Masking of the CD3γ di‐Leucine‐based Motif by ζ is Required for Efficient T‐Cell Receptor Expression

Abstract

The T-cell receptor (TCR) is a multimeric receptor composed of the Ti alpha beta heterodimer and the noncovalently associated CD3 gamma delta epsilon and zeta(2) chains. All of the TCR chains are required for efficient cell surface expression of the TCR. Previous studies on chimeric molecules containing the di-leucine-based endocytosis motif of the TCR subunit CD3 gamma have indicated that the zeta chain can mask this motif. In this study, we show that successive truncations of the cytoplasmic tail of zeta led to reduced surface expression levels of completely assembled TCR complexes. The reduced TCR expression levels were caused by an increase in the TCR endocytic rate constant in combination with an unaffected exocytic rate constant. Furthermore, the TCR degradation rate constant was increased in cells with truncated zeta. Introduction of a CD3 gamma chain with a disrupted di-leucine-based endocytosis motif partially restored TCR expression in cells with truncated zeta chains, indicating that the zeta chain masks the endocytosis motif in CD3 gamma and thereby stabilizes TCR cell surface expression.

T−B+NK+ severe combined immunodeficiency caused by complete deficiency of the CD3ζ subunit of the T-cell antigen receptor complex

“…Only incomplete TCR␣␤-CD3␥-CD3␦-CD3ε complexes lacking detectable CD3 were immunoprecipitated with anti-CD3ε Ab in MA5.8 cells transduced with vector alone ( Figure 7 lane 7) as expected, and CD3 was not identified in these cells (Figure 7 lanes 7-9). Consistent with the rescue of cell surface TCR expression noted in this study ( Figure 6A) and in a previous report, 85 newly synthesized CD3 assembled in complete TCR complexes was readily apparent in MA5.8 cells transduced with wild-type CD3 (Figure 7 lane 1). However, in cells transduced with the 411insC mutant CD3, ectopically expressed CD3 was not detected in any form ( Figure 7 lanes 4-6), thereby preventing the generation of complete TCR complexes (Figure 7 lane 4).…”

“…MA5.8 is a CD3-deficient murine T-cell hybridoma 34 in which expression of human CD3 efficiently restores surface TCR expression. 85 For these studies, retroviral expression constructs containing the patient C insertion mutation in each of 2 prevalent human CD3 transcript variants 74 were generated by site-directed mutagenesis using PCR. The longer CD3 transcript variant 1 (NM198053) 66,[72][73][74] uses an alternate 5Јsplice site in intron 4 and encodes a longer CD3 protein isoform including amino acid Q101 that is not present in canonical transcript variant 2 (NM000734).…”

Is there a place and role for endocytic TCR signaling?

“…This suggests that constitutively internalized CD3ε maintains a pool of receptors, susceptible to a polarized recycling, and that contributes to IS formation . At the plasma membrane, in fully assembled TCRs, the di‐Leucine motif of CD3γ is masked by the CD3ζ chain, which limits constitutive endocytosis of the TCR complex, as detected by the F101.01 conformational antibody and by anti‐TCRβ and anti‐CD3ε antibodies …”

“…CD3ζ is internalized together with pMHC from the APC, in a phagocytosis‐related process known as trogocytosis . The antigen‐triggered TCRα, TCRβ, CD3ε, and CD3ζ might be either recycled, especially at early time points, or, at least for CD3ε, directed to lysosomes and degraded …”

“…Both antigen‐triggered and bystander TCR endocytosis require Lck activity . The involvement of Lck activity in constitutive TCR endocytosis is not fully understood, yet it does not seem to regulate TCR complex constitutive recycling, as detected by the F101.01 conformational antibody . However, Lck by inducing the phosphorylation of the clathrin heavy chain has been shown to control endocytosis of CD3ε .…”