title: In vitro translation and assembly of a complete T cell receptor-CD3 complex.
abstract: The T cell receptor for antigen (TCR) is a multisubunit complex that consists of at least seven polypeptides: the clonotypic, disulfide-linked alpha/beta heterodimer that is noncovalently associated with the invariant polypeptides of the CD3 complex (CD3-gamma, -delta, -epsilon) and zeta, 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-alpha, -beta and CD3-gamma, -delta, -epsilon, and -zeta subunits. CD3-epsilon, 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-gamma or -delta, led to the expression of mainly monomeric and correctly folded epsilon 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-epsilon in a glycan-independent manner. A glycan-dependent interaction between CD3-epsilon and calnexin was mediated by CD3-gamma and concerned only monomeric CD3-epsilon complexed with CD3-gamma, but was dispensable for proper folding of CD3-epsilon. We suggest that in addition to its signaling function, CD3-epsilon serves as a monitor for proper subunit assembly of the TCR.
authors.affiliation: Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139, USA.
authors.forename: J B
authors.initials: JB
authors.lastname: Huppa
authors.forename: H L
authors.initials: HL
authors.lastname: Ploegh
mesh_terms: D000595:Amino Acid Sequence; D017252:CD3 Complex / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D037281:Calnexin; D004220:Disulfides / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000235:genetics / Q000276:immunology; D011108:Polymers / Q000378:metabolism; D014176:Protein Biosynthesis / Q000276:immunology*; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D017252:CD3 Complex; D002135:Calcium-Binding Proteins; D004220:Disulfides; D010455:Peptides; D011108:Polymers; D021382:Protein Sorting Signals; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D037281:Calnexin