New mechanisms of receptor crosstalk Shannon Lillian O’Brien BBiomedSc A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2018 Faculty of Medicine School of Biomedical Sciences ii 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 iii partially dependent upon the EGFR tyrosine kinase activity. This Gq/11 independence was confirmed using a G protein-uncoupled AT1R mutant that demonstrated transactivation comparable to that seen in the AT1R wild type. Finally, we provide evidence that both AngII- and EGF-stimulation promotes a physical association between AT1R-EGFR, however the molecular requirements vary for each receptor. In addition, I report in this thesis the concept of ‘reverse-transactivation’, whereby a significant proportion of the total EGF signalling is dependent upon the co-activation of the AT1R and its capacity to couple to G proteins. A BRET-based assay was used, both in transfected HEK293 cells and primary isolated vascular smooth muscle cells (VSMC), to demonstrate that EGF-stimulation of the EGFR leads to the activation and arrestin-binding of the AT1R. This EGF-mediated recruitment of -arrestin to the AT1R was completely dependent upon EGFR tyrosine kinase activation and apparently involves the active, Gq coupled state of the AT1R. Moreover, a truncated AT1R, lacking all carboxyl-terminal phosphorylation sites, was incapable of recruiting arrestin following EGF-stimulation, indicating reverse transactivation requires phosphorylation and binding of arrestin to the AT1R. The rapid internalisation of the AT1R following AngII-stimulation was not observed following EGF activation, instead causing increased recruitment of AT1R to the plasma membrane. In summary, I have developed and applied a sensor BRET based-assays to characterise the molecular, temporal and spatial aspects of AT1R-EGFR transactivation. We provide evidence of bi-directional receptor crosstalk, whereby both the AT1R and EGFR have the capacity to transactivate each other, however the molecular mechanisms governing this crosstalk vary for each receptor. Collectively, this powerful new platform, which interrogates the most proximal events of AT1R-EGFR transactivation, can be used to better define AngII influences on cardiovascular physiology and pathology, and therapeutically lead to the development of new drugs against cardiovascular disease. iv Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. 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 co- authors for any jointly authored works included in the thesis. v Publications included in this thesis No publications included Submitted manuscripts included in this thesis O’Brien, S.L., Johnstone E.K., Devost, D., Conroy, J., Reichelt, M.E., Purdue, B.W., Ayoub, M.A., Kawai, T., Inoue, A., Eguchi, S., Hébert, T.E., Pfleger, K.D.G., Thomas, W.G (2018). BRET-Based assay to monitor EGFR transactivation by the AT1R reveals Gq/11 protein-independent activation and AT1R-EGFR complexes. Biochemical Pharmacology [Under Review]. (Incorporated into Chapter 4.0) Impact Factor: 5.009 Other publications during candidature Peer-reviewed paper Kawai, T., Forrester, S. J., O’Brien, S., Baggett, A., Rizzo, V., & Eguchi, S. (2017). AT1 receptor signaling pathways in the cardiovascular system. Pharmacological Research, 125, 4-13. Impact Factor: 4.408 Ranking (Pharmacological Journals): 30/326 Peer-reviewed paper Reichelt, M. E., O’Brien, S., Thomas, W. G., & Headrick, J. P. (2016). Transactivation of the epidermal growth factor receptor in responses to myocardial stress and cardioprotection. The international Journal of Biochemistry & Cell Biology, 83, 97- 110. Impact Factor: 4.240 Ranking (Cell Biology Journals): 74/273 Peer-reviewed paper Forrester, S. J., Kawai, T., O'Brien, S., Thomas, W., Harris, R. C., & Eguchi, S. (2016). Epidermal growth factor receptor transactivation: mechanisms, pathophysiology, and vi potential therapies in the cardiovascular system. Annual review of pharmacology and toxicology, 56, 627-653 Impact Factor: 12.877 Ranking (Pharmacological Journals): 2/326 International presentations arising from thesis: O’Brien, S.L., Pfleger, K.D., Thomas, W.G. (2018) New mechanisms of AT1R-EGFR cross-talk. University of Otago Postgraduate symposium. Invited presentation. O’Brien, S.L., Pfleger, K.D., Thomas, W.G. (2018) Hijacking the EGFR by the type th 1 angiotensin receptor: Demonstration of AT1R-EGFR complexes in living cells. 7 Focused Meeting on Cell Signalling, Nottingham, United Kingdom. O’Brien, S.L., Purdue, B.W., Pfleger, K.D., Thomas, W.G. (2017) Development of a BRET-based assay for AT1R-EGR transactivation: Evidence for functional Heteromers. Australian society of Clinical and Experimental Pharmacologists and Toxicologists, Brisbane, Australia. Poster-433 Awarded best poster O’Brien, S.L., Purdue, B.W., Pfleger, K.D., Thomas, W.G. (2016) Use of BRET to demonstrate AT1R-EGFR complexes and AT1R-mediated EGFR transactivation. Australian society of Clinical and Experimental Pharmacologists and Toxicologists, Melbourne, Australia. Poster-694 National presentation arising from thesis: O’Brien, S.L., Purdue, B.W., Pfleger, K.D., Thomas, W.G. (2017) New mechanisms of AT1R-EGFR cross-talk. International Postgraduate Symposium in Biomedical Sciences. Oral. Awarded best oral O’Brien, S.L., Purdue, B.W., Pfleger, K.D., Thomas, W.G. (2017) Hijacking of the EGFR by the type 1 angiotensin receptor: Demonstration of AT1R-EGFR complexes in living cells. Cell and Developmental Biology