Biochemical and structural studies of the interaction between ARNO and the Epidermal Growth Factor Receptor

Abstract

Receptor tyrosine kinases (RTKs) and small GTP binding proteins (GTPases) are essential regulators of multiple cellular processes, making a tight control of their activity crucial for cellular homeostasis. Recently, it was shown that members of a class of guanine nucleotide exchange factors (GEFs), the cytohesin family, influence not only their canonical target proteins (small GTPases) but also the signaling of RTKs. Most prominently, they increase epidermal growth factor receptor (EGFR) activity by directly interacting with its intracellular domain. This mechanism is of pathophysiological relevance as demonstrated by in vivo studies in animal models and analysis of human tumor samples. In this study, I applied a combination of biochemical assays to further characterize the interaction between cytohesins and the EGFR. <br /> Using chemical crosslinking studies supplemented with microscale thermophoresis and fluorescence polarization experiments, I provide evidence for the direct interaction of the Sec7 domain of cytohesin 2 (ARNO) with the juxtamembrane (JM) domain of EGFR. Furthermore, the binding site was found to involve the C-terminus of ARNO Sec7 and the N-terminal region of EGFR JM. Together with functional data investigating the nucleotide exchange activity of ARNO, these results suggest a model in which the JM domain contacts the hydrophobic grove formed by helix F, G and H of ARNO Sec7. This interaction provides possible explanations for the positive regulation of EGFR kinase activity by ARNO. <br /> The discovery of non-canonical functions of cytohesins raises concerns about the use of GEF inhibitors like SecinH3 as specific inhibitors for Arf GTPases. Therefore, a high throughput screening (HTS) assay was established to identify small molecules that disrupt the interaction of active Arf6 with its specific effector protein JIP4. Due to very similar binding sites for different effector proteins on Arf6, molecules found in this assay are likely to exhibit a general inhibitory potential on GTPase signaling. The assay is based on fluorescence polarization (FP) and accurately tracks complex formation between Arf6 and the leucine zipper II (LZII) domain of JIP4. Tolerance towards detergents and organic solvents and an excellent Z’ value observed throughout the screening process indicate that the assay is suitable for future HTS applications using small molecule libraries optimized towards inhibitors for protein-protein-interactions.