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W2169165493 abstract "CD229 (Ly9) is a cell surface receptor selectively expressed on T and B lymphocytes, and it belongs to the CD150 receptor family. Like other receptors of this family, CD229 interacts with SAP/SH2D1a protein, mutation of which is responsible for the fatal X-linked lymphoproliferative disease. Receptors of the CD150 family function as costimulatory molecules, regulating cytokine production and cytotoxicity. Thus, their signaling and regulation in lymphocytes may be critical to an understanding of the pathogenesis of the X-linked lymphoproliferative disease. Here we show that CD229 interacts with the ॖ2 chain of the AP-2 adaptor complex that links transmembrane proteins to clathrin-coated pits. CD229 was the only member of the CD150 family associated with AP-2. We also show that the ॖ2 chain interacts with the Y470EKL motif of CD229. The integrity of this site was necessary for CD229 internalization, but it was not involved in SAP recruitment. Moreover, CD229 binds to the AP-2 complex in T and B cell lines, and it is internalized rapidly from the cell surface on T cells after antibody ligation. In contrast, cross-linking of CD229 receptors with intact antibody inhibited CD229 internalization on B cells. However, when F(ab′)2antibodies were used, CD229 internalization was similar on T and B cells, suggesting that Fcγ receptors control CD229 cell surface expression. Furthermore, CD229 was regulated by T cell receptor and B cell receptor signaling because coligation with antibodies against anti-CD3 and anti-IgM increased the rate of CD229 endocytosis. These data suggest that CD229 cell surface expression on lymphocytes surface is strongly and differentially regulated within the CD150 family members. CD229 (Ly9) is a cell surface receptor selectively expressed on T and B lymphocytes, and it belongs to the CD150 receptor family. Like other receptors of this family, CD229 interacts with SAP/SH2D1a protein, mutation of which is responsible for the fatal X-linked lymphoproliferative disease. Receptors of the CD150 family function as costimulatory molecules, regulating cytokine production and cytotoxicity. Thus, their signaling and regulation in lymphocytes may be critical to an understanding of the pathogenesis of the X-linked lymphoproliferative disease. Here we show that CD229 interacts with the ॖ2 chain of the AP-2 adaptor complex that links transmembrane proteins to clathrin-coated pits. CD229 was the only member of the CD150 family associated with AP-2. We also show that the ॖ2 chain interacts with the Y470EKL motif of CD229. The integrity of this site was necessary for CD229 internalization, but it was not involved in SAP recruitment. Moreover, CD229 binds to the AP-2 complex in T and B cell lines, and it is internalized rapidly from the cell surface on T cells after antibody ligation. In contrast, cross-linking of CD229 receptors with intact antibody inhibited CD229 internalization on B cells. However, when F(ab′)2antibodies were used, CD229 internalization was similar on T and B cells, suggesting that Fcγ receptors control CD229 cell surface expression. Furthermore, CD229 was regulated by T cell receptor and B cell receptor signaling because coligation with antibodies against anti-CD3 and anti-IgM increased the rate of CD229 endocytosis. These data suggest that CD229 cell surface expression on lymphocytes surface is strongly and differentially regulated within the CD150 family members. SLAM-associated protein R-phycoerythrin T cell receptor(s) B cell receptor(s) brefeldin A 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid enhanced green fluorescent protein green fluorescent protein monoclonal antibody mean fluorescence intensity wild-type CD229 (also termed Ly9) is a cell surface glycoprotein of relative mass 120 kDa found on T and B cells (1de la Fuente M.A. Tovar V. Villamor N. Zapater N. Pizcueta P. Campo E. Bosch J. Engel P. Blood. 2001; 97: 3513-3520Crossref PubMed Scopus (47) Google Scholar). It is a member of the immunoglobulin superfamily, and it belongs to the CD150 family of receptors (2Morra M. Howie D. Grande M.S. Sayos J. Wang N. Wu C. Engel P. Terhorst C. Annu. Rev. Immunol. 2001; 19: 657-682Crossref PubMed Scopus (173) Google Scholar, 3Sidorenko S.P. Clark E.A. Nat. Immunol. 2003; 4: 19-24Crossref PubMed Scopus (199) Google Scholar). Six members of the CD150 family (CD150, CD229, CD84, CD244, NTB-A, and CS1) have one or more cytoplasmic tyrosine motifs with the consensus sequence Thr-(Ile/Val)-Tyr(P)-X-X-(Ile/Val). This motif functions as a docking site for the SAP1/SH2D1a protein (4Sayos J. Wu C. Morra M. Wang N. Zhang X. Allen D. van Schaik S. Notarangelo L. Geha R. Roncarolo M.G. Oettgen H. de Vries J.E. Aversa G. Terhorst C. Nature. 1998; 395: 462-469Crossref PubMed Scopus (799) Google Scholar, 5Sayos J. Nguyen K.B. Wu C. Stepp S.E. Howie D. Schatzle J.D. Kumar V. Biron C.A. Terhorst C. Int. Immunol. 2000; 12: 1749-1757Crossref PubMed Scopus (92) Google Scholar, 6Tangye S.G. Lazetic S. Woollatt E. Sutherland G.R. Lanier L.L. Phillips J.H. J. Immunol. 1999; 162: 6981-6985PubMed Google Scholar, 7Sayos J. Martin M. Chen A. Simarro M. Howie D. Morra M. Engel P. Terhorst C. Blood. 2001; 97: 3867-3874Crossref PubMed Scopus (112) Google Scholar, 8Peck S.R. Ruley H.E. Immunogenetics. 2000; 52: 63-72Crossref PubMed Scopus (41) Google Scholar, 9Tovar V. Del Valle J. Zapater N. Martin M. Romero X. Pizcueta P. Bosch J. Terhorst C. Engel P. Immunogenetics. 2002; 54: 394-402Crossref PubMed Scopus (28) Google Scholar). Mutations on the SAP gene, sh2d1a, are the responsible for the fatal X-linked lymphoproliferative disease after Epstein-Barr virus infection, familial hemophagocytic lymphohistiocytosis, and some cases of combined variable immunodeficiency (2Morra M. Howie D. Grande M.S. Sayos J. Wang N. Wu C. Engel P. Terhorst C. Annu. Rev. Immunol. 