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Signaling efficiency of the T cell receptor controlled by a single amino acid in the beta chain constant region.

Bäckström BT, Hausmann BT, Palmer E - J. Exp. Med. (1997)

Bottom Line: TCRs in which this Cbeta residue is mutated to Phe, the residue found in TCR-gamma, are unresponsive to antigenic ligands.However, the biological response of the mutant TCR could be rescued with a calcium ionophore, implying that mutant TCRs are defective in generating a calcium-mediated signal.The implications of the differences between Cbeta and Cgamma are considered.

View Article: PubMed Central - PubMed

Affiliation: Basel Institute for Immunology, CH-4005 Basel, Switzerland.

ABSTRACT
A single amino acid residue, Gln136, located within the connecting peptide domain of Cbeta controls the ability of the alpha/beta TCR to transmit a full signal. TCRs in which this Cbeta residue is mutated to Phe, the residue found in TCR-gamma, are unresponsive to antigenic ligands. Interestingly, this Cbeta residue is either polar or charged in every species studied thus far, including the trout and the skate. In contrast, the analogous residue in Cgamma is always hydrophobic. In spite of their compromised antigen responsiveness, the mutant TCR complex contains the CD3-gamma, -delta, -epsilon, and -zeta chains, and undergoes zeta chain phosphorylation and ZAP-70 recruitment. However, the biological response of the mutant TCR could be rescued with a calcium ionophore, implying that mutant TCRs are defective in generating a calcium-mediated signal. The implications of the differences between Cbeta and Cgamma are considered.

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ζ chain phosphorylation of SEB-stimulated hybridomas expressing chimeric TCRs. Hybridomas (58hCD4) expressing wt or chimeric TCRs were stimulated using .221 cells with (10 μg/ml) or without  SEB. Cells were lysed in 1% Triton X-100, and the relevant chains were  immunoprecipitated with an anti-ζ mAb (A) or with an anti–ZAP-70 antiserum (B). After Western blotting, the presence of tyrosine phosphorylated proteins was detected with the antiphosphotyrosine mAb 4G10 as  described in Materials and Methods. The positions of phosphorylated ζ  chains, p34, unphosphorylated ζ chains, and ZAP-70 are indicated.
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Figure 3: ζ chain phosphorylation of SEB-stimulated hybridomas expressing chimeric TCRs. Hybridomas (58hCD4) expressing wt or chimeric TCRs were stimulated using .221 cells with (10 μg/ml) or without SEB. Cells were lysed in 1% Triton X-100, and the relevant chains were immunoprecipitated with an anti-ζ mAb (A) or with an anti–ZAP-70 antiserum (B). After Western blotting, the presence of tyrosine phosphorylated proteins was detected with the antiphosphotyrosine mAb 4G10 as described in Materials and Methods. The positions of phosphorylated ζ chains, p34, unphosphorylated ζ chains, and ZAP-70 are indicated.

Mentions: The tyrosine phosphorylation of the ζ chain was examined in these hybridomas as well. From the data in Fig. 3 A, it was apparent that the ζ chains in the αwt/βwt and αwt/βVI TCRs could be tyrosine phosphorylated and that both forms (p21 and p23) of the phosphorylated ζ chain could be generated. Although the tyrosine phosphorylation of ZAP-70 could not be demonstrated even in hybridomas expressing the αwt/βwt receptor (data not shown), an anti–ZAP-70 antiserum was used to evaluate ZAP-70 recruitment (Fig. 3 B). In superantigen-stimulated cells expressing either wt or mutant TCRs, the p21 and p23 forms of the ζ chain as well as the p34 phosphoprotein were coprecipitated with ZAP-70. These experiments indicated that even in hybridomas expressing the signaling-defective αwt/βVI TCR, ZAP-70 was nevertheless recruited to the phosphorylated ζ chains. Thus, the signaling defect in hybridomas expressing this mutant TCR was likely downstream from ZAP-70 recruitment.


