The C-terminal SH3 domain of the adapter protein Grb2 binds with high affinity to sequences in Gab1 and SLP-76 which lack the SH3-typical P-x-x-P core motif.
The adapter Grb2 is an important mediator of normal cell proliferation and oncogenic signal transduction events. It consists of a central SH2 domain flanked by two SH3 domains. While the binding specificities of the Grb2 SH2 and N-terminal SH3 domain [Grb2 SH3(N)] have been studied in detail, binding properties of the Grb2 SH3(C) domain remained poorly defined. Gab1, a receptor tyrosine kinase substrate which associates with Grb2 and the c-Met receptor, was previously shown to bind Grb2 via a region which lacks a Grb2 SH3(N)-typical motif (P-x-x-P-x-R). Precipitation experiments with the domains of Grb2 show now that Gab1 can bind stably to the Grb2 SH3(C) domain. For further analyses, Gab1 mutants were generated by PCR to test in vivo residues thought to be crucial for Grb2 SH3(C) binding. The Grb2 SH3(C) binding region of Gab1 has significant homology to a region of the adapter protein SLP-76. Peptides corresponding to epitopes SLP-76, Gab1, SoS and other proteins with related sequences, as well as mutant peptides were synthesized and analysed by tryptophan-fluorescence spectrometry and by in vitro competition experiments. These experiments define a 13 amino acid sequence with the unusual consensus motif P-x-x-x-R-x-x-K-P as required for a stable binding to the SH3(C) domain of Grb2. Additional analyses point to a distinct binding specificity of the Grb2-homologous adapter protein Mona (Gads), indicating that the proteins of the Grb2 adapter family may have partially overlapping, yet distinct protein binding properties.
Lewitzky
M
M
Laboratory of Molecular Oncology, MSZ, Universität Würzburg, Germany.
Kardinal
C
C
Gehring
N H
NH
Schmidt
E K
EK
Konkol
B
B
Eulitz
M
M
Birchmeier
W
W
Schaeper
U
U
Feller
S M
SM
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D016384:Consensus Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D051380:GRB2 Adaptor Protein; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011233:Precipitin Tests; D011392:Proline / Q000737:chemistry*; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C098587:GAB1 protein, human; C409587:GRAP2 protein, human; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D010750:Phosphoproteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C092947:SLP-76 signal Transducing adaptor proteins; D014364:Tryptophan; D011392:Proline; D005982:Glutathione Transferase