Stoichiometry and intracellular fate of TRIM-containing TCR complexes

Cell Communication and Signaling - 2010
Mahima Swamy1, Gabrielle M. Siegers1, Gina J. Fiala1, Éva D. Molnár1, Elaine P. Dopfer1, Paul Fisch2, Burkhart Schraven3, Wolfgang W. A. Schamel1
1Department of Molecular Immunology, Max Planck-Institute of Immunobiology and Institute for Biology III, Albert Ludwigs University Freiburg, Stübeweg 51, Freiburg, 79108, Germany
2Department of Pathology, University of Freiburg Medical Center, Freiburg, 79110, Germany
3Institute of Molecular and Clinical Immunology, Otto-von-Guericke-Universität Magdeburg, Leipziger Strasse 44, Magdeburg, 39120, Germany

Tóm tắt

AbstractBackgroundStudying the stoichiometry and intracellular trafficking of the T cell antigen receptor (TCR) is pivotal in understanding its mechanisms of activation. The αβTCR includes the antigen-binding TCRαβ heterodimer as well as the signal transducing CD3εγ, CD3εδ and ζ2subunits. Although the TCR-interacting molecule (TRIM) is also part of the αβTCR complex, it has not been included in most reports so far.ResultsWe used the native antibody-based mobility shift (NAMOS) assay in a first dimension (1D) blue native (BN)-PAGE and a 2D BN-/BN-PAGE to demonstrate that the stoichiometry of the digitonin-solublized TRIM-containing αβTCR is TCRαβCD3ε2γδζ2TRIM2. Smaller αβTCR complexes possess a TCRαβ CD3ε2γδζ2stoichiometry. Complexes of these sizes were detected in T cell lines as well as in primary human and mouse T cells. Stimulating the αβTCR with anti-CD3 antibodies, we demonstrate by confocal laser scanning microscopy that CD3ε colocalizes with ζ and both are degraded upon prolonged stimulation, possibly within the lysosomal compartment. In contrast, a substantial fraction of TRIM does not colocalize with ζ. Furthermore, TRIM neither moves to lysosomes nor is degraded. Immunoprecipitation studies and BN-PAGE indicate that TRIM also associates with the γδTCR.ConclusionsSmall αβTCR complexes have a TCRαβ CD3ε2γδζ2stoichiometry; whereas those associated with one TRIM dimer are TCRαβ CD3ε2γδζ2TRIM2. TRIM is differentially processed compared to CD3 and ζ subunits after T cell activation and is not degraded. The γδTCR also associates with TRIM.

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Tài liệu tham khảo

Hayday AC: γδ cells: a right time and a right place for a conserved third way of protection. Annu Rev Immunol. 2000, 18: 975-1026. 10.1146/annurev.immunol.18.1.975.

Siegers GM, Fernandez-Malave E, Fisch P, Regueiro JR, Schamel WW: From structure to disease: differential CD3 usage in the human and mouse gd T cell antigen receptor determines the impact of CD3 deficiencies on T cell development. Int J Med Biol Frontiers. 2008, 14: 115-132.

Alarcon B, Gil D, Delgado P, Schamel WWA: Initiation of TCR signaling: regulation within CD3 dimers. Immunol Rev. 2003, 191: 38-46. 10.1034/j.1600-065X.2003.00017.x.

Alarcon B, Swamy M, van Santen HM, Schamel WW: T-cell antigen-receptor stoichiometry: pre-clustering for sensitivity. EMBO Rep. 2006, 7: 490-495. 10.1038/sj.embor.7400682.

Blumberg RS, Alarcon B, Sancho J, McDermott FV, Lopez P, Breitmeyer J, Terhorst C: Assembly and function of the T cell antigen receptor. Requirement of either the lysine or arginine residues in the transmembrane region of the alpha chain. J Biol Chem. 1990, 265: 14036-14043.

Cosson P, Lankford SP, Bonifacino JS, Klausner RD: Membrane protein association by potential intramembrane charge pairs. Nature. 1991, 351: 414-416. 10.1038/351414a0.

