Limits...
T cell receptor engagement triggers its CD3epsilon and CD3zeta subunits to adopt a compact, locked conformation.

Risueño RM, Schamel WW, Alarcón B - PLoS ONE (2008)

Bottom Line: How the T cell antigen receptor (TCR) discriminates between molecularly related peptide/Major Histocompatibility Complex (pMHC) ligands and converts this information into different possible signaling outcomes is still not understood.Furthermore, the resistance to protease digestion suggests that CD3 cytoplasmic tails adopt a compact structure in the triggered TCR.These results are consistent with a model in which transduction of the conformational change induced upon TCR triggering promotes condensation and shielding of the CD3 cytoplasmic tails.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain.

ABSTRACT
How the T cell antigen receptor (TCR) discriminates between molecularly related peptide/Major Histocompatibility Complex (pMHC) ligands and converts this information into different possible signaling outcomes is still not understood. One current model proposes that strong pMHC ligands, but not weak ones, induce a conformational change in the TCR. Evidence supporting this comes from a pull-down assay that detects ligand-induced binding of the TCR to the N-terminal SH3 domain of the adapter protein Nck, and also from studies with a neoepitope-specific antibody. Both methods rely on the exposure of a polyproline sequence in the CD3epsilon subunit of the TCR, and neither indicates whether the conformational change is transmitted to other CD3 subunits. Using a protease-sensitivity assay, we now show that the cytoplasmic tails of CD3epsilon and CD3zeta subunits become fully protected from degradation upon TCR triggering. These results suggest that the TCR conformational change is transmitted to the tails of CD3epsilon and CD3zeta, and perhaps all CD3 subunits. Furthermore, the resistance to protease digestion suggests that CD3 cytoplasmic tails adopt a compact structure in the triggered TCR. These results are consistent with a model in which transduction of the conformational change induced upon TCR triggering promotes condensation and shielding of the CD3 cytoplasmic tails.

Show MeSH

Related in: MedlinePlus

The membrane-proximal ITAM of CD3ζ is constitutively protected from limited trypsin digestion.(A) Cartoon showing the potential trypsin cleavage sites in CD3ζ and in a truncated CD3ζ lacking ITAMs B and C. The position of a Flag epitope appended to the C-terminal end of both constructs is indicated. (B) The presence of ζFlag or ζAFlag does not alter the ligation-induced conformational switch in the TCR. GST-SH3.1 pull-down was performed as in Fig. 2C with lysates of Jurkat cells transfected either with ζFlag (JkζFlag) or ζAFlag (JkζAFlag). (C) ITAM A is constitutively condensed. JkζFlag and JkζAFlag were stimulated with antibodies and the lysates digested with trypsin as in Fig. 2C. Immunoblotting was performed with anti-Flag antibody.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2254190&req=5

pone-0001747-g003: The membrane-proximal ITAM of CD3ζ is constitutively protected from limited trypsin digestion.(A) Cartoon showing the potential trypsin cleavage sites in CD3ζ and in a truncated CD3ζ lacking ITAMs B and C. The position of a Flag epitope appended to the C-terminal end of both constructs is indicated. (B) The presence of ζFlag or ζAFlag does not alter the ligation-induced conformational switch in the TCR. GST-SH3.1 pull-down was performed as in Fig. 2C with lysates of Jurkat cells transfected either with ζFlag (JkζFlag) or ζAFlag (JkζAFlag). (C) ITAM A is constitutively condensed. JkζFlag and JkζAFlag were stimulated with antibodies and the lysates digested with trypsin as in Fig. 2C. Immunoblotting was performed with anti-Flag antibody.

