Structure-function analysis of Ia molecules: in-phase insertion mutagenesis of the amino-terminal domain of the E beta k polypeptide chain

To identify which segments of the beta 1 domain of the E beta k polypeptide control T cell recognition of antigen, E beta genes were constructed with in-phase insertion mutations. Five independent mutants, with insertions mapping to positions 24, 50 and 93 of the E beta k polypeptide, were obtained. Cell lines expressing these mutated genes were analysed by microfluorometry using a panel of 20 anti-Ek monoclonal antibodies. None of the tested in-phase insertions has resulted in the loss of antibody binding sites. In striking contrast, mutations at position 93 and at a lesser level 50 were indicative of a crucial role of the corresponding regions in T-cell recognition, because they led to significant or complete loss of antigen-presenting function with all but one of the T hybridomas tested. These data are discussed with regard to a model of the foreign antigen binding site of Ia molecules.     

Requirement for association of p56lck with CD4 in antigen-specific signal transduction in T cells.

The T cell-specific transmembrane glycoprotein CD4 interacts with class II MHC molecules via its external domain and is associated with tyrosine kinase p56lck via a cysteine motif in its cytoplasmic domain. We have assessed the ability of CD4 to synergize with the antigen-specific T cell receptor (TCR) for induction of transmembrane signals that result in lymphokine production. Mutant CD4 molecules were introduced into T cells that lacked endogenous CD4 but expressed TCRs specific for lysozyme peptides or the superantigen SEA bound to Ab or Abm12 class II MHC molecules. With either ligand, T cell activation occurred only when CD4 was associated with p56lck. These results demonstrate that residues within the cytoplasmic domain of CD4 are required for its coreceptor function in TCR-mediated signal transduction and strongly support the notion that the association of CD4 with p56lck is critical in this process.