Advancing our understanding of the EGFR-KRas-mediated activation of small G proteins RalA/B

University of Leicester • GB • 1w ago

Growth factor signalling is fundamental to cell proliferation and differentiation. Epidermal growth factor (EGF) signalling exemplifies this process by activating the small GTPase KRas at the plasma membrane (PM), where KRas serves as a central signalling hub that activates multiple effectors, including BRAF and Rgl2. The most extensively investigated KRas effector is BRAF that initiates a three-tiered kinase cascade (BRAF-MEK-ERK), which has been characterised in detail, including through cryo-electron microscopy (EM) structures of the KRas-BRAF-MEK complex. Another KRas effector, Rgl2, a guanine nucleotide exchange factor (GEF), activates the small GTPases RalA and RalB and is critical for membrane trafficking and cell motility. However, the mechanism by which KRas mediates Rgl2 activation remains poorly understood.

Rgl2 is a multidomain protein containing the Ras-associating (RA) domain and a domain responsible for its GEF activity, as well as intrinsically disordered regions (IDRs). We hypothesise that KRas binds the Rgl2-RA domain, causing a conformational change in the GEF domain and activating Rgl2. Furthermore, we predict that the IDRs interact with the scaffold protein, Grb2, to help Rgl2 cluster at the activated EGF receptor (EGFR), facilitating KRas-mediated activation. We will test these hypotheses through structural analyses of Rgl2 alone and the KRas-Rgl2-Grb2-RalA/B complex using cryo-EM, cross-link mass spectrometry, and small-angle X-ray scattering. Protein-protein interactions will be examined using biolayer interferometry, ITC and NMR. In parallel, we will examine the molecular clustering of these molecules in human cultured cells engineered to express a Halo-tag on Rgl2 for visualisation, using super-resolution microscopy. Finally, we will reconstitute the EGFR-KRAS-Rgl2-Grb2-RalA/B complex in lipid bilayer systems and examine clustering events using single-molecule optical imaging to obtain a comprehensive view of this signalling axis.

Techniques that will be undertaken during the project

• Recombinant protein purification using bacterial and insect cell expression systems

• Biolayer interferometry

• Guanine-nucleotide exchange assays

• Cryo-electron microscopy

• Cross-link mass spectrometry

• Small-angle X-ray scattering

• Nuclear magnetic resonance (NMR) analysis

• Lipid bilayer preparation

• CRISPR-Cas9-based gene editing of human cultured cells

• Fluorescence super-resolution microscopy and image data analysis

• Single-molecule microscopy and image data analysis