New mechanisms of receptor crosstalk

Abstract

Dysregulation of the Renin Angiotensin System (RAS), and its cognate receptor, the angiotensin (AngII) type 1 receptor (AT1R), has been implicated in the pathogenesis of several cardiovascular disorders including hypertension, fibrosis, congestive heart failure and stroke. Despite the development of pharmacological agents that target the RAS, the persistent burden of cardiovascular disease remains. My PhD focuses on the AT1R, specifically its ability to 'crosstalk' and transactivate signalling pathways downstream of the Epidermal Growth Factor Receptors (EGFR). This paradigm of EGFR transactivation thereby enables the AT1R to potentially regulate cell growth, differentiation, apoptosis and promote cancerous cell growth. Part of ongoing work in the Thomas laboratory is to better understand the roll of previously identified novel proteins in AT1R-EGFR transactivation, including CHKA (Choline Kinase Alpha), BMX (non-receptor tyrosine kinase) and TRIO (Triple Functional Domain PTPRF Interacting). It is therefore the focus of my research to use molecular and cellular approaches to interrogate the mechanistic basis for EGFR transactivation and better define the relative role of these novel proteins in AT1R-EGFR signalling. Although it is of great interest to identify and characterise the molecular, temporal and spatial mechanisms of AT1R-EGFR transactivation, demonstrating EGFR transactivation directly, in live cells, and in real-time has been challenging. Typically, end-point assays (e.g., phospho-ERK1/2) are used as a surrogate readout of EGFR activation, however they are often downstream readouts and are not applicable to live cells. Herein, I report the use of a Bioluminescence Resonance Energy Transfer (BRET) based assay to report the recruitment of the EGFR adaptor protein, growth factor receptor-bound protein 2 (Grb2), association with the EGFR. In live HEK293 cells, both epidermal growth factor (EGF) and AngII stimulation produced sustained ligand-dependent recruitment of Grb2 to the EGFR. The BRET assay was applied to a variety of cell lines and also used to screen a panel of 19 GPCRs, with the AT1R and vasopressin receptor showing distinct EGFR transactivation. Additionally, HER2, a member of the EGFR family (also known as ErbB2) and preferred dimerisation partner of the EGFR, was also transactivated by the AT1R. Mechanistically, we observed that the AT1R-mediated ERK1/2 activation was completely dependent on Gq/11 as well as EGFR tyrosine kinase activity, whereas EGFR-Grb2 recruitment was independent of Gq/11 and only I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from coauthors for any jointly authored works included in the thesis.