Involvement of the PPPGHR motif in T cell activation via CD2.
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.
Chang
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HC
Laboratory of Immunobiology, Dana-Farber Cancer Institute, Boston, Massachusetts.
Moingeon
P
P
Pedersen
R
R
Lucich
J
J
Stebbins
C
C
Reinherz
E L
EL
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000276:immunology; D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D018801:CD2 Antigens; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004247:DNA; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis; D008213:Lymphocyte Activation / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D011971:Receptors, Immunologic / Q000235:genetics / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*; D014162:Transfection
D000596:Amino Acids; D000945:Antigens, Differentiation, T-Lymphocyte; D018801:CD2 Antigens; D007376:Interleukin-2; D010446:Peptide Fragments; D011971:Receptors, Immunologic; D004247:DNA; D002118:Calcium