Signaling efficiency of the T cell receptor controlled by a single amino acid in the beta chain constant region.

Bäckström BT, Hausmann BT, Palmer E - J. Exp. Med. (1997)

ζ chain phosphorylation of SEB-stimulated hybridomas expressing chimeric TCRs. Hybridomas (58hCD4) expressing wt or chimeric TCRs were stimulated using .221 cells with (10 μg/ml) or without  SEB. Cells were lysed in 1% Triton X-100, and the relevant chains were  immunoprecipitated with an anti-ζ mAb (A) or with an anti–ZAP-70 antiserum (B). After Western blotting, the presence of tyrosine phosphorylated proteins was detected with the antiphosphotyrosine mAb 4G10 as  described in Materials and Methods. The positions of phosphorylated ζ  chains, p34, unphosphorylated ζ chains, and ZAP-70 are indicated.
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Related In: Results  -  Collection

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Figure 3: ζ chain phosphorylation of SEB-stimulated hybridomas expressing chimeric TCRs. Hybridomas (58hCD4) expressing wt or chimeric TCRs were stimulated using .221 cells with (10 μg/ml) or without SEB. Cells were lysed in 1% Triton X-100, and the relevant chains were immunoprecipitated with an anti-ζ mAb (A) or with an anti–ZAP-70 antiserum (B). After Western blotting, the presence of tyrosine phosphorylated proteins was detected with the antiphosphotyrosine mAb 4G10 as described in Materials and Methods. The positions of phosphorylated ζ chains, p34, unphosphorylated ζ chains, and ZAP-70 are indicated.
Mentions: The tyrosine phosphorylation of the ζ chain was examined in these hybridomas as well. From the data in Fig. 3 A, it was apparent that the ζ chains in the αwt/βwt and αwt/βVI TCRs could be tyrosine phosphorylated and that both forms (p21 and p23) of the phosphorylated ζ chain could be generated. Although the tyrosine phosphorylation of ZAP-70 could not be demonstrated even in hybridomas expressing the αwt/βwt receptor (data not shown), an anti–ZAP-70 antiserum was used to evaluate ZAP-70 recruitment (Fig. 3 B). In superantigen-stimulated cells expressing either wt or mutant TCRs, the p21 and p23 forms of the ζ chain as well as the p34 phosphoprotein were coprecipitated with ZAP-70. These experiments indicated that even in hybridomas expressing the signaling-defective αwt/βVI TCR, ZAP-70 was nevertheless recruited to the phosphorylated ζ chains. Thus, the signaling defect in hybridomas expressing this mutant TCR was likely downstream from ZAP-70 recruitment.

Bottom Line: TCRs in which this Cbeta residue is mutated to Phe, the residue found in TCR-gamma, are unresponsive to antigenic ligands.However, the biological response of the mutant TCR could be rescued with a calcium ionophore, implying that mutant TCRs are defective in generating a calcium-mediated signal.The implications of the differences between Cbeta and Cgamma are considered.

View Article: PubMed Central - PubMed

Affiliation: Basel Institute for Immunology, CH-4005 Basel, Switzerland.

ABSTRACT
A single amino acid residue, Gln136, located within the connecting peptide domain of Cbeta controls the ability of the alpha/beta TCR to transmit a full signal. TCRs in which this Cbeta residue is mutated to Phe, the residue found in TCR-gamma, are unresponsive to antigenic ligands. Interestingly, this Cbeta residue is either polar or charged in every species studied thus far, including the trout and the skate. In contrast, the analogous residue in Cgamma is always hydrophobic. In spite of their compromised antigen responsiveness, the mutant TCR complex contains the CD3-gamma, -delta, -epsilon, and -zeta chains, and undergoes zeta chain phosphorylation and ZAP-70 recruitment. However, the biological response of the mutant TCR could be rescued with a calcium ionophore, implying that mutant TCRs are defective in generating a calcium-mediated signal. The implications of the differences between Cbeta and Cgamma are considered.

Show MeSH