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High-efficiency expression/cloning of epidermal growth factor-receptor-binding proteins with Src homology 2 domains
B Margolis1, O Silvennoinen, F Comoglio
1Department of Pharmacology, New York University Medical Center, NY 10016.
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.
Insights
Researchers identified novel growth factor receptor-bound proteins (GRBs) using an improved cloning technique. This method enhances the discovery of proteins involved in growth factor signaling pathways.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Src homology 2 domains are crucial for binding tyrosine-phosphorylated growth factor receptors.
- Proteins with SH2 domains are substrates for tyrosine kinases, regulating cellular processes.
- Previous methods for identifying SH2 domain-containing proteins were less sensitive.
Purpose of the Study:
- To develop a more sensitive method for identifying proteins that bind to tyrosine-phosphorylated growth factor receptors.
- To discover novel growth factor receptor-bound proteins (GRBs) involved in signaling pathways.
Main Methods:
- Utilized T7 polymerase-based expression libraries for enhanced cloning sensitivity.
- Screened libraries using tyrosine-phosphorylated epidermal growth factor receptor as a probe.
- Identified and characterized novel Src homology 2 domain-containing proteins.
Main Results:
- Successfully identified five different murine Src homology 2 domain-containing proteins (GRBs).
- Included known proteins like tyrosine kinase fyn and phospholipase C-gamma 1 homologue.
- Discovered GRB-7, a novel protein with SH2 and ras GTPase-activating protein similarity domains, highly expressed in liver and kidney.
Conclusions:
- The T7 polymerase-based expression/cloning system significantly improves the identification of SH2 domain-containing proteins.
- This enhanced system facilitates the discovery of downstream modulators of growth factor action.
- GRB-7 represents a newly identified protein with potential roles in growth factor signaling.