2001; 19: 657-682Crossref PubMed Scopus (173) Google Scholar, 4Sayos J. Wu C. Morra M. Wang N. Zhang X. Allen D. van Schaik S. Notarangelo L. Geha R. Roncarolo M.G. Oettgen H. de Vries J.E. Aversa G. Terhorst C. Nature. 1998; 395: 462-469Crossref PubMed Scopus (799) Google Scholar, 10Coffey A.J. Brooksbank R.A. Brandau O. Oohashi T. Howell G.R. Bye J.M. Cahn A.P. Durham J. Heath P. Wray P. Pavitt R. Wilkinson J. Leversha M. Huckle E. Shaw-Smith C.J. Dunham A. Rhodes S. Schuster V. Porta G. Yin L. Serafini P. Sylla B. Zollo M. Franco B. Bolino A. Seri M. Lanyi A. Davis J.R. Webster D. Harris A. Lenoir G. de St. Basile G. Jones A. Behloradsky B.H. Achatz H. Murken J. Fassler R. Sumegi J. Romeo G. Vaudin M. Ross M.T. Meindl A. Bentley D.R. Nat. Genet. 1998; 20: 129-135Crossref PubMed Scopus (640) Google Scholar, 11Morra M. Silander O. Calpe S. Choi M. Oettgen H. Myers L. Etzioni A. Buckley R. Terhorst C. Blood. 2001; 98: 1321-1325Crossref PubMed Scopus (105) Google Scholar, 12Arico M. Imashuku S. Clementi R. Hibi S. Teramura T. Danesino C. Haber D.A. Nichols K.E. Blood. 2001; 97: 1131-1133Crossref PubMed Scopus (134) Google Scholar). These disease phenotypes point to distinct roles for the six CD150 family/SAP signaling pathways in the control of T, B, and natural killer cell activation and homeostasis. Studies on SAP knock-out mice reveal that T cells have an impaired ability to differentiate into T helper 2 cells, resulting in increased resistance to T helper 2-mediated disease such as infection withLeishmania major. In addition, SAP−/− mice fail to resolve the lymphocytic choriomeningitis virus, showing an increased number of interferon-γ-producing cells in the spleen and liver (13Wu C. Nguyen K.B. Pien G.C. Wang N. Gullo C. Howie D. Rodriguez-Sosa M. Edwards M.J. Borrow P. Satoskar A.R. Sharpe A.H. Biron C.A. Terhorst C. Nat. Immunol. 2001; 2: 410-414Crossref PubMed Scopus (9) Google Scholar) and a nearly complete absence of virus-specific long lived plasma cells and memory B cells (14Crotty S. Kersh E.N. Cannons J. Schwartzberg P.L. Ahmed R. Nature. 2003; 421: 282-287Crossref PubMed Scopus (342) Google Scholar). Altogether, these data indicate that SAP controls several distinct key T cell signal transduction pathways, which may be controlled differentially by the various CD150 receptors. It is crucial to elucidate the contribution of each member to these immune functions. Unlike other CD150 family members, CD229 has four rather than two extracellular Ig domains. Domains 1 and 3 are similar, as are domains 2 and 4, suggesting that CD229 arose from a progenitor with two domains (15Sandrin M.S. Gumley T.P. Henning M.M. Vaughan H.A. Gonez L.J. Trapani J.A. McKenzie I.F. J. Immunol. 1992; 149: 1636-1641PubMed Google Scholar). Although the function of CD229 is unknown, other members of the subfamily such as CD84, CD244, and CD150 are known to activate lymphocytes (16Martin M. Romero X. de la Fuente M.A. Tovar V. Zapater N. Esplugues E. Pizcueta P. Bosch J. Engel P. J. Immunol. 2001; 167: 3668-3676Crossref PubMed Scopus (104) Google Scholar, 17Valiante N.M. Trinchieri G. J. Exp. Med. 1993; 178: 1397-1406Crossref PubMed Scopus (192) Google Scholar, 18Nakajima H. Cella M. Langen H. Friedlein A. Colonna M. Eur. J. Immunol. 1999; 29: 1676-1683Crossref PubMed Scopus (202) Google Scholar, 19Aversa G. Carballido J. Punnonen J. Chang C.C. Hauser T. Cocks B.G. de Vries J.E. Immunol. Cell Biol. 1997; 75: 202-205Crossref PubMed Scopus (94) Google Scholar, 20Cocks B.G. Chang C.C. Carballido J.M. Yssel H. de Vries J.E. Aversa G. Nature. 1995; 376: 260-263Crossref PubMed Scopus (425) Google Scholar, 21Carballido J.M. Aversa G. Kaltoft K. Cocks B.G. Punnonen J. Yssel H. Thestrup-Pedersen K. de Vries J.E. J. Immunol. 1997; 159: 4316-4321PubMed Google Scholar, 22Castro A.G. Hauser T.M. Cocks B.G. Abrams J. Zurawski S. Churakova T. Zonin F. Robinson D. Tangye S.G. Aversa G. Nichols K.E. de Vries J.E. Lanier L.L. O'Garra A. J. Immunol. 1999; 163: 5860-5870PubMed Google Scholar), suggesting that the CD229 molecule is also involved in leukocyte activation. Recently, NTB-A and CS1 have also been shown to regulate cytotoxicity in natural killer cells (23Bottino C. Falco M. Parolini S. Marcenaro E. Augugliaro R. Sivori S. Landi E. Biassoni R. Notarangelo L.D. Moretta L. Moretta A. J. Exp. Med. 2001; 194: 235-246Crossref PubMed Scopus (263) Google Scholar, 24Kumaresan P.R. Lai W.C. Chuang S.S. Bennett M. Mathew P.A. Mol. Immunol. 2002; 39: 1-8Crossref PubMed Scopus (104) Google Scholar).In this study, we have investigated the biochemical mechanism involved in CD229 signaling and intracellular trafficking by identifying proteins that interact with the CD229 cytoplasmic domain. Two clones encoding human AP-2 ॖ2 chain were isolated using CD229 as bait in a three-hybrid screen of a human B cell library. ॖ2, also termed AP50, is the 50-kDa subunit of the AP-2 complex. AP-2 is a key component of the endocytic machinery that links cargo membrane proteins to the clathrin lattice. It is a heterotetramer consisting of two large (100–115 kDa) α and औ subunits or adaptins, one medium ॖ2 (50 kDa), and one small (17 kDa) ς2 subunit. The α-adaptin appears to contain the major membrane binding interface. The hinge domain of the औ subunit binds to the clathrin heavy chain, providing a mechanism for the formation of polyhedral lattices. In addition to its structural role in coat assembly, AP-2 is also involved in the transport of proteins from the plasma membrane to the endosomal and lysosomal compartments. For effective cargo into coated pits, receptors should contain specific internalization signals, namely tyrosine-based motifs such as NPXY or YXXφ (where X stands for any amino acid and φ for a bulky hydrophobic residue), dileucine motifs, and acidic clusters (25Kirchhausen T. Annu. Rev. Cell Dev. Biol. 1999; 15: 705-732Crossref PubMed Scopus (418) Google Scholar, 26Kirchhausen T. Cell. 2002; 109: 413-416Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The structural analysis of the AP-2 core reveals