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Involvement of the PPPGHR motif in T cell activation via CD2
H C Chang1, P Moingeon, R Pedersen
1Laboratory of Immunobiology, Dana-Farber Cancer Institute, Boston, Massachusetts.
Abstract:
Prior studies identified a segment of the CD2 cytoplasmic domain between amino acid (aa) residues 253 and 287 as important in T lymphocyte signal transduction. This region contains two repeats of the sequence motif PPPGHR, thought to form a "cage" structure involved in CD2-mediated signaling. To evaluate this segment, a series of mutant human CD2 molecules were produced by oligonucleotide-directed mutagenesis and inserted into the ovalbumin-specific, I-Ad-restricted murine T-T hybridoma 3DO54.8 using the DOL retroviral system. CD2 M1 (271-272), CD2 M2 (278-279), and CD2 M4 (264-265) mutants replaced the positively charged adjacent aa histidine and arginine (HR) in the wild-type CD2 sequence with aspartic and glutamic acid (DE) at positions 271-272, 278-279, and 264-265, respectively. In addition, a truncation mutant, CD2 M3 (268), containing only 57 of the 117 cytoplasmic aa and terminating before the second PPPGHR sequence, was generated. Stimulation of transfectants CD2 FL, CD2 M1 (271-272), and CD2 M2 (278-279) with anti-T11(2) + anti-T11(3) antibodies resulted in a rise in cytosolic-free calcium [( Ca2+]i) and subsequent interleukin 2 (IL-2) secretion. In contrast, CD2 M4 (264-265) transfectants could not be activated in either assay. Thus, alteration of histidine 264 and/or arginine 265 within the first PPPGHR motif affects the process of signal transduction via CD2, whereas identical mutations in residues at 271-272 or 278-279 were individually without effect. Consistent with these data, CD2 M3 (268) transfectants were able to generate a detectable amount of IL-2 via CD2 triggering. These data support the notion that the PPPGHR motif at aa 260-265 is important for activation of T lymphocytes via the CD2 molecule.
Insights
The PPPGHR motif in CD2 cytoplasmic domain is crucial for T lymphocyte activation. Mutations in the first motif (aa 260-265) block signaling, while later mutations have no effect, highlighting its importance in T cell response.
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- The CD2 cytoplasmic domain, specifically residues 253-287, is vital for T lymphocyte signal transduction.
- The PPPGHR sequence motif within this domain is hypothesized to form a structure critical for CD2-mediated signaling.
Purpose of the Study:
- To investigate the role of the PPPGHR motif in the CD2 cytoplasmic domain in T lymphocyte activation.
- To determine the functional significance of specific amino acid residues within the CD2 signaling motif.
Main Methods:
- Oligonucleotide-directed mutagenesis was used to create mutant human CD2 molecules.
- Mutant CD2 molecules were introduced into a murine T-T hybridoma cell line (3DO54.8) using the DOL retroviral system.
- Transfectants were stimulated with anti-T11(2) + anti-T11(3) antibodies to assess cytosolic-free calcium levels and interleukin-2 (IL-2) secretion.
Main Results:
- Mutations altering histidine 264 and/or arginine 265 in the first PPPGHR motif (CD2 M4) abolished T cell activation, indicated by lack of calcium flux and IL-2 secretion.
- Mutations at later positions (271-272 and 278-279) within the second PPPGHR motif did not individually impair CD2-mediated signaling.
- A truncation mutant (CD2 M3) lacking the second PPPGHR motif still allowed for detectable IL-2 secretion, suggesting the first motif is more critical.
Conclusions:
- The PPPGHR motif located at amino acid residues 260-265 is essential for CD2-mediated T lymphocyte activation.
- Specific residues within the first PPPGHR motif play a critical role in initiating the signal transduction cascade.
- The findings underscore the structural and functional importance of the initial PPPGHR repeat in CD2 signaling pathways.