Spatio-temporal assessment of β-arrestin-centred “signalosomes” – the impact of effector binding on the functional selectivity of protease-activated receptor 2.

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Cells are the most fundamental entities of life. To communicate with their surroundings, cells possess a multitude of different receptors on their most outer lining, the plasma membrane. In the human body, cell surface receptors control most physiological processes and not only elicit responses to hormones and neurotransmitters, but also enable the sensing of odours and light. However, large parts of the exact molecular processes which unfold inside a cell after receptor activation are unknown. From recent data, we can assume that the area surrounding active receptors rapidly populates with enzymes, supporting adaptors and effector proteins to induce a cellular function. These signalling competent complexes are then internalised and transported throughout the cell. However, we do not know of the exact protein composition of these complexes, whether they are subject to change, depending on their subcellular localisation and which consequences this has for cellular signalling. Here, the project aims to investigate how cell surface receptors and their effector proteins cluster and behave after an initial activation, while monitoring the cellular signalling machinery throughout the entire trafficking process. Focusing on the protease-activated receptor 2 (PAR2), as an important mediator of pain in different bowel diseases, this study will examine fundamental mechanisms of cellular signalling and how this relates to pain perception. Taking advantage of the cutting-edge imaging facilities provided by the University of Nottingham and the Centre of Membrane Proteins and Receptors (COMPARE), it is possible to track what happens inside a cell after receptor activation. Innovative technologies, such as NanoBRET signalling probes and single-molecule fluorescence microscopy will be used to advance our current understanding of “how”, “when and where” and “why” receptors associate with other signalling partners after their activation. Moreover, the aim is to provide an intracellular signalling “map” with high spatial and kinetic resolution. The expected results will add to our general understanding of cellular signalling mechanisms, while possibly revealing new pharmacological targets for pain relief.