that the binding site for YXXφ endocytic motifs is buried, indicating that a conformational change, probably triggered by phosphorylation in the disordered ॖ2 linker, is necessary to allow YXXφ motif binding (27Collins B.M. McCoy A.J. Kent H.M. Evans P.R. Owen D.J. Cell. 2002; 109: 523-535Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar). Here, we demonstrate that the only member of the CD150 family which specifically binds ॖ2 is CD229. We mapped the CD229-ॖ2interaction in Tyr470 of the CD229 cytoplasmic domain. Moreover, we show that TCR and BCR signaling regulates CD229 expression.DISCUSSIONIn this study, we have shown that CD229 binds to the ॖ2 chain of the AP-2 complex. It is well established that the ॖ2 subunit binds to the consensus sorting signal NPXY or YPPφ (25Kirchhausen T. Annu. Rev. Cell Dev. Biol. 1999; 15: 705-732Crossref PubMed Scopus (418) Google Scholar). Although it has been suggested that ॖ2 interacts with dileucine motifs (34Bremnes T. Lauvrak V. Lindqvist B. Bakke O. J. Biol. Chem. 1998; 273: 8638-8645Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), peptides containing dileucine motifs bind only to the औ subunit of AP1 and AP-2 complexes and not to the other subunits (35Marks M.S. Woodruff L. Ohno H. Bonifacino J.S. J. Cell Biol. 1996; 135: 341-354Crossref PubMed Scopus (274) Google Scholar, 36Rapoport I. Chen Y.C. Cupers P. Shoelson S.E. Kirchhausen T. EMBO J. 1998; 17: 2148-2155Crossref PubMed Scopus (257) Google Scholar). Analysis of CD229 revealed only one sequence that follows the YPPφ consensus (Y470EKL), which is conserved in mouse (37Tovar V. de la Fuente M.A. Pizcueta P. Bosch J. Engel P. Immunogenetics. 2000; 51: 788-793Crossref PubMed Scopus (19) Google Scholar). Using site-directed mutagenesis, we mapped the ॖ2interaction to CD229 in this motif. The single replacement of tyrosine by phenylalanine abrogates ॖ2 binding to CD229 in yeast. It has been proposed that the hydroxyl group of tyrosine contributes to a network of interactions with ॖ2 residues that form the hydrophobic pocket, explaining why phenylalanine does not efficiently replace tyrosine in YPPφ signals (38Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (700) Google Scholar, 39Sauter M.M. Pelchen-Matthews A. Bron R. Marsh M. LaBranche C.C. Vance P.J. Romano J. Haggarty B.S. Hart T.K. Lee W.M. Hoxie J.A. J. Cell Biol. 1996; 132: 795-811Crossref PubMed Scopus (127) Google Scholar). As expected, the recruitment of SAP in the mutant is not altered because both molecules do not compete for the same motif (7Sayos J. Martin M. Chen A. Simarro M. Howie D. Morra M. Engel P. Terhorst C. Blood. 2001; 97: 3867-3874Crossref PubMed Scopus (112) Google Scholar). CD229 binds to ॖ2 when Fyn is active, or kinase activity of Fyn is completely abrogated by depletion of its catalytic region in the yeast system. This suggests various possibilities: CD229-ॖ2interaction occurs when Tyr470 is phosphorylated, or phosphorylation of the CD229 tail is partial, allowing the screening of interactions dependent and independent of phosphorylation at the same time. Our results point to this last possibility because the CD229-ॖ2 interaction only occurred when the receptor was not phosphorylated. This agrees with literature data, which show that this type of interactions does not depend on phosphorylation. The crystal structure of ॖ2 indicates that phosphotyrosine does not fit into its hydrophobic pocket (40Owen D.J. Evans P.R. Science. 1998; 282: 1327-1332Crossref PubMed Scopus (450) Google Scholar).We have also demonstrated that the cytoplasmic tail of CD229 but not that of any of the other members of CD150 family can interact with the AP-2 complex via its ॖ2 chain. This intriguing difference suggests that the expression and internalization of CD229 are tightly and differentially regulated. The structure of CD229 differs from that of the rest of the CD150 family of receptors. It is the only member that has four extracellular Ig-like domains, and it has the longest cytoplasmic tail, comprising 180 amino acids, 16 threonines, 17 serines, and 8 tyrosines. Two unique tyrosine-based motifs (with the consensus amino acid sequence TV/I YXXV/I) are critical for the binding to the SAP/SH2D1a protein and the Eat-2 SH2 domain protein homolog to SAP in B cells (41Morra M. Lu J. Poy F. Martin M. Sayos J. Calpe S. Gullo C. Howie D. Rietdijk S. Thompson A. Coyle A.J. Denny C. Yaffe M.B. Engel P. Eck M.J. Terhorst C. EMBO J. 2001; 20: 5840-5852Crossref PubMed Scopus (122) Google Scholar).Furthermore, CD229 can bind to endogenous AP-2 in COS cells, ॖ2-EGFP chimera can efficiently integrate in the endogenous AP-2 complex, and CD229 can coprecipitate with ॖ2-EGFP and the AP-2 endogenous complex. The complex is functional because CD229 internalization and CD229·ॖ2-EGFP colocalization were detected in COS cells after antibody-induced endocytosis. In agreement with our mapping data, CD229Y470F and CD229Y470A endocytosis was severely impaired. Although in our model a phenylalanine substitution abrogates the binding to ॖ2, the FXXφ motif can fit into the hydrophobic pocket of ॖ2 (42Marsh B.J. Alm R.A. McIntosh S.R. James D.E. J. Cell Biol. 1995; 130: 1081-1091Crossref PubMed Scopus (76) Google Scholar) but leads to a weaker interaction and slower endocytosis. Thus, Tyr470 is essential for CD229 endocytosis and may inhibit the internalization of this receptor after its phosphorylation in vivo. CD229 can be phosphorylated by Src-kinase Fyn in COS cells, and its possible interaction with a SH2 domain protein may further regulate the signaling and trafficking of the molecule, as proposed for CTLA-4 (43Shiratori T. Miyatake S. Ohno H. Nakaseko C. Isono K. Bonifacino J.S. Saito T. Immunity. 