In Vitro Translation and Assembly of a Complete T Cell Receptor–CD3 Complex

The T cell receptor for antigen (TCR) is a multisubunit complex that consists of at least seven polypeptides: the clonotypic, disulfide-linked α/β heterodimer that is noncovalently associated with the invariant polypeptides of the CD3 complex (CD3-γ, -δ, -ε) and ζ, a disulfide-linked homodimer. We achieved the complete assembly of the human TCR in an in vitro transcription/translation system supplemented with dog pancreas microsomes by simultaneous translation of the messenger RNAs encoding the TCR-α, -β and CD3-γ, -δ, -ε, and -ζ subunits. CD3-ε, one of the subunits that initiates the assembly of the TCR in living cells, forms misfolded, disulfide-linked homooligomers when translated alone. However, co-translation of one of its first binding partners in the course of assembly, CD3-γ or -δ, led to the expression of mainly monomeric and correctly folded ε subunits, the only form we could detect as part of a properly assembled TCR complex. In the absence of these subunits, the ER-resident chaperone calnexin interacted with oligomeric, i.e. misfolded, structures of CD3-ε in a glycan-independent manner. A glycan-dependent interaction between CD3-ε and calnexin was mediated by CD3-γ and concerned only monomeric CD3-ε complexed with CD3-γ, but was dispensable for proper folding of CD3-ε. We suggest that in addition to its signaling function, CD3-ε serves as a monitor for proper subunit assembly of the TCR.

• Publication year

  1997

• Venue

  Journal of Experimental Medicine

• Publication date

  1997-08-04

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1084/JEM.186.3.393PMID 9236191PMCID 2198996
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Conformation-independent binding of monoglucosylated ribonuclease B to calnexin.

  1997cited by this paper
• Glycan-dependent and -independent Association of Vesicular Stomatitis Virus G Protein with Calnexin*

  1996cited by this paper
• The molecular chaperone calnexin facilitates folding and assembly of class I histocompatibility molecules.

  1996cited by this paper
• Role of CD3 gamma in T cell receptor assembly

  1996cited by this paper
• Physical dissociation of the TCR-CD3 complex accompanies receptor ligation

  1995cited by this paper
• The molecular chaperone calnexin is expressed on the surface of immature thymocytes in association with clonotype‐independent CD3 complexes.

  1995cited by this paper
• TCR alpha-CD3 delta epsilon association is the initial step in alpha beta dimer formation in murine T cells and is limiting in immature CD4+ CD8+ thymocytes.

  1995cited by this paper
• Glucose trimming and reglucosylation determine glycoprotein association with calnexin in the endoplasmic reticulum.

  1995cited by this paper
• TCRα-CD3δϵ association is the initial step in αβ dimer formation in murine T cells and is limiting in immature CD4+CD8+ thymocytes

  1995cited by this paper
• The Molecular Chaperone Calnexin Binds Glc1Man9GlcNAc2 Oligosaccharide as an Initial Step in Recognizing Unfolded Glycoproteins (*)

  1995cited by this paper
• Regulation of MHC class I transport by the molecular chaperone, calnexin (p88, IP90).

  1994cited by this paper
• Mapping functional regions in the lumenal domain of the class II- associated invariant chain

  1994cited by this paper
• Retention of unassembled components of integral membrane proteins by calnexin.

  1994influential reference
• Persistence of glucose residues on core oligosaccharides prevents association of TCR alpha and TCR beta proteins with calnexin and results specifically in accelerated degradation of nascent TCR alpha proteins within the endoplasmic reticulum.

  1994cited by this paper
• Stoichiometry of the T cell antigen receptor (TCR) complex: each TCR/CD3 complex contains one TCR alpha, one TCR beta, and two CD3 epsilon chains

  1994cited by this paper
• Introduction of oxygen into the alkyl chain of N-decyl-dNM decreases lipophilicity and results in increased retention of glucose residues on N-linked oligosaccharides.

  1994cited by this paper
• Interaction with newly synthesized and retained proteins in the endoplasmic reticulum suggests a chaperone function for human integral membrane protein IP90 (calnexin).

  1993cited by this paper
• Assembly of the T-cell antigen receptor. Participation of the CD3 omega chain.