Call ME, Pyrdol J, Wiedmann M, Wucherpfennig KW: The organizing principle in the formation of the T cell receptor-CD3 complex. Cell. 2002, 111: 967-979. 10.1016/S0092-8674(02)01194-7.

Reth M: Antigen receptor tail clue. Nature. 1989, 338: 383-10.1038/338383b0.

Minguet S, Swamy M, Dopfer EP, Dengler E, Alarcon B, Schamel WWA: The extracellular part of zeta is buried in the T cell antigen receptor complex. Immunol Lett. 2008, 116: 203-210. 10.1016/j.imlet.2007.11.024.

Minguet S, Swamy M, Schamel WWA: The short length of the extracellular domain of zeta is crucial for T cell antigen receptor function. Immunol Lett. 2008, 116: 195-202. 10.1016/j.imlet.2007.11.020.

Gil D, Schamel WW, Montoya M, Sanchez-Madrid F, Alarcon B: Recruitment of Nck by CD3 epsilon reveals a ligand-induced conformational change essential for T cell receptor signaling and synapse formation. Cell. 2002, 109: 901-912. 10.1016/S0092-8674(02)00799-7.

Minguet S, Swamy M, Alarcon B, Luescher IF, Schamel WW: Full activation of the T cell receptor requires both clustering and conformational changes at CD3. Immunity. 2007, 26: 43-54. 10.1016/j.immuni.2006.10.019.

Risueno RM, Schamel WW, Alarcon B: T cell receptor engagement triggers its CD3epsilon and CD3zeta subunits to adopt a compact, locked conformation. PLoS ONE. 2008, 3: e1747-10.1371/journal.pone.0001747.

Minguet S, Schamel WWA: A permissive geometry model for TCR-CD3 activation. Trends Biochem Sci. 2008, 33: 51-57. 10.1016/j.tibs.2007.10.008.

Schraven B, Ratnofsky S, Gaumont Y, Lindegger H, Kirchgessner H, Bruyns E, Moebius U, Meuer SC: Identification of a novel dimeric phosphoprotein (PP29/30) associated with signaling receptors in human T lymphocytes and natural killer cells. J Exp Med. 1994, 180: 897-906. 10.1084/jem.180.3.897.

Bruyns E, Marie-Cardine A, Kirchgessner H, Sagolla K, Shevchenko A, Mann M, Autschbach F, Bensussan A, Meuer S, Schraven B: T cell receptor (TCR) interacting molecule (TRIM), a novel disulfide-linked dimer associated with the TCR-CD3-zeta complex, recruits intracellular signaling proteins to the plasma membrane. J Exp Med. 1998, 188: 561-575. 10.1084/jem.188.3.561.

Horejsi V, Zhang W, Schraven B: Transmembrane adaptor proteins: organizers of immunoreceptor signalling. Nat Rev Immunol. 2004, 4: 603-616. 10.1038/nri1414.

Kirchgessner H, Dietrich J, Scherer J, Isomaki P, Korinek V, Hilgert I, Bruyns E, Leo A, Cope AP, Schraven B: The transmembrane adaptor protein TRIM regulates T cell receptor (TCR) expression and TCR-mediated signaling via an association with the TCR zeta chain. J Exp Med. 2001, 193: 1269-1284. 10.1084/jem.193.11.1269.

Berkhout B, Alarcon B, Terhorst C: Transfection of genes encoding the T cell receptor-associated CD3 complex into COS cells results in assembly of the macromolecular structure. J Biol Chem. 1988, 263: 8528-8536.

Hall C, Berkhout B, Alarcon B, Sancho J, Wileman T, Terhorst C: Requirements for cell surface expression of the human TCR/CD3 complex in non-T cells. Int Immunol. 1991, 3: 359-368. 10.1093/intimm/3.4.359.

Carson GR, Kuestner RE, Ahmed A, Pettey CL, Concino MF: Six chains of the human T cell antigen receptor. CD3 complex are necessary and sufficient for processing the receptor heterodimer to the cell surface. J Biol Chem. 1991, 266: 7883-7887.