1997; 6: 583-589Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar).Because CD229 is expressed in T and B lymphocytes, we have defined the mechanism of CD229 internalization and demonstrated that it can be regulated differentially in T and B cell lines. CD229 was endocytosed rapidly in response to antibody ligation in Jurkat T cells, whereas no internalization was observed in B cells. The same results were obtained in lymphocytes from peripheral blood. CD229 in B cells was inhibited by the Fcγ receptor coligation because F(ab′)2 antibodies induce CD229 endocytosis in B cells. The CD229 uptake rate was similar using the intact and F(ab′)2 antibodies in Jurkat cells lacking Fcγ receptors. In agreement with our results, the coligation of Fcγ receptors and BCR has been suggested to inhibit endocytosis by blocking the processing and presentation of the BCR-bound antigen (44Minskoff S.A. Matter K. Mellman I. J. Immunol. 1998; 161: 2079-2083PubMed Google Scholar). Using BFA and cycloheximide, we have determined that CD229, unlike transferrin receptor (CD71), exhibits a low constitutive rate of turnover, and therefore, the rapid internalization of CD229 represents ligand-induced endocytosis. After endocytosis, CD229 was segregated into two distinguishable pools, one that is shuttled to a nonrecycling degradation pathway and another that is recycled to the cell surface. The results suggest that ∼75–807 of CD229 in T cells become degraded via lysosomal compartments, and the remaining 20–257 of complexes are recycled to the cell surface. In contrast, most CD229 is trafficked to the lysosomal degradation pathway in B cells.Moreover, TCR and anti-IgM coligation enhances CD229 endocytosis, suggesting that the antigen receptor signaling regulates the availability of CD229 at the plasma membrane. Thus, endocytosis may represent a critical mechanism that modulates CD229 cell surface expression rapidly. After TCR activation, phosphatidylinositol 3-kinase may become activated and catalyze the conversion of phosphatidylinositol 4,5-biphosphate to phosphatidylinositol 3,4,5-trisphosphate. The pleckstrin homology domain in the α2 subunit of the AP-2 adaptor complex binds with high affinity to phosphatidylinositol 3,4,5-trisphosphate. This binding mediates two effects: localization of AP-2 in the membrane and increase in the affinity of ॖ2 binding to endocytosis sorting signals (45Rapoport I. Miyazaki M. Boll W. Duckworth B. Cantley L.C. Shoelson S. Kirchhausen T. EMBO J. 1997; 16: 2240-2250Crossref PubMed Scopus (182) Google Scholar, 46Jost M. Simpson F. Kavran J.M. Lemmon M.A. Schmid S.L. Curr. Biol. 1998; 8: 1399-1402Abstract Full Text Full Text PDF PubMed Google Scholar).Because CD229 endocytosis is impaired when Fcγ receptors are triggered and enhanced when the BCR is activated, this molecule may have a differential role in these different events. The ability to regulate CD229 internalization in T and B cells differentially may allow for different net effects of CD229 signaling which may be cell-specific. Recent studies indicate that CD229, unlike CD150 and CD84, inhibits interferon-γ secretion after TCR activation in T cells. 2J. M. Del Valle, P. Engel, and M. Martı́n, unpublished result. TCR and BCR may increase the rate of CD229 endocytosis to prevent CD229 from delivering negative signals to the cell and Fcγ receptor ligation retains the CD229 on the cell surface, thus favoring signaling through this receptor and the maintenance of an inhibitory response. The development of CD229 knock-out mice may be a key step to define the physiological role of CD229. CD229 (also termed Ly9) is a cell surface glycoprotein of relative mass 120 kDa found on T and B cells (1de la Fuente M.A. Tovar V. Villamor N. Zapater N. Pizcueta P. Campo E. Bosch J. Engel P. Blood. 2001; 97: 3513-3520Crossref PubMed Scopus (47) Google Scholar). It is a member of the immunoglobulin superfamily, and it belongs to the CD150 family of receptors (2Morra M. Howie D. Grande M.S. Sayos J. Wang N. Wu C. Engel P. Terhorst C. Annu. Rev. Immunol. 2001; 19: 657-682Crossref PubMed Scopus (173) Google Scholar, 3Sidorenko S.P. Clark E.A. Nat. Immunol. 2003; 4: 19-24Crossref PubMed Scopus (199) Google Scholar). Six members of the CD150 family (CD150, CD229, CD84, CD244, NTB-A, and CS1) have one or more cytoplasmic tyrosine motifs with the consensus sequence Thr-(Ile/Val)-Tyr(P)-X-X-(Ile/Val). This motif functions as a docking site for the SAP1/SH2D1a protein (4Sayos J. Wu C. Morra M. Wang N. Zhang X. Allen D. van Schaik S. Notarangelo L. Geha R. Roncarolo M.G. Oettgen H. de Vries J.E. Aversa G. Terhorst C. Nature. 1998; 395: 462-469Crossref PubMed Scopus (799) Google Scholar, 5Sayos J. Nguyen K.B. Wu C. Stepp S.E. Howie D. Schatzle J.D. Kumar V. Biron C.A. Terhorst C. Int. Immunol. 2000; 12: 1749-1757Crossref PubMed Scopus (92) Google Scholar, 6Tangye S.G. Lazetic S. Woollatt E. Sutherland G.R. Lanier L.L. Phillips J.H. J. Immunol. 1999; 162: 6981-6985PubMed Google Scholar, 7Sayos J. Martin M. Chen A. Simarro M. Howie D. Morra M. Engel P. Terhorst C. Blood. 2001; 97: 3867-3874Crossref PubMed Scopus (112) Google Scholar, 8Peck S.R. Ruley H.E. Immunogenetics. 2000; 52: 63-72Crossref PubMed Scopus (41) Google Scholar, 9Tovar V. Del Valle J. Zapater N. Martin M. Romero X. Pizcueta P. Bosch J. Terhorst C. Engel P. Immunogenetics. 2002; 54: 394-402Crossref PubMed Scopus (28) Google Scholar). Mutations on the SAP gene, sh2d1a, are the responsible for the fatal X-linked lymphoproliferative disease after Epstein-Barr virus infection, familial hemophagocytic lymphohistiocytosis, and some cases of combined variable immunodeficiency (2Morra M. Howie D. Grande M.S. Sayos J. Wang N. Wu C. Engel P. Terhorst C. Annu. Rev. Immunol. 2001; 19: 657-682Crossref PubMed Scopus (173) Google Scholar, 4Sayos J. Wu C. Morra M. Wang N. Zhang X. Allen D. van Schaik S. Notarangelo L. Geha R. Roncarolo M.G. Oettgen H. de Vries J.E. Aversa G. Terhorst C. Nature. 1998; 395: 462-469Crossref PubMed Scopus (799) Google Scholar, 10Coffey A.J. Brooksbank R.A. Brandau O. Oohashi T. Howell G.R. Bye J.M. Cahn A.P. Durham J. Heath P. Wray P. Pavitt R. Wilkinson J. Leversha M. Huckle E. Shaw-Smith C.J. Dunham A. Rhodes S. Schuster V. Porta G. Yin L. Serafini P. Sylla B. Zollo M. Franco B. Bolino A. Seri M. Lanyi A. Davis J.R. Webster D. Harris A. Lenoir G. de St. Basile G. Jones A. Behloradsky B.H. Achatz H. Murken J. Fassler R. Sumegi J. Romeo G. Vaudin M. Ross M.T. Meindl A. Bentley D.R. Nat. Genet. 