The CD4 and CD3δε Cytosolic Juxtamembrane Regions Are Proximal within a Compact TCR–CD3–pMHC–CD4 Macrocomplex

TCRs relay information about peptides embedded within MHC molecules (pMHC) to the ITAMs of the associated CD3γε, CD3δε, and CD3ζζ signaling modules. CD4 then recruits the Src kinase p56(Lck) (Lck) to the TCR-CD3 complex to phosphorylate the ITAMs, initiate intracellular signaling, and drive CD4(+) T cell fate decisions. Whereas the six ITAMs of CD3ζζ are key determinants of T cell development, activation, and the execution of effector functions, multiple models predict that CD4 recruits Lck proximal to the four ITAMs of the CD3 heterodimers. We tested these models by placing FRET probes at the cytosolic juxtamembrane regions of CD4 and the CD3 subunits to evaluate their relationship upon pMHC engagement in mouse cell lines. The data are consistent with a compact assembly in which CD4 is proximal to CD3δε, CD3ζζ resides behind the TCR, and CD3γε is offset from CD3δε. These results advance our understanding of the architecture of the TCR-CD3-pMHC-CD4 macrocomplex and point to regions of high CD4-Lck + ITAM concentrations therein. The findings thus have implications for TCR signaling, as phosphorylation of the CD3 ITAMs by CD4-associated Lck is important for CD4(+) T cell fate decisions.