  1993cited by this paper
• Associations between subunit ectodomains promote T cell antigen receptor assembly and protect against degradation in the ER

  1993cited by this paper
• The assembly of H2‐Kb class I molecules translated in vitro requires oxidized glutathione and peptide

  1993cited by this paper
• Failure to synthesize the CD3-gamma chain. Consequences for T cell antigen receptor assembly, processing, and expression.

  1992cited by this paper
• Role of ATP and disulphide bonds during protein folding in the endoplasmic reticulum

  1992cited by this paper
• Early disulfide bond formation prevents heterotypic aggregation of membrane proteins in a cell-free translation system

  1992cited by this paper
• Manipulating disulfide bond formation and protein folding in the endoplasmic reticulum.

  1992cited by this paper
• Endoplasmic reticulum resident protein of 90 kilodaltons associates with the T- and B-cell antigen receptors and major histocompatibility complex antigens during their assembly.

  1992cited by this paper
• Major histocompatibility complex independent clonal T cell anergy by direct interaction of Staphylococcus aureus enterotoxin B with the T cell antigen receptor

  1992cited by this paper
• Generation of Monoclonal Antibodies Against a Human T Cell Receptor β Chain Expressed in Transgenic Mice

  1992cited by this paper
• Role of potentially charged transmembrane residues in targeting proteins for retention and degradation within the endoplasmic reticulum.

  1991cited by this paper
• Six chains of the human T cell antigen receptor.CD3 complex are necessary and sufficient for processing the receptor heterodimer to the cell surface.

  1991cited by this paper
• A conformational epitope expressed upon association of CD3-epsilon with either CD3-delta or CD3-gamma is the main target for recognition by anti-CD3 monoclonal antibodies.

  1991cited by this paper
• Requirements for cell surface expression of the human TCR/CD3 complex in non-T cells.

  1991cited by this paper
• Membrane protein association by potential intrarnembrane charge pairs

  1991cited by this paper
• The T cell antigen receptor: insights into organelle biology.

  1990cited by this paper
• The implications of subunit interactions for the structure of the T cell receptor‐CD3 complex

  1990cited by this paper
• A fraction of CD3 epsilon subunits exists as disulfide-linked dimers in both human and murine T lymphocytes.

  1990cited by this paper
• Human natural killer cells and mature T lymphocytes express identical CD3 zeta subunits as defined by cDNA cloning and sequence analysis.

  1990cited by this paper
• Human natural killer cells and mature T lymphocytes express identical CD3ζ subunits as defined by cDNA cloning and sequence analysis

  1990cited by this paper
• Pre-Golgi degradation of newly synthesized T-cell antigen receptor chains: intrinsic sensitivity and the role of subunit assembly

  1989cited by this paper
• Monoclonal antibodies reactive with the T cell receptor zeta chain: production and characterization using a new method.

  1989cited by this paper
• T-cell antigen receptor (TCR)-alpha/beta heterodimer formation is a prerequisite for association of CD3-zeta 2 into functionally competent TCR.CD3 complexes.

  1989cited by this paper
• Selective degradation of T cell antigen receptor chains retained in a pre-Golgi compartment

  1988cited by this paper
• Failure to synthesize the T cell CD3-zeta chain: structure and function of a partial T cell receptor complex.

  1988cited by this paper
• Transfection of genes encoding the T cell receptor-associated CD3 complex into COS cells results in assembly of the macromolecular structure.

  1988cited by this paper
• Structural model of HLA-DR1 restricted T cell antigen recognition.

  1988influential reference
• Molecular cloning and chromosomal localization of the human T-cell receptor zeta chain: distinction from the molecular CD3 complex.

  1988cited by this paper
• Evolutionary relationship between the T3 chains of the T-cell receptor complex and the immunoglobulin supergene family.

  1987cited by this paper
• The T cell receptor/T3 complex.

  1986cited by this paper
• Primary structure of the T3 gamma subunit of the T3/T cell antigen receptor complex deduced from cDNA sequences: evolution of the T3 gamma and delta subunits.

  1986cited by this paper
• Isolation of cDNA clones encoding the 20K non-glycosylated polypeptide chain of the human T-cell receptor/T3 complex

  1986cited by this paper
• DNA sequence and characterization of human class II major histocompatibility complex beta chains from the DR1 haplotype.

  1985cited by this paper
• Specificity of monoclonal antibodies directed against human and murine class II histocompatibility antigens as analyzed by binding to HLA-deletion mutant cell lines.