1998; 20: 129-135Crossref PubMed Scopus (640) Google Scholar, 11Morra M. Silander O. Calpe S. Choi M. Oettgen H. Myers L. Etzioni A. Buckley R. Terhorst C. Blood. 2001; 98: 1321-1325Crossref PubMed Scopus (105) Google Scholar, 12Arico M. Imashuku S. Clementi R. Hibi S. Teramura T. Danesino C. Haber D.A. Nichols K.E. Blood. 2001; 97: 1131-1133Crossref PubMed Scopus (134) Google Scholar). These disease phenotypes point to distinct roles for the six CD150 family/SAP signaling pathways in the control of T, B, and natural killer cell activation and homeostasis. Studies on SAP knock-out mice reveal that T cells have an impaired ability to differentiate into T helper 2 cells, resulting in increased resistance to T helper 2-mediated disease such as infection withLeishmania major. In addition, SAP−/− mice fail to resolve the lymphocytic choriomeningitis virus, showing an increased number of interferon-γ-producing cells in the spleen and liver (13Wu C. Nguyen K.B. Pien G.C. Wang N. Gullo C. Howie D. Rodriguez-Sosa M. Edwards M.J. Borrow P. Satoskar A.R. Sharpe A.H. Biron C.A. Terhorst C. Nat. Immunol. 2001; 2: 410-414Crossref PubMed Scopus (9) Google Scholar) and a nearly complete absence of virus-specific long lived plasma cells and memory B cells (14Crotty S. Kersh E.N. Cannons J. Schwartzberg P.L. Ahmed R. Nature. 2003; 421: 282-287Crossref PubMed Scopus (342) Google Scholar). Altogether, these data indicate that SAP controls several distinct key T cell signal transduction pathways, which may be controlled differentially by the various CD150 receptors. It is crucial to elucidate the contribution of each member to these immune functions. Unlike other CD150 family members, CD229 has four rather than two extracellular Ig domains. Domains 1 and 3 are similar, as are domains 2 and 4, suggesting that CD229 arose from a progenitor with two domains (15Sandrin M.S. Gumley T.P. Henning M.M. Vaughan H.A. Gonez L.J. Trapani J.A. McKenzie I.F. J. Immunol. 1992; 149: 1636-1641PubMed Google Scholar). Although the function of CD229 is unknown, other members of the subfamily such as CD84, CD244, and CD150 are known to activate lymphocytes (16Martin M. Romero X. de la Fuente M.A. Tovar V. Zapater N. Esplugues E. Pizcueta P. Bosch J. Engel P. J. Immunol. 2001; 167: 3668-3676Crossref PubMed Scopus (104) Google Scholar, 17Valiante N.M. Trinchieri G. J. Exp. Med. 1993; 178: 1397-1406Crossref PubMed Scopus (192) Google Scholar, 18Nakajima H. Cella M. Langen H. Friedlein A. Colonna M. Eur. J. Immunol. 1999; 29: 1676-1683Crossref PubMed Scopus (202) Google Scholar, 19Aversa G. Carballido J. Punnonen J. Chang C.C. Hauser T. Cocks B.G. de Vries J.E. Immunol. Cell Biol. 1997; 75: 202-205Crossref PubMed Scopus (94) Google Scholar, 20Cocks B.G. Chang C.C. Carballido J.M. Yssel H. de Vries J.E. Aversa G. Nature. 1995; 376: 260-263Crossref PubMed Scopus (425) Google Scholar, 21Carballido J.M. Aversa G. Kaltoft K. Cocks B.G. Punnonen J. Yssel H. Thestrup-Pedersen K. de Vries J.E. J. Immunol. 1997; 159: 4316-4321PubMed Google Scholar, 22Castro A.G. Hauser T.M. Cocks B.G. Abrams J. Zurawski S. Churakova T. Zonin F. Robinson D. Tangye S.G. Aversa G. Nichols K.E. de Vries J.E. Lanier L.L. O'Garra A. J. Immunol. 1999; 163: 5860-5870PubMed Google Scholar), suggesting that the CD229 molecule is also involved in leukocyte activation. Recently, NTB-A and CS1 have also been shown to regulate cytotoxicity in natural killer cells (23Bottino C. Falco M. Parolini S. Marcenaro E. Augugliaro R. Sivori S. Landi E. Biassoni R. Notarangelo L.D. Moretta L. Moretta A. J. Exp. Med. 2001; 194: 235-246Crossref PubMed Scopus (263) Google Scholar, 24Kumaresan P.R. Lai W.C. Chuang S.S. Bennett M. Mathew P.A. Mol. Immunol. 2002; 39: 1-8Crossref PubMed Scopus (104) Google Scholar). In this study, we have investigated the biochemical mechanism involved in CD229 signaling and intracellular trafficking by identifying proteins that interact with the CD229 cytoplasmic domain. Two clones encoding human AP-2 ॖ2 chain were isolated using CD229 as bait in a three-hybrid screen of a human B cell library. ॖ2, also termed AP50, is the 50-kDa subunit of the AP-2 complex. AP-2 is a key component of the endocytic machinery that links cargo membrane proteins to the clathrin lattice. It is a heterotetramer consisting of two large (100–115 kDa) α and औ subunits or adaptins, one medium ॖ2 (50 kDa), and one small (17 kDa) ς2 subunit. The α-adaptin appears to contain the major membrane binding interface. The hinge domain of the औ subunit binds to the clathrin heavy chain, providing a mechanism for the formation of polyhedral lattices. In addition to its structural role in coat assembly, AP-2 is also involved in the transport of proteins from the plasma membrane to the endosomal and lysosomal compartments. For effective cargo into coated pits, receptors should contain specific internalization signals, namely tyrosine-based motifs such as NPXY or YXXφ (where X stands for any amino acid and φ for a bulky hydrophobic residue), dileucine motifs, and acidic clusters (25Kirchhausen T. Annu. Rev. Cell Dev. Biol. 1999; 15: 705-732Crossref PubMed Scopus (418) Google Scholar, 26Kirchhausen T. Cell. 2002; 109: 413-416Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The structural analysis of the AP-2 core reveals that the binding site for YXXφ endocytic motifs is buried, indicating that a conformational change, probably triggered by phosphorylation in the disordered ॖ2 linker, is necessary to allow YXXφ motif binding (27Collins B.M. McCoy A.J. Kent H.M. Evans P.R. Owen D.J. Cell. 2002; 109: 523-535Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar). Here, we demonstrate that the only member of the CD150 family which specifically binds