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Title: Elucidation of activation mechanisms of adhesion GPCRs
Author: Holdich, Rebecca Fern
ISNI:       0000 0004 7655 0260
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2018
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The sensing of mechanical forces by cells is an essential process in physiological systems. If perturbed, it can result in various pathologies including cancer, vascular disease, and deafness. Despite its obvious importance, only a limited number of cell receptors are currently implicated in mediating responses to mechanical stimuli. One group of receptors routinely exposed to physical forces within their microenvironments, including the circulation, inner ear, and epididymis, and with great potential in mechanosensing, are the adhesion GPCR family. Adhesion GPCRs comprise 33 human transmembrane signalling receptors with particularly large extracellular domains, composed of several protein repeats known to facilitate adhesive interactions. However, despite their involvement in key physiological and pathological processes the exact mechanisms of signal transduction for these receptors remains unspecified. The work undertaken for this research therefore aimed to provide mechanistic insights into the signalling of adhesion GPCRs using the following experimental approaches: 1) elucidation of the atomic structure of a crucial signalling domain, 2) characterisation of signal induction mechanisms and intracellular signalling pathways and 3) interrogation of the mechanical sensing properties of the extracellular domain. In summary, the results of this work have generated a 2.2Å resolution native X-ray diffraction dataset of the extracellular GAIN domain of the adhesion GPCR, CD97. In addition, using luciferase reporter assays this research has also generated robust readouts for the dissection of adhesion GPCR signalling. These readouts have for the first time established Gα12/13 as a major signalling pathway for GPR97, shown vibration-induced signalling of GPR56, and have demonstrated that small protein scaffolds known as Affimers can be used as specific modulators of adhesion GPCR receptor signalling. Finally, atomic force microscopy was utilised to dissect the force required to induce the removal of the extracellular domains of adhesion-GPCR, a key potential mechanism involved in mechanosensing. Together, these results provide structural information essential for the future atomic resolution of CD97 GAIN domain, demonstrate that members of the adhesion GPCR family are indeed mechanosensors, and that modulation of receptor signalling via Affimers can be a useful potential strategy for future mechanistic and pharmacological studies.
Supervisor: Stacey, Martin ; Trinh, Chi Sponsor: BBSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available