Koelsch U, Arndt B, Reinhold D, Lindquist JA, Juling N, Kliche S, Pfeffer K, Bruyns E, Schraven B, Simeoni L: Normal T-cell development and immune functions in TRIM-deficient mice. Mol Cell Biol. 2006, 26: 3639-3648. 10.1128/MCB.26.9.3639-3648.2006.

Koelsch U, Schraven B, Simeoni L: SIT and TRIM determine T cell fate in the thymus. J Immunol. 2008, 181: 5930-5939.

Zapata DA, Schamel WW, Torres PS, Alarcon B, Rossi NE, Navarro MN, Toribio ML, Regueiro J: Biochemical differences in the alphabeta T cell receptor. CD3 surface complex between CD8+ and CD4+ human mature T lymphocytes. J Biol Chem. 2004, 279: 24485-24492. 10.1074/jbc.M311455200.

Schamel WW, Arechaga I, Risueno RM, van Santen HM, Cabezas P, Risco C, Valpuesta JM, Alarcon B: Coexistence of multivalent and monovalent TCRs explains high sensitivity and wide range of response. J Exp Med. 2005, 202: 493-503. 10.1084/jem.20042155.

Swamy M, Minguet S, Siegers GM, Alarcon B, Schamel WW: A native antibody-based mobility-shift technique (NAMOS-assay) to determine the stoichiometry of multiprotein complexes. J Immunol Methods. 2007, 324: 74-83. 10.1016/j.jim.2007.05.003.

Swamy M, Dopfer EP, Molnar E, Alarcon B, Schamel WW: The 450 kDa TCR Complex has a Stoichiometry of alphabetagammaepsilondeltaepsilonzetazeta. Scand J Immunol. 2008, 67: 418-420. 10.1111/j.1365-3083.2008.02082.x. author reply 421.

Siegers GM, Swamy M, Fernandez-Malave E, Minguet S, Rathmann S, Guardo AC, Perez-Flores V, Regueiro JR, Alarcon B, Fisch P, Schamel WW: Different composition of the human and the mouse gammadelta T cell receptor explains different phenotypes of CD3gamma- and CD3delta-immunodeficiencies. J Exp Med. 2007, 204: 2537-2544. 10.1084/jem.20070782.

Reth M, Imanishi-Kari T, Rajewsky K: Analysis of the repertoire of anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies in C 57 BL/6 mice by cell fusion. II. Characterization of idiotopes by monoclonal anti-idiotope antibodies. Eur J Immunol. 1979, 12: 1004-1013. 10.1002/eji.1830091216.

Huynh T, Wurch A, Bruyns E, Korinek V, Schraven B, Eichmann K: Developmentally regulated expression of the transmembrane adaptor protein trim in fetal and adult T cells. Scand J Immunol. 2001, 54: 146-154. 10.1046/j.1365-3083.2001.00953.x.

Alcover A, Alarcon B: Internalization and intracellular fate of TCR-CD3 complexes. Crit Rev Immunol. 2000, 20: 325-346.

Niedergang F, San Jose E, Rubin B, Alarcon B, Dautry-Varsat A, Alcover A: Differential cytosolic tail dependence and intracellular fate of T-cell receptors internalized upon activation with superantigen or phorbol ester. Res Immunol. 1997, 148: 231-245. 10.1016/S0923-2494(97)80865-6.

Valitutti S, Muller S, Salio M, Lanzavecchia A: Degradation of T cell receptor (TCR)-CD3-zeta complexes after antigenic stimulation. J Exp Med. 1997, 185: 1859-1864. 10.1084/jem.185.10.1859.

Ouchida R, Yamasaki S, Hikida M, Masuda K, Kawamura K, Wada A, Mochizuki S, Tagawa M, Sakamoto A, Hatano M, Tokuhisa T, Koseki H, Saito T, Kurosaki T, Wang JY: A lysosomal protein negatively regulates surface T cell antigen receptor expression by promoting CD3zeta-chain degradation. Immunity. 2008, 29: 33-43. 10.1016/j.immuni.2008.04.024.

