Growth Factor Receptor-Bound Protein 2

1. Prateek Agarwal Growth Factor Receptor-Bound Protein 2

2. Introduction • Mammalian growth factor receptor-binding protein, Grb2, is an adaptor that mediates activation of guanine nucleotide exchange on Ras signalling pathway. • Plays a significant role in the signal transduction pathway by binding to cell surface receptors through SH2 domain and two SH3 domains. • Gene expression takes place in nearly all embryonic and adult tissues. • Localization of the protein takes place mainly in the cytosol, but a few nuclear localizations have been found.

3. Structure • Viewed in 3.1 angstrom resolution • Embedded dimer makes up asymmetric unit of protein • Interlaced junctions between the SH2 and SH3 domains bring the two adjacent faces of the SH3 domains in van der Waals contact

4. SH3 Domain of Grb2 N • Detailed look at SH3 domain • Binds to cell surface receptors • N-terminal and C-terminal of domain labeled C

5. Primary Functions • Signaling of cell surface receptors that takes place in cytosol. • Endocytosis of receptors and regulation of the internalization of certain receptors. • Direct participant in the regulation of actin-based cell motility and actin filament formation.

6. Transduction Pathway • Growth factor binds to a receptor • GRB2 and SOS complex binds to the receptor bound to growth factor • SOS exchanges GDP for GTP • SOS associates with Ras protein • Kinases are phosphorylated by the complex • Transcription factors are then phosphorylated, which results in gene expression (detailed diagram of pathway shown on next slide)‏

8. Ras/MAP Kinase Pathway • Shc protein activates Ras/MAP kinase cascade • Binds to adaptor protein Grb2 • Grb2 binds through its SH3 domain to the guanine nucleotide-releasing factor SOS • Ras protein becomes activated through GDP/GTP exchange • GTP-bound Ras activates the so-called MAP kinase cascade • Transcription factors are phosphorylated, resulting in expression of proteins required for a given response (detailed diagram of pathway shown on next slide)‏

10. Different Signaling Pathways Downstream of Ras-GTP • Numerous cascades that can be signaled by Ras-GTP in Ras/MAP kinase pathway • Pathways include Raf-MEK-ERK kinase, phosphatidylinositol-3-kinase (PI3-kinase), RalGDS and phoshpholipase C (PLC) cascades • Expression of these pathways involves cell proliferation and differentiation, transcription, cell-cycle regulation, and many others (detailed diagram of different signaling pathways shown on next slide)

12. Involvement in Normal Embryogenesis • Grb2 is necessary for embryogenesis because the protein is required for the differentiation of endodermal cells and epiblast formation. • The protein is more necessary in the regulation of differentiation in the early embryo of the mouse.

13. Cardiac Hypertrophy • Abnormal condition where the heart becomes enlarged • Heart must work harder under stresses such as high blood pressure, or reduced blood flow through the coronary arteries • Excessive hypertrophy can kill more cells, which increases the stress on the heart, causing surviving cells to grow even larger, which in turn leads to an ever accelerating cycle that can eventually result in heart failure • Cardiac hypertrophy can also cause diseases such as myocardial infarction and arrhythmia

14. Involvement in Cardiac Hypertrophy • Mice have been used to investigate the activity of Grb2 in relation to cardiac hypertrophy • Findings have linked the necessity of Grb2 activity for the pressure overload response by cardiac hypertrophy and fibrosis • Pathways that the protein is involved in for the cell surface receptors are important in regulating fibrosis, fetal gene induction, and cardiomyocyte growth

15. Involvement with Cancer • Pivotal activator of cell-cycle control and motility pathways downstream of several growth factor receptors, GRB2 is involved in oncogenic signaling in a wide variety of human tumors. • GRB2 directly interacts with SOS-1 and the Bcr portion of the Bcr-Abl fusion protein, a tyrosine kinase oncoprotein which has been implicated in the pathogenesis of Philadelphia chromosome positive leukemias. • GRB2 over expression has been reported in human breast, bladder and prostate cancer cell lines. • In animals, the GRB2 has been inhibited by small molecules that block solid tumor metastasis.

16. Tyrosine Phosphorylation of Grb2 in Bcr/Abl-Expressing Cells

17. High-Affinity Binding • High-affinity binding of the growth factor receptor-bound protein 2 (Grb2) SH2 domain to tyrosine-phosphorylated cytosolic domains of receptor tyrosine kinases (RTKs) is an attractive target for therapeutic intervention in many types of cancer. • Two crystal forms of a complex between the Grb2 SH2 domain and a potent non-phosphorus containing macrocyclic peptide mimetic that exhibits significant antiproliferative effects against erbB-2-dependent breast cancers • The mode of binding and specific interactions between the protein and the inhibitor provide insight into the high potency of this class of macrocylic compounds and may aid in further optimization as part of the iterative rational drug design process.