Biochemical Evidence for the Presence of a Single CD3δ and CD3γ Chain in the Surface T Cell Receptor/CD3 Complex

Abstract

The T cell antigen receptor (TCR) consists of an ␣␤ heterodimer and associated invariant CD3␥, ␦, ⑀, and chains (TCR/CD3 complex). The general stoichiometry of the receptor complex, which is believed to be one molecule each of TCR␣, TCR␤, CD3␥, and CD3␦ and two molecules each of CD3⑀ and CD3, is not clearly understood. Although it has been shown that there are two chains of CD3⑀ and CD3, the stoichiometry of CD3␥ or CD3␦ chains in the surface antigen receptor complex has not been determined. In the present study, transgenic mice expressing an altered form of mouse CD3␦ and CD3␥ were employed to show that the surface TCR complexes contain one molecule each of CD3␦ and CD3␥. Thymocytes from wild type and CD3 chain transgenic mice on the appropriate knockout background were surface-biotinylated and immunoprecipitated using a specific antibody. The immunoprecipitates were resolved in two dimensions under nonreducing/reducing conditions to determine the stoichiometry of CD3␦ and CD3␥ in the surface antigen receptor complex. Our data clearly show the presence of one molecule each of CD3␦ and CD3␥ in the surface TCR/CD3 complex. The TCR1 /CD3 complex is multimeric and consists of a covalently linked heterodimer of polymorphic TCR␣ and ␤ glycoproteins and noncovalently associated invariant CD3␥, ␦, ⑀, and chains. Individual CD3 chains contain immunoreceptor tyrosine-based activation motifs in their cytoplasmic domain. The TCR/CD3 receptor complex recognizes a peptide antigen presented by MHC molecules expressed on the surface of antigen-presenting cells. The engagement of the surface receptor complex to its ligand results in signal transduction and is most likely mediated by CD3 chains via phosphorylation of immunoreceptor tyrosine-based activation motifs (1, 2). The presence of multiple CD3 chains has been suggested to amplify a signal generated by TCR-peptide/MHC interactions resulting in diverse outcomes, for instance, positive versus negative selection of developing thymocytes. In addition to signal transduction, the CD3 components play a crucial role in the receptor complex assembly, stability, and transport to the cell surface as well as ligand-induced surface receptor internalization. Furthermore, it has been suggested that engagement of the surface receptor complex by a single molecule of peptide/MHC is sufficient to elicit a cytotoxic T cell response (3). A single peptide/MHC complex could engage multiple TCR complexes in a serial manner, as suggested by Lanzavecchia and colleagues (4,5). This would imply that CD3 chains present in a single TCR complex are capable of recruiting sufficient numbers of intracellular signaling molecules, leading to T cell activation. This could be achieved by altering the conformation of the receptor, as has recently been shown by Alarcon and colleagues (6). Following TCR/ligand engagement, recruitment of an adaptor protein, Nck, to the proline-rich sequence in the cytoplasmic domain of CD3⑀ has been suggested to alter the conformation of the receptor complex, leading to T cell act...

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