  1985cited by this paper
• The isolation and nucleotide sequence of a cDNA encoding the T cell surface protein T4: a new member of the immunoglobulin gene family.

  1985cited by this paper
• Production and characterization of antibody probes directed at constant regions of the α and β subunit of the human T cell receptor

  1985cited by this paper
• Isolation of cDNA clones encoding the 20K T3 glycoprotein of human T-cell receptor complex

  1984cited by this paper
• Preparation of microsomal membranes for cotranslational protein translocation.

  1983cited by this paper
• Alpha chain of HLA-DR transplantation antigens is a member of the same protein superfamily as the immunoglobulins.

  1982cited by this paper
• Separation of functional subsets of human T cells by a monoclonal antibody.

  1979influential reference
• Monoclonal antibodies defining distinctive human T cell surface antigens.

  1979cited by this paper
• Purification of HLA-linked B lymphocyte alloantigens in immunologically active form by preparative sodium dodecyl sulfate-gel electrophoresis and studies on their subunit association.

  1977cited by this paper
• Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4

  1970cited by this paper

• Modulating T Cell Responses by Targeting CD3

  2023cites this paper
• TCR/CD3-based synthetic antigen receptors (TCC) convey superior antigen sensitivity combined with high fidelity of activation

  2023cites this paper
• T cell and B cell antigen receptors share a conserved core transmembrane structure

  2022cites this paper
• DNA-encoded immunoassay in picoliter drops: a minimal cell-free approach

  2022cites this paper
• T‐Cell Antigen Recognition – Lessons from the Past and Projections into the Future

  2020cites this paper
• Pathogenic Effects of Impaired Retrieval between the Endoplasmic Reticulum and Golgi Complex

  2019cites this paper
• Synthetic Biology Goes Cell-Free

  2019cites this paper
• Amphipol-Assisted Cell-Free Expression of Membrane Proteins

  2018cites this paper
• Perspective: Solidifying the impact of cell-free synthetic biology through lyophilization

  2018cites this paper
• Monomeric TCRs drive T cell antigen recognition

  2018cites this paper
• A Cholesterol-Based Allostery Model of T Cell Receptor Phosphorylation.

  2016cites this paper
• Molecular cloning and characterization of CD3ε in Chinese domestic goose (Anser cygnoides).

  2015cites this paper
• Development and characterization of monoclonal antibodies against goose CD3ɛ extracellular domain and their application in detection of CD3(+) T lymphocytes.

  2015cites this paper
• Folding membrane proteins in vitro: a table and some comments.

  2014cites this paper
• Structure of the Chicken CD3ϵδ/γ Heterodimer and Its Assembly with the αβT Cell Receptor*

  2014cites this paper
• Detection of Constant Domain of Human T Cell Antigen Receptor Alpha-Chain Via Novel Monoclonal Antibody 7F18

  2014cites this paper
• Orchestration of secretory protein folding by ER chaperones.

  2013cites this paper
• Protein nanomachines assembly modes: cell-free expression and biochip perspectives.

  2013cites this paper
• The interdisciplinary science of T-cell recognition.

  2013cites this paper
• An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease

  2012cites this paper
• Synthetic immune signaling.

  2012cites this paper
• Molecular Mechanisms Of Neuroinflammation Following Global Cerebral Ischemia: The Role of Hypothermia Therapy

  2011cites this paper
• Characterization of Early EDEM1 Protein Maturation Events and Their Functional Implications*

  2011cites this paper
• Production of protein complexes via co-expression.

  2011cites this paper
• Control of cholesterol synthesis through regulated ER-associated degradation of HMG CoA reductase

  2010cites this paper
• The first transmembrane region of the beta-chain stabilizes the tetrameric Fc epsilon RI complex.

  2009cites this paper
• Protein degradation from the mammalian endoplasmic reticulum

  2009cites this paper
• THE FIRST TRANSMEMBRANE REGION OF THE BETA CHAIN STABILIZES THE TETRAMERIC Fc ε RI COMPLEX

  2009cites this paper
• Transmembrane Domains in Membrane Protein Folding, Oligomerization, and Function

  2008cites this paper
• Proteolytic cleavage in an endolysosomal compartment is required for activation of Toll-like receptor 9

