Importance of the CD3γ Ectodomain Terminal β-Strand and Membrane Proximal Stalk in Thymic Development and Receptor Assembly

Abstract

CD3εγ and CD3εδ are noncovalent heterodimers; each consists of Ig-like extracellular domains associated side-to-side via paired terminal β-strands that are linked to individual subunit membrane proximal stalk segments. CD3ε, CD3γ, and CD3δ stalks contain the RxCxxCxE motif. To investigate the functional importance of a CD3 stalk and terminal β-strand, we created a CD3γ double mutant CD3γC82S/C85S and a CD3γ β-strand triple mutant CD3γQ76S/Y78A/Y79A for use in retroviral transduction of lymphoid progenitors for comparison with CD3γwt. Although both mutant CD3γ molecules reduced association with CD3ε in CD3εγ heterodimers, CD3γQ76S/Y78A/Y79A abrogated surface TCR expression whereas CD3γC82S/C85S did not. Furthermore, CD3γC82S/C85S rescued thymic development in CD3γ−/− fetal thymic organ culture. However, the numbers of double-positive and single-positive thymocytes after CD3γC82S/C85S transduction were significantly reduced despite surface pre-TCR and TCR expression comparable to that of CD3γ−/− thymocytes transduced in fetal thymic organ culture with a retrovirus harboring CD3γwt cDNA. Furthermore, double-negative thymocyte development was perturbed with attenuated double-negative 3/double-negative 4 maturation and altered surface-expressed CD3εγ, as evidenced by the loss of reactivity with CD3γ N terminus-specific antisera. Single histidine substitution of either CD3γ stalk cysteine failed to restore CD3εγ association and conformation in transient COS-7 cell transfection studies. Thus, CD3γC82 and CD3γC85 residues likely are either reduced or form a tight intrachain disulfide loop rather than contribute to a metal coordination site in conjunction with CD3εC80 and CD3εC83. The implications of these results for CD3εγ and TCR structure and signaling function are discussed.

Constitutively Oxidized CXXC Motifs within the CD3 Heterodimeric Ectodomains of the T Cell Receptor Complex Enforce the Conformation of Juxtaposed Segments

“…These results substantiate the hypothesis that the CXXC motifs are oxidized during folding in the endoplasmic reticulum and are, therefore, oxidized on the T cell surface. Previous data probing cell surface CD3␥ using anti-CD3␥-antibody specific for its N terminus showed reactivity with WT CD3␥ on the surface TCR complex but not of a SXXS motif variant of CD3␥ in which both cysteines of CXXC were mutated to serine (19). Collectively, these data suggest that the normal, active state of CD3-CXXC in CD3␥, CD3␦, and CD3⑀ on the cellular surface is oxidized and that this state supports proper domain topology.…”

“…The CD3 CXXC motif is important for CD3⑀␥ and CD3⑀␦ heterodimer association (17)(18)(19)(20). Moreover, mutation of the cysteine residues in the CXXC motif has a significant effect on pre-TCR and ␣␤ TCR development and signaling (19,21).…”

“…The CD3 CXXC motif is important for CD3⑀␥ and CD3⑀␦ heterodimer association (17)(18)(19)(20). Moreover, mutation of the cysteine residues in the CXXC motif has a significant effect on pre-TCR and ␣␤ TCR development and signaling (19,21). Although the physiological state of the CXXC motif has not been definitively characterized, it is known that the cysteines residues neither participate in intermolecular disulfide bonding interactions nor in metal ion coordination (19,20).…”

“…Moreover, mutation of the cysteine residues in the CXXC motif has a significant effect on pre-TCR and ␣␤ TCR development and signaling (19,21). Although the physiological state of the CXXC motif has not been definitively characterized, it is known that the cysteines residues neither participate in intermolecular disulfide bonding interactions nor in metal ion coordination (19,20). It has been postulated that the adjacent, closely spaced cysteines might form an intramolecular disulfide bond to provide structural stability to the membrane proximal region and TM domain (18).…”

Pre-TCR ligand binding impacts thymocyte development before αβTCR expression

“…The OP9-DL4 stromal cell findings were validated in a separate assay using a two-color retroviral internal ribosome entry site (IRES) system, allowing control of interlobe variability within the fetal thymic organ culture system, which recapitulates a more complete thymic inductive environment (Fig. S9F) (38). In this assay as well, the M2 2 β-chain attained lesser cellularity than WT N15β, whereas the M2 3 β-chain did not differ significantly from WT (Fig.…”