High-efficiency expression/cloning of epidermal growth factor-receptor-binding proteins with Src homology 2 domains.
Src homology 2 domains bind to tyrosine-phosphorylated growth factor receptors and are found in proteins that serve as substrates for tyrosine kinases, such as phospholipase C-gamma 1 and ras GTPase-activating protein. We have previously described the cloning of phosphatidylinositol 3'-kinase-associated p85 from expression libraries with the tyrosine-phosphorylated epidermal growth factor receptor as a probe. We have now modified this technique by using T7 polymerase-based expression libraries, which significantly improves sensitivity of the method. In one screening of such a library, we identified five different murine Src homology 2 domain-containing proteins, which we call GRBs (growth factor receptor-bound proteins). Two of these proteins represented the tyrosine kinase fyn and the mouse homologue of phospholipase C-gamma 1, whereas two genes encoded proteins similar to v-crk and NCK. We also isolated the gene for GRB-7, which encodes a protein of 535 amino acids. In addition to a Src homology 2 domain, GRB-7 also has a region of similarity to the noncatalytic domain of ras GTPase-activating protein and is highly expressed in liver and kidney. Use of this expression/cloning system should increase our ability to identify downstream modulators of growth factor action.
Margolis
B
B
Department of Pharmacology, New York University Medical Center, NY 10016.
Silvennoinen
O
O
Comoglio
F
F
Roonprapunt
C
C
Skolnik
E
E
Ullrich
A
A
Schlessinger
J
J
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D066246:ErbB Receptors / Q000235:genetics*; D005260:Female; D051382:GRB7 Adaptor Protein; D015870:Gene Expression; D015723:Gene Library; D016391:Genes, src; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics*; D009928:Organ Specificity; D016133:Polymerase Chain Reaction / Q000379:methods; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D010738:Type C Phospholipases / Q000235:genetics
C495955:GRB7 protein, human; C495956:Grb7 protein, mouse; D011506:Proteins; D012333:RNA, Messenger; D051382:GRB7 Adaptor Protein; D066246:ErbB Receptors; D015688:Oncogene Protein pp60(v-src); D010738:Type C Phospholipases