  2008cites this paper
• 13C- and 15N-Isotopic Labeling of Proteins

  2008cites this paper
• Transmemebrane Domains in Proteins

  2008cites this paper
• UNIVERSITY OF ZAGREB FACULTY OF FOOD TECHNOLOGY AND BIOTECHNOLOGY

  2007cites this paper
• Common themes in the assembly and architecture of activating immune receptors

  2007cites this paper
• N-linked carbohydrates act as lumenal maturation and quality control protein tags

  2007cites this paper
• Etude des interactions moléculaires à l'aire de contact formée entre les lymphocytes T et les cellules présentatrices d'antigène

  2007cites this paper
• Influence des lymphocytes T CD4+ CD25+ régulateurs sur la dynamique de formation de la synapse immunologique entre un lymphocyte T CD4+ effecteur et une cellule présentatrice d'antigène

  2007cites this paper
• The Assembly of Diverse Immune Receptors Is Focused on a Polar Membrane-Embedded Interaction Site

  2006cites this paper
• Reconstitution of Allogeneic Hemopoietic Stem Cells: The Essential Role of FcRγ and the TCR β-Chain-FCp33 Complex1

  2006cites this paper
• A Membrane-proximal Tetracysteine Motif Contributes to Assembly of CD3δϵ and CD3γϵ Dimers with the T Cell Receptor*

  2006cites this paper
• SEL1L, the homologue of yeast Hrd3p, is involved in protein dislocation from the mammalian ER

  2006cites this paper
• Molecular characterization of the Japanese flounder, Paralichthys olivaceus, CD3epsilon and evolution of the CD3 cluster.

  2005cites this paper
• Intravenous catecholamine administration affects mouse intestinal lymphocyte number and apoptosis.

  2005cites this paper
• Cotranslational endoplasmic reticulum assembly of FcɛRI controls the formation of functional IgE-binding receptors

  2005influential citation
• EBAG 9 Adds a New Layer of Control on Large Dense-Core Vesicle Exocytosis via Interaction with Snapin □ D

  2005cites this paper
• The cotranslational maturation of the type I membrane glycoprotein tyrosinase: the heat shock protein 70 system hands off to the lectin-based chaperone system.

  2005influential citation
• The activating NKG2D receptor assembles in the membrane with two signaling dimers into a hexameric structure.

  2005cites this paper
• EBAG9 adds a new layer of control on large dense-core vesicle exocytosis via interaction with Snapin.

  2005cites this paper
• Coupled tertiary folding and oligomerization of the T1 domain of Kv channels.

  2005cites this paper
• The T cell receptor: critical role of the membrane environment in receptor assembly and function.

  2005cites this paper
• Cloning and Characterization of Two Distinct CD3 Genes from Olive Flounder Paralichthys olivaceus

  2005cites this paper
• Fukutin-related protein mutations that cause congenital muscular dystrophy result in ER-retention of the mutant protein in cultured cells.

  2005cites this paper
• Convergence on a distinctive assembly mechanism by unrelated families of activating immune receptors.

  2005cites this paper
• High level cell-free expression and specific labeling of integral membrane proteins.

  2004cites this paper
• Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatin.

  2004cites this paper
• A novel emulsion mixture for in vitro compartmentalization of transcription and translation in the rabbit reticulocyte system.

  2004cites this paper
• Stoichiometry of the T‐cell receptor–CD3 complex and key intermediates assembled in the endoplasmic reticulum

  2004influential citation
• Beta-adrenergic receptor blockade during exercise decreases intestinal lymphocyte apoptosis but not cell loss in mice.

  2004cites this paper
• Crystal structure of the human T cell receptor CD3 epsilon gamma heterodimer complexed to the therapeutic mAb OKT3.

  2004cites this paper
• Molecular mechanisms for the assembly of the T cell receptor-CD3 complex.

  2004cites this paper
• Crystal structure of the human T cell receptor CD3εγ heterodimer complexed to the therapeutic mAb OKT3

  2004cites this paper
• Mutational analysis of conserved amino acids in the T cell receptor alpha-chain transmembrane region: a critical role of leucine 112 and phenylalanine 127 for assembly and surface expression.

