Protease-activated receptor 1 (PAR1) is a G protein coupled-receptor expressed on the surface of endothelial cells and platelets. It has a critical role in maintaining vascular homeostasis and its dysregulated activation has been proposed to contribute to a range of cardiovascular and inflammatory diseases. Unlike most receptors, PARs become activated not when bound by ligands, but when cleaved by specific proteases. Recent studies indicate PAR1 activation can occur via a number of different proteases, which can promote either pro-inflammatory, prohemostatic signalling activity or antiinflammatory, cell-protective effects, depending on the activating protease. Analogously, PAR1 activation in platelets can either potentiate platelet aggregation or aid thrombus consolidation.
Despite growing recognition of its physiological importance, the molecular basis for how PAR1 signalling ‘bias’ is achieved by different proteases remains poorly understood. An enhanced understanding of the molecular parameters that control signalling bias is important given the early promise of drugs that tilt PAR1 towards ‘cytoprotective’ signalling for the treatment of inflammatory vascular disease. The objective of this study is to define how different proteases confer distinct PAR1 cell signaling outputs. To achieve this, we will utilize state-of-the-art genome engineering approaches combined with advanced microscopy techniques to decipher the structural determinants required to mediate PAR1 signaling bias in different cell types. We will assess whether specific PAR1 molecular regions are differentially altered in response to activation with different proteases, using mutant recombinant versions of PAR1 in new assays of PAR1 signaling that have been developed in our lab. In addition, we will use fluorescence resonance energy transfer (FRET) live cell imaging and super-resolution microscopy to determine PAR1 interactions with other cell surface receptors that we hypothesize contribute to skewing of PAR1 signaling output. Finally, we propose to utilize genome-wide screening approaches to identify and subsequently characterize novel modifiers of PAR1 signaling outcomes. Collectively, the proposed study is anticipated to reveal novel insights as to how PAR1 structure, activation status and molecular interactions with co-receptors impact upon downstream signaling outcomes.
Ultimately, this will enable generation of new pharmacological strategies to facilitate preferential skewing of PAR1 signaling output for therapeutic benefit. The project will be performed under the supervision of Dr. Roger Preston and project co-supervisor Dr. Ingmar Schoen. The Preston lab (www.prestonlab.com) is a large multi-disciplinary research group that is currently funded by prestigious awards from Science Foundation Ireland, Bayer Healthcare and the National Children’s Research Centre. It has well-established expertise and an international reputation in the study of the mechanistic basis of PAR1 proteolysis and subsequent downstream signaling (Gleeson et al Blood 2015, Gleeson et al JTH 2017). The Schoen lab (http://schoenlab.strikingly.com
) is the only dedicated super-resolution microscopy lab in Ireland and has specific expertise in FRET imaging and super-resolution microscopy techniques (Li et al Nature Methods 2018, Früh et al Nature Communications 2015) with a research focus on platelet mechanobiology. Consequently, the PhD student would benefit from a multi-disciplinary approach, with expert tuition provided in molecular biology techniques, recombinant protein generation, CRISPR-Cas9 mediated genome modification, advanced microscopy and cell labeling techniques.