The CD4 transmembrane GGXXG and juxtamembrane (C/F)CV+C motifs mediate pMHCII-specific signaling independently of CD4-Lck interactions

CD4(+) T cell activation is driven by 5-module receptor complexes. The T cell receptor (TCR) is the receptor module that binds composite surfaces of peptide antigens embedded within MHCII molecules (pMHCII). It associates with three signaling modules (CD3γε, CD3δε, and CD3ζζ) to form TCR-CD3 complexes. CD4 is the coreceptor module. It reciprocally associates with TCR-CD3-pMHCII assemblies on the outside of a CD4(+) T cells and with the Src kinase, Lck, on the inside. Previously, we reported that the CD4 transmembrane GGXXG motif and cytoplasmic juxtamembrane (C/F)CV+C motif found in eutherian CD4 (placental mammals) have constituent residues that evolved under purifying selection. Mutating these motifs together increased CD4-Lck association but reduced CD3ζ, Zap70, and Plcγ1 phosphorylation levels, as well as IL-2 production, in response to agonist pMHCII. Because these mutants preferentially localized CD4-Lck pairs to non-raft membrane fractions, one explanation for our results was that they impaired proximal signaling by sequestering Lck away from TCR-CD3. An alternative hypothesis is that the mutations directly impacted signaling because the motifs normally play a Lck-independent role in signaling. The goal of this study was to discriminate between these possibilities. Our results indicate that: intracellular CD4-Lck interactions are not necessary for pMHCII-specific signal initiation; the GGXXG and (C/F)CV+C motifs are key determinants of CD4-mediated pMHCII-specific signal amplification; the GGXXG and (C/F)CV+C motifs exert their functions independently of direct CD4-Lck association. These data provide a mechanistic explanation for why residues within these motifs are under purifying selection, and thus functionally important for CD4(+) T cells in vivo. The results are also important to consider for biomimetic engineering of synthetic receptors.