title: 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.
abstract: The T cell receptor (TCR) for antigen consists, on the majority of peripheral lymphocytes, of an immunoglobulin-like, disulfide-linked heterodimeric glycoprotein: the alpha and beta chain. These proteins are noncovalently linked to at least four nonvariant proteins which comprise the CD3 complex: CD3 gamma, delta, epsilon, and zeta. Whereas the TCR alpha and beta proteins have positively charged residues in the transmembrane region, all the CD3 proteins have similarly placed negatively charged amino acid residues. It has been suggested that these basic and acidic amino acid residues may play an important role in TCR.CD3 complex assembly and/or function. In this paper, the structural and functional role of the lysine and arginine residues of the TCR alpha chain was addressed using oligonucleotide mediated site directed mutagenesis. The Arg256 and Lys261 residues of the TCR alpha cDNA of the HPB-ALL cell line were mutated to either Gly256 and/or Ile261. The altered cDNAs were transfected into a TCR alpha negative recipient mutant cell line of REX, clone 20A. Metabolic labeling of the T cell transfectants showed that mutation of either the Arg256 or Lys261 amino acid residues had no effect on the ability of the TCR alpha chain to form either a heterodimer with the TCR beta chain or a complex with the CD3 gamma, delta, and epsilon proteins. Consequently, the Arg256 to Gly256 and Lys261 to Ile261 mutations did not prevent the formation of a mature, functional TCR.CD3 complex on the cell surface as determined by immunofluorescence, cell surface radioiodination, and the ability of the transfectants to mobilize intracellular calcium after stimulation with a mitogenic anti-CD3 epsilon monoclonal antibody. In contrast, a mutant cDNA in which both the Arg256 and Lys261 residues were mutated to Gly256 and Ile261, respectively, failed to reconstitute the cell surface expression of the TCR.CD3 complex and, consequently, the ability to respond to mitogenic stimuli. In the absence of both the Arg256 and Lys261 residues, TCR alpha beta heterodimer formation was not observed. Cotransfection studies in COS cells showed that the failure of assembly of a heterodimer was likely due to an inability of the mutated TCR alpha chain to form a subcomplex with either the CD3 gamma, delta, epsilon, or zeta proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
authors: Laboratory of Molecular Immunology, Dana Farber Cancer Institute, Boston, Massachusetts 02115.
authors: R S
authors: RS
authors: Blumberg
authors: B
authors: B
authors: Alarcon
authors: J
authors: J
authors: Sancho
authors: F V
authors: FV
authors: McDermott
authors: P
authors: P
authors: Lopez
authors: J
authors: J
authors: Breitmeyer
authors: C
authors: C
authors: Terhorst
mesh_terms: D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000235:genetics; D000945:Antigens, Differentiation, T-Lymphocyte / Q000235:genetics; D001120:Arginine; D001483:Base Sequence; D017252:CD3 Complex; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D005822:Genetic Vectors; D006801:Humans; D008239:Lysine; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D013601:T-Lymphocytes / Q000276:immunology; D014162:Transfection
chemical_list: D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D046911:Macromolecular Substances; D011948:Receptors, Antigen, T-Cell; D001120:Arginine; D008239:Lysine