Quantitative analysis by surface plasmon resonance of CD28 interaction with cytoplasmic adaptor molecules Grb2, Gads and p85 PI3K.
CD28 surface receptors provide co-stimulatory signals that are required for full T cell activation. The CD28 cytoplasmic region has one YMNM and two PXXP motifs as a functional motif. Upon CD28 ligation, Grb2, Gads, and the p85 subunit of PI3 kinase are recruited to the CD28 cytoplasmic region. Here, the interactions between these adaptor proteins and CD28 cytoplasmic domains were analyzed using a Biacore surface plasmon resonance biosensor. For all three adaptor proteins, entire molecules bound more tightly to CD28 than did their isolated SH2 domains. For each adaptor, different outcomes of mutation of CD28's PXXP motifs on binding affinity indicated that only the SH3 domain of Grb2 bound directly. Regarding binding of SH2s to CD28, the SH2 domains of p85 bound more strongly than those of both Grb2 and Gads. Since intact p85 had a 50-fold higher binding affinity than its fragments, and yet the p85-CD28 interaction does not involve SH3-PXXP binding, binding of both N-terminal and C-terminal SH2s to YMNM may create an "avidity" effect. In contrast, when Grb2 and Gads interact with CD28, binding of their SH3 domains may be important. These results suggest that all these interactions are multivalent, through both SH2 and SH3 domains.
Division of Immunobiology, Research Institute for Biomedical Sciences, Tokyo University of Science , Noda, Chiba 278-0022 , Japan .
Kunitake
K
Higo
Masayuki
M
Oda
Hisayuki
H
Morii
Jun
J
Takahashi
Yohsuke
Y
Harada
Shuhei
S
Ogawa
Ryo
R
Abe
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs / Q000235:genetics; D018106:CD28 Antigens / Q000235:genetics / Q000378:metabolism*; D058543:Class Ia Phosphatidylinositol 3-Kinase / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D051380:GRB2 Adaptor Protein / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008213:Lymphocyte Activation; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics; D020349:Surface Plasmon Resonance / Q000379:methods; D013601:T-Lymphocytes / Q000276:immunology*; D018909:src Homology Domains / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; D018106:CD28 Antigens; C409587:GRAP2 protein, human; D051380:GRB2 Adaptor Protein; D011993:Recombinant Fusion Proteins; D058543:Class Ia Phosphatidylinositol 3-Kinase