipitation. We do not know why we can successfully detect the physical association of the TCR to TSA-1 among many GPI-anchored proteins. Since none of mAbs against Thy-1, Ly-6A/E, and Ly-6C co-precipitated CD3ζ in an experiment in which both mAbs, PRST1 and GR12, against TSA-1 clearly co-precipitated CD3ζ (Fig. 2), the association between TSA-1 and CD3ζ is considered to be specific. Given that TSA-1 is a GPI-anchored protein and CD3ζ has a very short extracellular portion, the interaction between TSA-1 and CD3ζ could be mediated by an as yet undefined membrane protein, which could serve as a linker between these two proteins (27Brown D. Curr. Opin. Immunol. 1993; 5: 349-354Crossref PubMed Scopus (189) Google Scholar). This “linker” protein presumably functions not only in T cells but in COS-7 cells (Fig. 4). Moreover, the association between TSA-1 and the “linker” protein could not be dependent on the GPI anchor, but on primary sequence motifs of TSA-1. An effort should be made to identify the “linker” protein in biochemical analysis. Alternatively, another possibility may account for the mechanism underlying the physical association between TSA-1 and CD3ζ. GPI-anchored proteins are known to be localized to caveolae, glycosphingolipid-rich areas in the cell membrane (28Bamezai A. Goldmacher V. Reiser H. Rock K.L. J. Immunol. 1989; 143: 3107-3116PubMed Google Scholar, 29Anderson R.G.W. Kamen B.A. Rothberg K.G. Lacey S.W. Science. 1992; 255: 410-411Crossref PubMed Scopus (657) Google Scholar, 30Parton R.G. Simons K. Science. 1995; 269: 1398-1399Crossref PubMed Scopus (295) Google Scholar). Caveolae are also enriched in signal-transducing molecules, such as GTP-binding proteins, small G proteins, and nonreceptor-type tyrosine kinases (31Anderson R.G.W. Curr. Opin. Cell Biol. 1993; 5: 647-652Crossref PubMed Scopus (171) Google Scholar). It has been proposed that caveolae could represent a specialized signaling compartment at the cell surface (32Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8019) Google Scholar). Although lymphocytes do not have caveolae due to the lack of caveolin, there is the same membrane microdomain that are enriched in glycosphingolipids in lymphocytes (33Fra A.M. Williamson E. Simons K. Parton R.G. J. Biol. Chem. 1994; 269: 30745-30748Abstract Full Text PDF PubMed Google Scholar). Thus, if the TCR complex or the CD3ζ may reside in this microdomain, GPI-anchored proteins could be associated with CD3ζ by lipid-protein interactions, thereby forming a signaling compartment at the surface of T cells. Stimulation of GPI-anchored proteins with mAbs results in the delivery of a signal through this signaling compartment. If this possibility is correct, TSA-1 is not special among other GPI-anchored proteins, but our mAbs against TSA-1 could be special among other mAbs against GPI-anchored proteins. Although we can not thus far explain whether and how a transmembrane TSA-1 could be localized in this signaling compartment, the above hypothesis is very attractive, given that most of GPI-anchored proteins have a signal-transducing ability when cross-linked with mAbs. Although the mechanism is not fully understood, our previous finding that a signal through TSA-1 down-modulates the TCR signaling pathway could be explained by the physical association between TSA-1 and CD3ζ. Cross-linking of TSA-1 with the mAb induces the phosphorylation of tyrosine residues in CD3ζ chains (Fig. 9) through the activation of the Src family tyrosine kinases, which may subsequently cause recruitment of another protein tyrosine kinase, ZAP-70 (34Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1944) Google Scholar). Thus, intracellular signal-transducing molecules could be sequestered from TCR-signaling pathways to the TSA-1/CD3ζ complex, resulting in down-modulation of TCR signaling. Studies are in progress to elucidate a molecular mechanism for the TSA-1-signaling pathway. We are grateful to Dr. Yasuhiro Minami for helpful discussions." @default.
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