FIGURE 5.
Identification of surface-exposed and buried residues in human CD3ϵ in the TCR-CD3 complex. A, FACS analysis of cells stably transduced with a lentiviral vector encoding both HA-tagged CD3ϵ and GFP, separated by an IRES sequence. The level of GFP fluorescence (x axis) indicates the efficiency of transduction. Anti-HA antibody staining (y axis) for transduced J.RT3-T3.5 cells lacking a functional TCRβ chain and for transduced J.RT3-T3.5 cells stably expressing a TCRβ/luciferase chimera (Jluchi cells) is shown in the left and right panels, respectively. B, a schematic showing the assay for CD3ϵ incorporation. C, three views of the surface of CD3ϵ (from Protein Data Bank code 1XIW), each related by a 120° rotation about the vertical axis. Residues colored blue are buried by the monoclonal antibodies UCHT1 (as in Protein Data Bank code 1XIW) or OKT3 (as in Protein Data Bank code 1SY6). Other residues are colored according to whether their mutation reduces CD3ϵ expression by more than 85% (red), by 60–85% (orange), or by less than 60% (green) versus wild-type CD3ϵ expression. D, the same views of CD3ϵ as in C colored according to whether the residues are buried in the heterodimeric interfaces with CD3γ (purple; as in Protein Data Bank code 1SY6), CD3δ (red; as in Protein Data Bank code 1XIW), or both (blue). See also supplemental Fig. S4.