Mellman I: Endocytosis and molecular sorting. Annu Rev Cell Dev Biol. 1996, 12: 575-625. 10.1146/annurev.cellbio.12.1.575.

Wang HY, Altman Y, Fang D, Elly C, Dai Y, Shao Y, Liu YC: Cbl promotes ubiquitination of the T cell receptor zeta through an adaptor function of Zap-70. J Biol Chem. 2001, 276: 26004-26011. 10.1074/jbc.M010738200.

Naramura M, Jang IK, Kole H, Huang F, Haines D, Gu H: c-Cbl and Cbl-b regulate T cell responsiveness by promoting ligand-induced TCR down-modulation. Nat Immunol. 2002, 3: 1192-1199. 10.1038/ni855.

Myers MD, Dragone LL, Weiss A: Src-like adaptor protein down-regulates T cell receptor (TCR)-CD3 expression by targeting TCRzeta for degradation. J Cell Biol. 2005, 170: 285-294. 10.1083/jcb.200501164.

Myers MD, Sosinowski T, Dragone LL, White C, Band H, Gu H, Weiss A: Src-like adaptor protein regulates TCR expression on thymocytes by linking the ubiquitin ligase c-Cbl to the TCR complex. Nat Immunol. 2006, 7: 57-66. 10.1038/ni1291.

Dietrich J, Backstrom T, Lauritsen JP, Kastrup J, Christensen MD, von Bulow F, Palmer E, Geisler C: The phosphorylation state of CD3gamma influences T cell responsiveness and controls T cell receptor cycling. J Biol Chem. 1998, 273: 24232-24238. 10.1074/jbc.273.37.24232.

San Jose E, Borroto A, Niedergang F, Alcover A, Alarcon B: Triggering the TCR complex causes the downregulation of nonengaged receptors by a signal transduction-dependent mechanism. Immunity. 2000, 12: 161-170. 10.1016/S1074-7613(00)80169-7.

Borroto A, Lama J, Niedergang F, Dautry-Varsat A, Alarcon B, Alcover A: The CD3 epsilon subunit of the TCR contains endocytosis signals. J Immunol. 1999, 163: 25-31.

Schraven B, Roux M, Hutmacher B, Meuer SC: Triggering of the alternative pathway of human T cell activation involves members of the T 200 family of glycoproteins. Eur J Immunol. 1989, 19: 397-403. 10.1002/eji.1830190226.

Fernandez-Malave E, Wang N, Pulgar M, Schamel WW, Alarcon B, Terhorst C: Overlapping functions of human CD3{delta} and mouse CD3{gamma} in {alpha}beta T-cell development revealed in a humanized CD3{gamma}-deficient mouse. Blood. 2006, 108: 3420-3427. 10.1182/blood-2006-03-010850.

San Jose E, Sahuquillo AG, Bragado R, Alarcon B: Assembly of the TCR/CD3 complex: CD3 epsilon/delta and CD3 epsilon/gamma dimers associate indistinctly with both TCR alpha and TCR beta chains. Evidence for a double TCR heterodimer model. Eur J Immunol. 1998, 28: 12-21. 10.1002/(SICI)1521-4141(199801)28:01<12::AID-IMMU12>3.0.CO;2-9.

Schägger H, von Jagow G: Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem. 1991, 199: 223-231. 10.1016/0003-2697(91)90094-A.

Schamel WW, Reth M: Monomeric and oligomeric complexes of the B cell antigen receptor. Immunity. 2000, 13: 5-14. 10.1016/S1074-7613(00)00003-0.

Swamy M, Siegers GM, Minguet S, Wollscheid B, Schamel WW: Blue native polyacrylamide gel electrophoresis (BN-PAGE) for the identification and analysis of multiprotein complexes. Sci STKE. 2006, 2006: l4-10.1126/stke.3452006pl4.

Schamel WW: Two-dimensional blue native polyacrylamide gel electrophoresis. Curr Protoc Cell Biol. 2008, Chapter 6 (Unit 6): 10.

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