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. 1992 Oct 1;89(19):8894–8898. doi: 10.1073/pnas.89.19.8894

High-efficiency expression/cloning of epidermal growth factor-receptor-binding proteins with Src homology 2 domains.

B Margolis 1, O Silvennoinen 1, F Comoglio 1, C Roonprapunt 1, E Skolnik 1, A Ullrich 1, J Schlessinger 1
PMCID: PMC50030  PMID: 1409582

Abstract

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.

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Selected References

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