  2003cites this paper
• Prospects for High-Throughput Structure Determination of Proteins by NMR Spectroscopy

  2003cites this paper
• Ubiquitinylation of the Cytosolic Domain of a Type I Membrane Protein Is Not Required to Initiate Its Dislocation from the Endoplasmic Reticulum*

  2003cites this paper
• Surface T‐cell antigen receptor expression and availability for long‐term antigenic signaling

  2003cites this paper
• Folding of the Voltage-gated K+ Channel T1 Recognition Domain*

  2003cites this paper
• Membrane-specific, Host-derived Factors Are Required for US2- and US11-mediated Degradation of Major Histocompatibility Complex Class I Molecules*

  2002cites this paper
• Expression and characterization of recombinant soluble human CD3 molecules: presentation of antigenic epitopes defined on the native TCR-CD3 complex.

  2002cites this paper
• Jcb: Article Introduction

  2002cites this paper
• In Vitro Production and Characterization of Partly Assembled Human CD3 Complexes

  2002cites this paper
• Human Cytomegalovirus Gene Products US2 and US11 Differ in Their Ability To Attack Major Histocompatibility Class I Heavy Chains in Dendritic Cells

  2002cites this paper
• Structural and thermodynamic correlates of T cell signaling.

  2002cites this paper
• US2, a Human Cytomegalovirus-encoded Type I Membrane Protein, Contains a Non-cleavable Amino-terminal Signal Peptide* 210

  2002cites this paper
• The organizing principle in the formation of the T cell receptor-CD3 complex.

  2002cites this paper
• The TCR/CD3 complex: molecular interactions in a changing structure.

  2002cites this paper
• Early TCR αβ Expression Promotes Maturation of T Cells Expressing FcεRIγ Containing TCR/CD3 Complexes1

  2001cites this paper
• The Transmembrane Adaptor Protein Trim Regulates T Cell Receptor (Tcr) Expression and Tcr-Mediated Signaling via an Association with the Tcr ζ Chain

  2001cites this paper
• Quality control of transmembrane domain assembly in the tetraspanin CD82

  2001cites this paper
• Molecular characterisation of the tammar wallaby (Macropus eugenii) CD3 epsilon chain cDNA.

  2001cites this paper
• Early TCR ab Expression Promotes Maturation of T Cells Expressing FceRIg Containing TCR/CD3 Complexes

  2001cites this paper
• Four-color flow cytometric detection of retrovirally expressed red, yellow, green, and cyan fluorescent proteins.

  2001cites this paper
• Translation Rate of Human Tyrosinase Determines ItsN-Linked Glycosylation Level*

  2001cites this paper
• Folding of Hepatitis C Virus E1 Glycoprotein in a Cell-Free System

  2001cites this paper
• Kinetics of T-cell Receptor Binding by Bivalent HLA-DR·Peptide Complexes That Activate Antigen-specific Human T-cells*

  2000cites this paper
• A New Monoclonal Anti-CD3∊ Antibody Reactive on Paraffin Sections

  2000cites this paper
• Evidence for a Stepwise Evolution of the CD3 Family1

  2000cites this paper
• A new monoclonal anti-CD3epsilon antibody reactive on paraffin sections.

  2000cites this paper
• Generation of catalytically active 6-phosphofructokinase from Saccharomyces cerevisiae in a cell-free system.

  2000cites this paper
• Cytotoxic cells : basic mechanisms and medical applications

  2000cites this paper
• Evolution of the T cell receptor signal transduction units.

  2000cites this paper
• Heterodimeric CD3epsilongamma extracellular domain fragments: production, purification and structural analysis.

  2000cites this paper
• Processing and Maturation of Flavocytochromeb 558 Include Incorporation of Heme as a Prerequisite for Heterodimer Assembly*

  2000cites this paper
• T cell receptor assembly and expression in the absence of calnexin.

  2000cites this paper
• The CD3ε Subunit of the TCR Contains Endocytosis Signals

  1999cites this paper
• Bypassing immunization: optimized design of "designer T cells" against carcinoembryonic antigen (CEA)-expressing tumors, and lack of suppression by soluble CEA.

  1999cites this paper
• Expression of the T cell antigen receptor zeta chain following activation is controlled at distinct checkpoints. Implications for cell surface receptor down-modulation and re-expression.

  1999cites this paper
• TCRζ is transported to and retained in the Golgi apparatus independently of other TCR chains: implications for TCR assembly

  1999cites this paper
• The CD3 epsilon subunit of the TCR contains endocytosis signals.

  1999cites this paper
• Family Evidence for a Stepwise Evolution of the CD 3

  1999cites this paper
• Biosynthesis of inositol trisphosphate receptors: selective association with the molecular chaperone calnexin.

  1999cites this paper