The transmembrane orientation of the epsilon chain of the TcR/CD3 complex.
The antigen receptor of the T lymphocytes is one of the most complex eukaryotic membrane structures studied to date. The T cell receptor (TcR) consists of two disulfide-linked glycoprotein chains (alpha/beta or gamma/delta) and is noncovalently associated with a group of small and invariable CD 3 proteins. Four CD 3 chains have been recognized: two highly homologous glycoproteins CD 3 gamma and delta, the more distantly related nonglycosylated CD 3 epsilon chain, and the nonglycosylated CD 3 zeta, the latter being present as a homodimer. The unraveling of the architecture of the TcR/CD 3 complex is crucial to our understanding of the processes underlying its assembly, recognition and transmembrane signaling. The transmembrane orientation of the TcR chains and of CD 3 gamma and CD 3 delta can be directly inferred from their primary structure, based on the presence of concensus N-linked glycosylation sites N-terminal of their transmembrane domains. This prediction can not be made, however, for nonglycosylated molecules like the CD 3 epsilon chain. In order to determine the transmembrane orientation of CD 3 epsilon, anti-peptide antisera directed against the N-termini of the human and murine CD 3 epsilon chains were generated in rabbits. Both antisera stained intact T cells, demonstrating that the N-terminus of the CD 3 epsilon chain was located at the outer surface of the plasma membrane. The anti-human CD 3 epsilon peptide antiserum was found to be mitogenic for peripheral blood T cells, a finding previously reported only for monoclonal anti-TcR/CD 3 reagents. Using a novel transient expression system in murine T lymphocytes, the human CD 3 epsilon chain could be expressed on the surface of CD 3+, but not CD 3- murine T cells, as indicated by fluorescence staining with the anti-peptide antiserum. This experiment confirmed the specificity of the anti-peptide antiserum and, perhaps more importantly, indicated that the human CD 3 epsilon chain was correctly assembled in the murine CD 3 complex. Moreover, the anti-human CD 3 monoclonal antibody UCHT1 was found to stain T cells expressing the human CD 3 epsilon chain.
Clevers
H
H
Laboratory of Molecular Immunology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115.
Dunlap
S
S
Terhorst
C
C
D000595:Amino Acid Sequence; D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis* / Q000235:genetics* / Q000276:immunology; D017252:CD3 Complex; D004247:DNA / Q000235:genetics; D006801:Humans; D008213:Lymphocyte Activation; D008565:Membrane Proteins / Q000032:analysis* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000032:analysis* / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012689:Sequence Homology, Nucleic Acid; D013601:T-Lymphocytes / Q000276:immunology
D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D008565:Membrane Proteins; D010446:Peptide Fragments; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D004247:DNA