Abstract
The CD28 family molecules, CD28, and inducible costimulator (ICOS) all provide positive costimulatory signals. However, unlike CD28, ICOS does not costimulate IL-2 secretion. The YMNM motif that exists in the CD28 cytoplasmic domain is a known binding site for phosphatidylinositol 3-kinase (PI3-K) and Grb2. ICOS possesses the YMFM motif in the corresponding region of CD28 that binds PI3-K but not Grb2. We postulated that the reason that ICOS does not have the ability to induce IL-2 production is because it fails to recruit Grb2. To verify this hypothesis, we generated a mutant ICOS gene that contains the CD28 YMNM motif and measured IL-2 promoter activation after ICOS ligation. The results indicated that ICOS became competent to activate the IL-2 promoter by this single alteration. Further analysis demonstrated that Grb2 binding to ICOS was sufficient to activate the NFAT/AP-1 site in the IL-2 promoter and that the cytoplasmic domain of CD28 outside of the YMNM motif is required for activation of the CD28RE/AP-1 and NF-kappaB sites. Together, these observations lead us to believe that the difference of a single amino acid, which affects Grb2 binding ability, may define a functional difference between the CD28- and ICOS-mediated costimulatory signals.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing*
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Amino Acid Motifs
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Amino Acid Substitution
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Animals
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Antigens, Differentiation, T-Lymphocyte / chemistry
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Antigens, Differentiation, T-Lymphocyte / genetics
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Antigens, Differentiation, T-Lymphocyte / metabolism*
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Binding Sites / genetics
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CD28 Antigens / chemistry*
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CD28 Antigens / genetics
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CD28 Antigens / metabolism*
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DNA-Binding Proteins / metabolism
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GRB2 Adaptor Protein
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Gene Expression Regulation
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Humans
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Inducible T-Cell Co-Stimulator Protein
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Interleukin-2 / genetics*
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Jurkat Cells
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Mice
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Mutagenesis, Site-Directed
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NF-kappa B / metabolism
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NFATC Transcription Factors
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Nuclear Proteins*
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Phosphatidylinositol 3-Kinases / metabolism
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Promoter Regions, Genetic*
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Protein Structure, Tertiary
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Proteins / metabolism
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Transcription Factor AP-1 / metabolism
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Transcription Factors / metabolism
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Transfection
Substances
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Adaptor Proteins, Signal Transducing
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Antigens, Differentiation, T-Lymphocyte
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CD28 Antigens
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DNA-Binding Proteins
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GRB2 Adaptor Protein
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GRB2 protein, human
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Grb2 protein, mouse
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ICOS protein, human
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Icos protein, mouse
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Inducible T-Cell Co-Stimulator Protein
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Interleukin-2
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NF-kappa B
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NFATC Transcription Factors
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Nuclear Proteins
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Proteins
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Recombinant Fusion Proteins
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Transcription Factor AP-1
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Transcription Factors
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Phosphatidylinositol 3-Kinases