Mentions: From the mobilities of the digestion products (Fig. 1A and 1C) it appeared that the cytoplasmic tail of CD3ε from non-triggered TCRs was completely digested, whereas the tail of CD3ζ was only partly accessible to trypsin. The estimated loss of relative mass in the CD3ζ2 dimer after digestion was 16 kDa (from 32 to 16 kDa), which is below the 24 kDa loss that would be expected if all the CD3ζ tail were digested (Supplemental material Fig. S2). Upon stimulation, partial proteolytic products were detected at 22 and 27 kDa (Fig. 1C). The sequences that separate the three Immune receptor Tyrosine-based Activation Motifs (ITAM) in CD3ζ are particularly rich in basic amino acids, and therefore in potential trypsin-cleavage sites (Fig. 1A, 3A). Inspection of ITAM distribution in CD3ζ suggests that the 27 kDa fragment could derive from a cleavage between ITAMs B and C, and the 22 kDa fragment from cleavage within ITAM B (Suppl. Fig. S2). The 16 kDa product resulting from digestion of CD3ζ in resting TCRs could derive from cleavage between ITAMs A and B. If these calculations are correct, they would indicate that compared with the more membrane-distal ITAMs, ITAM A might be constitutively protected from trypsin attack in the resting TCR. No antibody specific for ITAM A was available, so to test this we generated a truncated CD3ζ mutant with a Flag epitope appended immediately after ITAM A (Fig. 3A, construct ζAflag). Transfection of this construct into Jurkat cells generated disulfide-linked ζAflag homodimers and heterodimers of ζAflag and endogenous CD3ζ (Fig. 3B, total lysates, TL). TCR complexes containing either one (ζζAflag) or two ζAflag constructs (ζAflag2) underwent the conformational change after TCR triggering, as indicated by a positive reaction in the GST-Nck pull-down assay (Fig. 3B). However, ζAflag was completely resistant to trypsin digestion even in non-triggered TCRs, whereas the Flag epitope was completely digested when appended at the C-terminal end of full-length CD3ζ (Fig. 3C). These results suggest that the CD3ζ ITAM closest to the membrane is permanently protected from digestion and that the conformational change in the TCR modifies the exposure of the second and third ITAMs of CD3ζ to trypsin cleavage.


T cell receptor engagement triggers its CD3epsilon and CD3zeta subunits to adopt a compact, locked conformation.

Risueño RM, Schamel WW, Alarcón B - PLoS ONE (2008)

The membrane-proximal ITAM of CD3ζ is constitutively protected from limited trypsin digestion.(A) Cartoon showing the potential trypsin cleavage sites in CD3ζ and in a truncated CD3ζ lacking ITAMs B and C. The position of a Flag epitope appended to the C-terminal end of both constructs is indicated. (B) The presence of ζFlag or ζAFlag does not alter the ligation-induced conformational switch in the TCR. GST-SH3.1 pull-down was performed as in Fig. 2C with lysates of Jurkat cells transfected either with ζFlag (JkζFlag) or ζAFlag (JkζAFlag). (C) ITAM A is constitutively condensed. JkζFlag and JkζAFlag were stimulated with antibodies and the lysates digested with trypsin as in Fig. 2C. Immunoblotting was performed with anti-Flag antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2254190&req=5