ॖ2 is CD229. We mapped the CD229-ॖ2interaction in Tyr470 of the CD229 cytoplasmic domain. Moreover, we show that TCR and BCR signaling regulates CD229 expression. DISCUSSIONIn this study, we have shown that CD229 binds to the ॖ2 chain of the AP-2 complex. It is well established that the ॖ2 subunit binds to the consensus sorting signal NPXY or YPPφ (25Kirchhausen T. Annu. Rev. Cell Dev. Biol. 1999; 15: 705-732Crossref PubMed Scopus (418) Google Scholar). Although it has been suggested that ॖ2 interacts with dileucine motifs (34Bremnes T. Lauvrak V. Lindqvist B. Bakke O. J. Biol. Chem. 1998; 273: 8638-8645Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), peptides containing dileucine motifs bind only to the औ subunit of AP1 and AP-2 complexes and not to the other subunits (35Marks M.S. Woodruff L. Ohno H. Bonifacino J.S. J. Cell Biol. 1996; 135: 341-354Crossref PubMed Scopus (274) Google Scholar, 36Rapoport I. Chen Y.C. Cupers P. Shoelson S.E. Kirchhausen T. EMBO J. 1998; 17: 2148-2155Crossref PubMed Scopus (257) Google Scholar). Analysis of CD229 revealed only one sequence that follows the YPPφ consensus (Y470EKL), which is conserved in mouse (37Tovar V. de la Fuente M.A. Pizcueta P. Bosch J. Engel P. Immunogenetics. 2000; 51: 788-793Crossref PubMed Scopus (19) Google Scholar). Using site-directed mutagenesis, we mapped the ॖ2interaction to CD229 in this motif. The single replacement of tyrosine by phenylalanine abrogates ॖ2 binding to CD229 in yeast. It has been proposed that the hydroxyl group of tyrosine contributes to a network of interactions with ॖ2 residues that form the hydrophobic pocket, explaining why phenylalanine does not efficiently replace tyrosine in YPPφ signals (38Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (700) Google Scholar, 39Sauter M.M. Pelchen-Matthews A. Bron R. Marsh M. LaBranche C.C. Vance P.J. Romano J. Haggarty B.S. Hart T.K. Lee W.M. Hoxie J.A. J. Cell Biol. 1996; 132: 795-811Crossref PubMed Scopus (127) Google Scholar). As expected, the recruitment of SAP in the mutant is not altered because both molecules do not compete for the same motif (7Sayos J. Martin M. Chen A. Simarro M. Howie D. Morra M. Engel P. Terhorst C. Blood. 2001; 97: 3867-3874Crossref PubMed Scopus (112) Google Scholar). CD229 binds to ॖ2 when Fyn is active, or kinase activity of Fyn is completely abrogated by depletion of its catalytic region in the yeast system. This suggests various possibilities: CD229-ॖ2interaction occurs when Tyr470 is phosphorylated, or phosphorylation of the CD229 tail is partial, allowing the screening of interactions dependent and independent of phosphorylation at the same time. Our results point to this last possibility because the CD229-ॖ2 interaction only occurred when the receptor was not phosphorylated. This agrees with literature data, which show that this type of interactions does not depend on phosphorylation. The crystal structure of ॖ2 indicates that phosphotyrosine does not fit into its hydrophobic pocket (40Owen D.J. Evans P.R. Science. 1998; 282: 1327-1332Crossref PubMed Scopus (450) Google Scholar).We have also demonstrated that the cytoplasmic tail of CD229 but not that of any of the other members of CD150 family can interact with the AP-2 complex via its ॖ2 chain. This intriguing difference suggests that the expression and internalization of CD229 are tightly and differentially regulated. The structure of CD229 differs from that of the rest of the CD150 family of receptors. It is the only member that has four extracellular Ig-like domains, and it has the longest cytoplasmic tail, comprising 180 amino acids, 16 threonines, 17 serines, and 8 tyrosines. Two unique tyrosine-based motifs (with the consensus amino acid sequence TV/I YXXV/I) are critical for the binding to the SAP/SH2D1a protein and the Eat-2 SH2 domain protein homolog to SAP in B cells (41Morra M. Lu J. Poy F. Martin M. Sayos J. Calpe S. Gullo C. Howie D. Rietdijk S. Thompson A. Coyle A.J. Denny C. Yaffe M.B. Engel P. Eck M.J. Terhorst C. EMBO J. 2001; 20: 5840-5852Crossref PubMed Scopus (122) Google Scholar).Furthermore, CD229 can bind to endogenous AP-2 in COS cells, ॖ2-EGFP chimera can efficiently integrate in the endogenous AP-2 complex, and CD229 can coprecipitate with ॖ2-EGFP and the AP-2 endogenous complex. The complex is functional because CD229 internalization and CD229·ॖ2-EGFP colocalization were detected in COS cells after antibody-induced endocytosis. In agreement with our mapping data, CD229Y470F and CD229Y470A endocytosis was severely impaired. Although in our model a phenylalanine substitution abrogates the binding to ॖ2, the FXXφ motif can fit into the hydrophobic pocket of ॖ2 (42Marsh B.J. Alm R.A. McIntosh S.R. James D.E. J. Cell Biol. 1995; 130: 1081-1091Crossref PubMed Scopus (76) Google Scholar) but leads to a weaker interaction and slower endocytosis. Thus, Tyr470 is essential for CD229 endocytosis and may inhibit the internalization of this receptor after its phosphorylation in vivo. CD229 can be phosphorylated by Src-kinase Fyn in COS cells, and its possible interaction with a SH2 domain protein may further regulate the signaling and trafficking of the molecule, as proposed for CTLA-4 (43Shiratori T. Miyatake S. Ohno H. Nakaseko C. Isono K. Bonifacino J.S. Saito T. Immunity. 1997; 6: 583-589Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar).Because CD229 is expressed in T and B lymphocytes, we have defined the mechanism of CD229 internalization and demonstrated that it can be regulated differentially in T and B cell lines. CD229 was endocytosed rapidly in response to antibody ligation in Jurkat T cells, whereas no internalization was observed in B cells. The same results were obtained in lymphocytes from peripheral blood. CD229 in B cells was inhibited by the Fcγ receptor coligation because F(ab′)2 antibodies induce CD229 endocytosis in B cells. The CD229 uptake rate was similar using the intact and F(ab′)2 antibodies in Jurkat cells lacking Fcγ receptors. In agreement with our results, the coligation of Fcγ receptors and BCR has been suggested to inhibit endocytosis by blocking the processing and presentation of the BCR-bound antigen (44Minskoff S.A. Matter K. Mellman I. J. Immunol. 