A high-affinity Arg-X-X-Lys SH3 binding motif confers specificity for the interaction between Gads and SLP-76 in T cell signaling.
A critical event in T cell receptor (TCR)-mediated signaling is the recruitment of hematopoietic-specific adaptor proteins that collect and transmit signals downstream of the TCR. Gads, a member of the Grb2 family of SH2 and SH3 domain-containing adaptors, mediates the formation of a complex between LAT and SLP-76 that is essential for signal propagation from the TCR. Here we examine the binding specificity of the Gads and Grb2 SH3 domains using peptide arrays and find that a nonproline-based R-X-X-K motif found in SLP-76 binds to the Gads carboxy-terminal SH3 domain with high affinity (K(D) = 240 +/- 45 nM). The Grb2 C-terminal SH3 domain also binds this motif, but with a 40-fold lower affinity than Gads. Single point mutations in either the relevant R (237) or K (240) completely abrogated SLP-76 association with Gads in vivo and impaired SLP-76 function. A chimeric Grb2 protein, possessing the C-terminal SH3 domain of Gads, was able to partially substitute for Gads in signaling downstream of the T cell receptor. These results provide a molecular explanation for the specific role of Gads in T cell receptor signaling, and identify a discrete subclass of SH3 domains whose binding is dependent on a core R-X-X-K motif.
Berry
Donna M
DM
The Arthur and Sonia Labatt, Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, M5G1X8, Ontario, Canada.
Nash
Piers
P
Liu
Stanley K-W
SK
Pawson
Tony
T
McGlade
C Jane
CJ
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D007376:Interleukin-2 / Q000235:genetics; D007700:Kinetics; D008239:Lysine; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology*; D014158:Transcription, Genetic; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C409587:GRAP2 protein, human; D051380:GRB2 Adaptor Protein; D007376:Interleukin-2; D010446:Peptide Fragments; D010750:Phosphoproteins; D011506:Proteins; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins; C092947:SLP-76 signal Transducing adaptor proteins; D001120:Arginine; D008239:Lysine