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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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Amino acid sequences and surface expression  of chimeric TCR-β chains. (A)  The sequences of the wt TCR-β  chain (Cβ), wt TCR-γ chain  (Cγ), and the 3 chimeric TCR-β  γ chains (βV-βVII) are shown  using the single letter amino acid  code. The boxes indicate the  TCR-γ chain–derived amino acids. Only the CP, transmembrane,  and Cyto domains of the TCR  constant regions are shown. The  complete α and β chain cDNAs  have been previously described  (16). The NH2-terminal amino  acid in A represents the interchain Cys127 of the TCR-β constant region. The dotted lines indicate the approximate boundary  of the TM domain, defined using  the Lasergene Navigator Protean  Software program (DNASTAR,  Inc., Madison, WI). (B) 58hCD4  (α−/β−)T cell hybridomas expressing wt α chains and wt β or  chimeric TCR-β γ chains were stained with the biotinylated anti-Vβ8 mAb, F23.1, and SAPE, and then analyzed by flow cytometry. Dashed lines represent fluorescence of the same cells stained with streptavidin-phycoerythrin alone. The solid vertical lines indicate the mean fluorescence intensity of  58hCD4 cells expressing the wt α/β TCR. Similar results were obtained using anti-Vα2, anti–CD3-ε, or anti–Cβ specific mAbs (data not shown).
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Figure 1: Amino acid sequences and surface expression of chimeric TCR-β chains. (A) The sequences of the wt TCR-β chain (Cβ), wt TCR-γ chain (Cγ), and the 3 chimeric TCR-β γ chains (βV-βVII) are shown using the single letter amino acid code. The boxes indicate the TCR-γ chain–derived amino acids. Only the CP, transmembrane, and Cyto domains of the TCR constant regions are shown. The complete α and β chain cDNAs have been previously described (16). The NH2-terminal amino acid in A represents the interchain Cys127 of the TCR-β constant region. The dotted lines indicate the approximate boundary of the TM domain, defined using the Lasergene Navigator Protean Software program (DNASTAR, Inc., Madison, WI). (B) 58hCD4 (α−/β−)T cell hybridomas expressing wt α chains and wt β or chimeric TCR-β γ chains were stained with the biotinylated anti-Vβ8 mAb, F23.1, and SAPE, and then analyzed by flow cytometry. Dashed lines represent fluorescence of the same cells stained with streptavidin-phycoerythrin alone. The solid vertical lines indicate the mean fluorescence intensity of 58hCD4 cells expressing the wt α/β TCR. Similar results were obtained using anti-Vα2, anti–CD3-ε, or anti–Cβ specific mAbs (data not shown).

Mentions: The Vα2.1 and Vβ8.1 TCR cDNAs were isolated from the T cell hybridoma, 3BBM74, and confer reactivity to the I-Abm12 alloantigen and the staphylococcal enterotoxin B (SEB) superantigen (16). The wild-type (wt) and mutated TCR constructs were generated using overlapping oligo nucleotides and PCR as previously described (15). All constructs were verified by DNA sequencing using the SequiTherm™ cycle sequencing kit (Epicentre Technologies Corp., Madison, WI) and the deduced amino acid sequences of the mutant β chains are shown in Fig. 1.


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)

Amino acid sequences and surface expression  of chimeric TCR-β chains. (A)  The sequences of the wt TCR-β  chain (Cβ), wt TCR-γ chain  (Cγ), and the 3 chimeric TCR-β  γ chains (βV-βVII) are shown  using the single letter amino acid  code. The boxes indicate the  TCR-γ chain–derived amino acids. Only the CP, transmembrane,  and Cyto domains of the TCR  constant regions are shown. The  complete α and β chain cDNAs  have been previously described  (16). The NH2-terminal amino  acid in A represents the interchain Cys127 of the TCR-β constant region. The dotted lines indicate the approximate boundary  of the TM domain, defined using  the Lasergene Navigator Protean  Software program (DNASTAR,  Inc., Madison, WI). (B) 58hCD4  (α−/β−)T cell hybridomas expressing wt α chains and wt β or  chimeric TCR-β γ chains were stained with the biotinylated anti-Vβ8 mAb, F23.1, and SAPE, and then analyzed by flow cytometry. Dashed lines represent fluorescence of the same cells stained with streptavidin-phycoerythrin alone. The solid vertical lines indicate the mean fluorescence intensity of  58hCD4 cells expressing the wt α/β TCR. Similar results were obtained using anti-Vα2, anti–CD3-ε, or anti–Cβ specific mAbs (data not shown).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199154&req=5

Figure 1: Amino acid sequences and surface expression of chimeric TCR-β chains. (A) The sequences of the wt TCR-β chain (Cβ), wt TCR-γ chain (Cγ), and the 3 chimeric TCR-β γ chains (βV-βVII) are shown using the single letter amino acid code. The boxes indicate the TCR-γ chain–derived amino acids. Only the CP, transmembrane, and Cyto domains of the TCR constant regions are shown. The complete α and β chain cDNAs have been previously described (16). The NH2-terminal amino acid in A represents the interchain Cys127 of the TCR-β constant region. The dotted lines indicate the approximate boundary of the TM domain, defined using the Lasergene Navigator Protean Software program (DNASTAR, Inc., Madison, WI). (B) 58hCD4 (α−/β−)T cell hybridomas expressing wt α chains and wt β or chimeric TCR-β γ chains were stained with the biotinylated anti-Vβ8 mAb, F23.1, and SAPE, and then analyzed by flow cytometry. Dashed lines represent fluorescence of the same cells stained with streptavidin-phycoerythrin alone. The solid vertical lines indicate the mean fluorescence intensity of 58hCD4 cells expressing the wt α/β TCR. Similar results were obtained using anti-Vα2, anti–CD3-ε, or anti–Cβ specific mAbs (data not shown).
Mentions: The Vα2.1 and Vβ8.1 TCR cDNAs were isolated from the T cell hybridoma, 3BBM74, and confer reactivity to the I-Abm12 alloantigen and the staphylococcal enterotoxin B (SEB) superantigen (16). The wild-type (wt) and mutated TCR constructs were generated using overlapping oligo nucleotides and PCR as previously described (15). All constructs were verified by DNA sequencing using the SequiTherm™ cycle sequencing kit (Epicentre Technologies Corp., Madison, WI) and the deduced amino acid sequences of the mutant β chains are shown in Fig. 1.

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