pone-0001747-g003: The membrane-proximal ITAM of CD3ζ is constitutively protected from limited trypsin digestion.(A) Cartoon showing the potential trypsin cleavage sites in CD3ζ and in a truncated CD3ζ lacking ITAMs B and C. The position of a Flag epitope appended to the C-terminal end of both constructs is indicated. (B) The presence of ζFlag or ζAFlag does not alter the ligation-induced conformational switch in the TCR. GST-SH3.1 pull-down was performed as in Fig. 2C with lysates of Jurkat cells transfected either with ζFlag (JkζFlag) or ζAFlag (JkζAFlag). (C) ITAM A is constitutively condensed. JkζFlag and JkζAFlag were stimulated with antibodies and the lysates digested with trypsin as in Fig. 2C. Immunoblotting was performed with anti-Flag antibody.
Mentions: From the mobilities of the digestion products (Fig. 1A and 1C) it appeared that the cytoplasmic tail of CD3ε from non-triggered TCRs was completely digested, whereas the tail of CD3ζ was only partly accessible to trypsin. The estimated loss of relative mass in the CD3ζ2 dimer after digestion was 16 kDa (from 32 to 16 kDa), which is below the 24 kDa loss that would be expected if all the CD3ζ tail were digested (Supplemental material Fig. S2). Upon stimulation, partial proteolytic products were detected at 22 and 27 kDa (Fig. 1C). The sequences that separate the three Immune receptor Tyrosine-based Activation Motifs (ITAM) in CD3ζ are particularly rich in basic amino acids, and therefore in potential trypsin-cleavage sites (Fig. 1A, 3A). Inspection of ITAM distribution in CD3ζ suggests that the 27 kDa fragment could derive from a cleavage between ITAMs B and C, and the 22 kDa fragment from cleavage within ITAM B (Suppl. Fig. S2). The 16 kDa product resulting from digestion of CD3ζ in resting TCRs could derive from cleavage between ITAMs A and B. If these calculations are correct, they would indicate that compared with the more membrane-distal ITAMs, ITAM A might be constitutively protected from trypsin attack in the resting TCR. No antibody specific for ITAM A was available, so to test this we generated a truncated CD3ζ mutant with a Flag epitope appended immediately after ITAM A (Fig. 3A, construct ζAflag). Transfection of this construct into Jurkat cells generated disulfide-linked ζAflag homodimers and heterodimers of ζAflag and endogenous CD3ζ (Fig. 3B, total lysates, TL). TCR complexes containing either one (ζζAflag) or two ζAflag constructs (ζAflag2) underwent the conformational change after TCR triggering, as indicated by a positive reaction in the GST-Nck pull-down assay (Fig. 3B). However, ζAflag was completely resistant to trypsin digestion even in non-triggered TCRs, whereas the Flag epitope was completely digested when appended at the C-terminal end of full-length CD3ζ (Fig. 3C). These results suggest that the CD3ζ ITAM closest to the membrane is permanently protected from digestion and that the conformational change in the TCR modifies the exposure of the second and third ITAMs of CD3ζ to trypsin cleavage.

Bottom Line: How the T cell antigen receptor (TCR) discriminates between molecularly related peptide/Major Histocompatibility Complex (pMHC) ligands and converts this information into different possible signaling outcomes is still not understood.Furthermore, the resistance to protease digestion suggests that CD3 cytoplasmic tails adopt a compact structure in the triggered TCR.These results are consistent with a model in which transduction of the conformational change induced upon TCR triggering promotes condensation and shielding of the CD3 cytoplasmic tails.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain.

ABSTRACT
How the T cell antigen receptor (TCR) discriminates between molecularly related peptide/Major Histocompatibility Complex (pMHC) ligands and converts this information into different possible signaling outcomes is still not understood. One current model proposes that strong pMHC ligands, but not weak ones, induce a conformational change in the TCR. Evidence supporting this comes from a pull-down assay that detects ligand-induced binding of the TCR to the N-terminal SH3 domain of the adapter protein Nck, and also from studies with a neoepitope-specific antibody. Both methods rely on the exposure of a polyproline sequence in the CD3epsilon subunit of the TCR, and neither indicates whether the conformational change is transmitted to other CD3 subunits. Using a protease-sensitivity assay, we now show that the cytoplasmic tails of CD3epsilon and CD3zeta subunits become fully protected from degradation upon TCR triggering. These results suggest that the TCR conformational change is transmitted to the tails of CD3epsilon and CD3zeta, and perhaps all CD3 subunits. Furthermore, the resistance to protease digestion suggests that CD3 cytoplasmic tails adopt a compact structure in the triggered TCR. These results are consistent with a model in which transduction of the conformational change induced upon TCR triggering promotes condensation and shielding of the CD3 cytoplasmic tails.

Show MeSH
Related in: MedlinePlus