Potent inhibitory ligands of the GRB2 SH2 domain from recombinant peptide libraries.
We cloned and expressed the SH2 domain of human GRB2 as glutathione S-transferase and maltose binding protein fusion proteins. We screened three phagemid-based fd pVIII-protein phage display libraries against SH2 domain fusion proteins. Sequence analysis of the peptide extensions yielded a variety of related peptides. By examining the ability of the phage clones to bind other SH2 domains, we demonstrated that the phage were specific for the SH2 domain of GRB2. Based on the sequence motif identified in the "random" library screening experiment, we also built and screened a phage display library based on a Tyr-X-Asn motif (X5-Tyr-X-Asn-X8). To examine the affinity of the phage derived peptides for GRB2, we set up a radioligand competition binding assay based on immobilized GRB2 and radiolabelled autophosphorylated EGFR ICD as the radioligand. Results obtained with peptide competitors derived from the phage sequences demonstrated that nonphosphotyrosine-containing peptides identified with the phage display technology had an affinity for the receptor similar to tyrosine-phosphorylated peptides derived from the EGFR natural substrate. Interestingly, when the phage display peptides were then phosphorylated on tyrosine, their affinity for GRB2 increased dramatically. We also demonstrated the ability of the peptides to block the binding of the GRB2 SH2 domain to EGFR in a mammalian cell-based binding assay.
Hart
C P
CP
Affymax Research Institute, Santa Clara, CA 95051, USA. charles_hart@affymax.com
Martin
J E
JE
Reed
M A
MA
Keval
A A
AA
Pustelnik
M J
MJ
Northrop
J P
JP
Patel
D V
DV
Grove
J R
JR
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001216:Asparagine; D001435:Bacteriophages; D019556:COS Cells; D003001:Cloning, Molecular; D051380:GRB2 Adaptor Protein; D015870:Gene Expression; D006801:Humans; D008024:Ligands; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017421:Sequence Analysis; D014443:Tyrosine; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D001216:Asparagine