1998; 161: 2079-2083PubMed Google Scholar). Using BFA and cycloheximide, we have determined that CD229, unlike transferrin receptor (CD71), exhibits a low constitutive rate of turnover, and therefore, the rapid internalization of CD229 represents ligand-induced endocytosis. After endocytosis, CD229 was segregated into two distinguishable pools, one that is shuttled to a nonrecycling degradation pathway and another that is recycled to the cell surface. The results suggest that ∼75–807 of CD229 in T cells become degraded via lysosomal compartments, and the remaining 20–257 of complexes are recycled to the cell surface. In contrast, most CD229 is trafficked to the lysosomal degradation pathway in B cells.Moreover, TCR and anti-IgM coligation enhances CD229 endocytosis, suggesting that the antigen receptor signaling regulates the availability of CD229 at the plasma membrane. Thus, endocytosis may represent a critical mechanism that modulates CD229 cell surface expression rapidly. After TCR activation, phosphatidylinositol 3-kinase may become activated and catalyze the conversion of phosphatidylinositol 4,5-biphosphate to phosphatidylinositol 3,4,5-trisphosphate. The pleckstrin homology domain in the α2 subunit of the AP-2 adaptor complex binds with high affinity to phosphatidylinositol 3,4,5-trisphosphate. This binding mediates two effects: localization of AP-2 in the membrane and increase in the affinity of ॖ2 binding to endocytosis sorting signals (45Rapoport I. Miyazaki M. Boll W. Duckworth B. Cantley L.C. Shoelson S. Kirchhausen T. EMBO J. 1997; 16: 2240-2250Crossref PubMed Scopus (182) Google Scholar, 46Jost M. Simpson F. Kavran J.M. Lemmon M.A. Schmid S.L. Curr. Biol. 1998; 8: 1399-1402Abstract Full Text Full Text PDF PubMed Google Scholar).Because CD229 endocytosis is impaired when Fcγ receptors are triggered and enhanced when the BCR is activated, this molecule may have a differential role in these different events. The ability to regulate CD229 internalization in T and B cells differentially may allow for different net effects of CD229 signaling which may be cell-specific. Recent studies indicate that CD229, unlike CD150 and CD84, inhibits interferon-γ secretion after TCR activation in T cells. 2J. M. Del Valle, P. Engel, and M. Martı́n, unpublished result. TCR and BCR may increase the rate of CD229 endocytosis to prevent CD229 from delivering negative signals to the cell and Fcγ receptor ligation retains the CD229 on the cell surface, thus favoring signaling through this receptor and the maintenance of an inhibitory response. The development of CD229 knock-out mice may be a key step to define the physiological role of CD229. In this study, we have shown that CD229 binds to the ॖ2 chain of the AP-2 complex. It is well established that the ॖ2 subunit binds to the consensus sorting signal NPXY or YPPφ (25Kirchhausen T. Annu. Rev. Cell Dev. Biol. 1999; 15: 705-732Crossref PubMed Scopus (418) Google Scholar). Although it has been suggested that ॖ2 interacts with dileucine motifs (34Bremnes T. Lauvrak V. Lindqvist B. Bakke O. J. Biol. Chem. 1998; 273: 8638-8645Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), peptides containing dileucine motifs bind only to the औ subunit of AP1 and AP-2 complexes and not to the other subunits (35Marks M.S. Woodruff L. Ohno H. Bonifacino J.S. J. Cell Biol. 1996; 135: 341-354Crossref PubMed Scopus (274) Google Scholar, 36Rapoport I. Chen Y.C. Cupers P. Shoelson S.E. Kirchhausen T. EMBO J. 1998; 17: 2148-2155Crossref PubMed Scopus (257) Google Scholar). Analysis of CD229 revealed only one sequence that follows the YPPφ consensus (Y470EKL), which is conserved in mouse (37Tovar V. de la Fuente M.A. Pizcueta P. Bosch J. Engel P. Immunogenetics. 2000; 51: 788-793Crossref PubMed Scopus (19) Google Scholar). Using site-directed mutagenesis, we mapped the ॖ2interaction to CD229 in this motif. The single replacement of tyrosine by phenylalanine abrogates ॖ2 binding to CD229 in yeast. It has been proposed that the hydroxyl group of tyrosine contributes to a network of interactions with ॖ2 residues that form the hydrophobic pocket, explaining why phenylalanine does not efficiently replace tyrosine in YPPφ signals (38Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (700) Google Scholar, 39Sauter M.M. Pelchen-Matthews A. Bron R. Marsh M. LaBranche C.C. Vance P.J. Romano J. Haggarty B.S. Hart T.K. Lee W.M. Hoxie J.A. J. Cell Biol. 1996; 132: 795-811Crossref PubMed Scopus (127) Google Scholar). As expected, the recruitment of SAP in the mutant is not altered because both molecules do not compete for the same motif (7Sayos J. Martin M. Chen A. Simarro M. Howie D. Morra M. Engel P. Terhorst C. Blood. 2001; 97: 3867-3874Crossref PubMed Scopus (112) Google Scholar). CD229 binds to ॖ2 when Fyn is active, or kinase activity of Fyn is completely abrogated by depletion of its catalytic region in the yeast system. This suggests various possibilities: CD229-ॖ2interaction occurs when Tyr470 is phosphorylated, or phosphorylation of the CD229 tail is partial, allowing the screening of interactions dependent and independent of phosphorylation at the same time. Our results point to this last possibility because the CD229-ॖ2 interaction only occurred when the receptor was not phosphorylated. This agrees with literature data, which show that this type of interactions does not depend on phosphorylation. The crystal structure of ॖ2 indicates that phosphotyrosine does not fit into its hydrophobic pocket (40Owen D.J. Evans P.R. Science. 