We previously reported that the transmembrane GGXXG motif co-arose with the cytoplasmic juxtamembrane (C/F)CV+C motif in eutherian (placental mammal) CD4 and that constituent residues therein evolve under purifying selection (Lee, et al., 2022). These data led us to discover that mutating these motifs together increased CD4-Lck association but reduced CD3z, Zap70, and Plcg1 phosphorylation levels as well as IL-2 production. Because these mutants preferentially localized CD4-Lck pairs to non-membrane raft fractions, one potential 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 TCR-CD3 signaling because the wild type motifs play a Lck-independent role in signaling. Here we used a reductionist approach to discriminate between these possibilities (Lee, et al., 2023). Our current data lead us to conclude 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. The results are also important to consider for biomimetic engineering of synthetic receptors.

Methods are described in the manuscript

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