Conserved transmembrane tyrosine residues of the TCR beta chain are required for TCR expression and function in primary T cells and hybridomas.
The T cell receptor (TCR) beta chain transmembrane domain contains two evolutionarily conserved tyrosines (Y). In this study, the functional basis for the evolutionary conservation is addressed by mutation of the residues, expression of the mutants in hybridoma and primary T cells, and examination of TCR signaling function. We find that the phenotype of the mutants, both surface expression and ability to signal for IL-2 production, is highly variable in different mouse T hybridoma lines. Although we have not been able to determine the basis for these differences in the hybridomas, expression of the mutants in primary T cells provides a definitive assessment of mutant phenotype. We show that mutation of the N-terminal Y to either leucine (L) or alanine (A) results in low surface expression in primary T cells, while mutation of both N- and C-terminal Y to A or L abrogates surface expression. However, the more conservative mutation of both transmembrane Y to phenylalanine maintained receptor surface expression and assembly while severely disrupting signaling in primary T cells. Our data demonstrate that TCR beta chain transmembrane Y are essential for TCR signal transduction as well as complex assembly. These findings suggest that protein-protein interactions involving membrane-spanning domains are likely relevant for TCR signal transduction mechanisms.
Kunjibettu
S
S
Department of Immunology, Jerome H. Holland Laboratory for the Biomedical Sciences and George Washington University School of Medicine, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855, USA.
Fuller-Espie
S
S
Carey
G B
GB
Spain
L M
LM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D017124:Conserved Sequence / Q000276:immunology*; D005786:Gene Expression Regulation / Q000276:immunology; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism*; D016130:Immunophenotyping; D008213:Lymphocyte Activation / Q000235:genetics; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000172:deficiency / Q000235:genetics; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000096:biosynthesis* / Q000172:deficiency / Q000235:genetics / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D013154:Spleen / Q000276:immunology / Q000378:metabolism; D016176:T-Lymphocyte Subsets / Q000276:immunology / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000235:genetics / Q000502:physiology*
D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta; C054979:antigen T cell receptor, zeta chain; D014443:Tyrosine