1998; 282: 1327-1332Crossref PubMed Scopus (450) Google Scholar). We have also demonstrated that the cytoplasmic tail of CD229 but not that of any of the other members of CD150 family can interact with the AP-2 complex via its ॖ2 chain. This intriguing difference suggests that the expression and internalization of CD229 are tightly and differentially regulated. The structure of CD229 differs from that of the rest of the CD150 family of receptors. It is the only member that has four extracellular Ig-like domains, and it has the longest cytoplasmic tail, comprising 180 amino acids, 16 threonines, 17 serines, and 8 tyrosines. Two unique tyrosine-based motifs (with the consensus amino acid sequence TV/I YXXV/I) are critical for the binding to the SAP/SH2D1a protein and the Eat-2 SH2 domain protein homolog to SAP in B cells (41Morra M. Lu J. Poy F. Martin M. Sayos J. Calpe S. Gullo C. Howie D. Rietdijk S. Thompson A. Coyle A.J. Denny C. Yaffe M.B. Engel P. Eck M.J. Terhorst C. EMBO J. 2001; 20: 5840-5852Crossref PubMed Scopus (122) Google Scholar). Furthermore, CD229 can bind to endogenous AP-2 in COS cells, ॖ2-EGFP chimera can efficiently integrate in the endogenous AP-2 complex, and CD229 can coprecipitate with ॖ2-EGFP and the AP-2 endogenous complex. The complex is functional because CD229 internalization and CD229·ॖ2-EGFP colocalization were detected in COS cells after antibody-induced endocytosis. In agreement with our mapping data, CD229Y470F and CD229Y470A endocytosis was severely impaired. Although in our model a phenylalanine substitution abrogates the binding to ॖ2, the FXXφ motif can fit into the hydrophobic pocket of ॖ2 (42Marsh B.J. Alm R.A. McIntosh S.R. James D.E. J. Cell Biol. 1995; 130: 1081-1091Crossref PubMed Scopus (76) Google Scholar) but leads to a weaker interaction and slower endocytosis. Thus, Tyr470 is essential for CD229 endocytosis and may inhibit the internalization of this receptor after its phosphorylation in vivo. CD229 can be phosphorylated by Src-kinase Fyn in COS cells, and its possible interaction with a SH2 domain protein may further regulate the signaling and trafficking of the molecule, as proposed for CTLA-4 (43Shiratori T. Miyatake S. Ohno H. Nakaseko C. Isono K. Bonifacino J.S. Saito T. Immunity. 1997; 6: 583-589Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Because CD229 is expressed in T and B lymphocytes, we have defined the mechanism of CD229 internalization and demonstrated that it can be regulated differentially in T and B cell lines. CD229 was endocytosed rapidly in response to antibody ligation in Jurkat T cells, whereas no internalization was observed in B cells. The same results were obtained in lymphocytes from peripheral blood. CD229 in B cells was inhibited by the Fcγ receptor coligation because F(ab′)2 antibodies induce CD229 endocytosis in B cells. The CD229 uptake rate was similar using the intact and F(ab′)2 antibodies in Jurkat cells lacking Fcγ receptors. In agreement with our results, the coligation of Fcγ receptors and BCR has been suggested to inhibit endocytosis by blocking the processing and presentation of the BCR-bound antigen (44Minskoff S.A. Matter K. Mellman I. J. Immunol. 1998; 161: 2079-2083PubMed Google Scholar). Using BFA and cycloheximide, we have determined that CD229, unlike transferrin receptor (CD71), exhibits a low constitutive rate of turnover, and therefore, the rapid internalization of CD229 represents ligand-induced endocytosis. After endocytosis, CD229 was segregated into two distinguishable pools, one that is shuttled to a nonrecycling degradation pathway and another that is recycled to the cell surface. The results suggest that ∼75–807 of CD229 in T cells become degraded via lysosomal compartments, and the remaining 20–257 of complexes are recycled to the cell surface. In contrast, most CD229 is trafficked to the lysosomal degradation pathway in B cells. Moreover, TCR and anti-IgM coligation enhances CD229 endocytosis, suggesting that the antigen receptor signaling regulates the availability of CD229 at the plasma membrane. Thus, endocytosis may represent a critical mechanism that modulates CD229 cell surface expression rapidly. After TCR activation, phosphatidylinositol 3-kinase may become activated and catalyze the conversion of phosphatidylinositol 4,5-biphosphate to phosphatidylinositol 3,4,5-trisphosphate. The pleckstrin homology domain in the α2 subunit of the AP-2 adaptor complex binds with high affinity to phosphatidylinositol 3,4,5-trisphosphate. This binding mediates two effects: localization of AP-2 in the membrane and increase in the affinity of ॖ2 binding to endocytosis sorting signals (45Rapoport I. Miyazaki M. Boll W. Duckworth B. Cantley L.C. Shoelson S. Kirchhausen T. EMBO J. 1997; 16: 2240-2250Crossref PubMed Scopus (182) Google Scholar, 46Jost M. Simpson F. Kavran J.M. Lemmon M.A. Schmid S.L. Curr. Biol. 1998; 8: 1399-1402Abstract Full Text Full Text PDF PubMed Google Scholar). Because CD229 endocytosis is impaired when Fcγ receptors are triggered and enhanced when the BCR is activated, this molecule may have a differential role in these different events. The ability to regulate CD229 internalization in T and B cells differentially may allow for different net effects of CD229 signaling which may be cell-specific. Recent studies indicate that CD229, unlike CD150 and CD84, inhibits interferon-γ secretion after TCR activation in T cells. 2J. M. Del Valle, P. Engel, and M. Martı́n, unpublished result. TCR and BCR may increase the rate of CD229 endocytosis to prevent CD229 from delivering negative signals to the cell and Fcγ receptor ligation retains the CD229 on the cell surface, thus favoring signaling through this receptor and the maintenance of an inhibitory response. The development of CD229 knock-out mice may be a key step to define the physiological role of CD229. We thank Joan Sayós for a critical review of this manuscript." @default.
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W2169165493 title "The Cell Surface Expression of SAP-binding Receptor CD229 Is Regulated via Its Interaction with Clathrin-associated Adaptor Complex 2 (AP-2)" @default.
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