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Department of Pharmacology

Carvalho, S

G protein-coupled receptors (GPCRs) possess an inherent ability to bind to an extremely diverse range of ligands and activate multiple downstream signalling pathways. It is becomingly increasingly evident, however, that for many GPCRs, the exact downstream signalling cascade(s), and the extent of activation, often varies in an agonist- and cell type-dependent manner. Receptor signalling can be further influenced by interacting proteins, such as receptor activity-modifying proteins (RAMPs), or internalisation, whereby distinct responses may be propagated by intracellular GPCR populations. Hence, the signalling repertoire subsequent to receptor activation is extremely complex, varying between individual GPCRs and the biological systems in which they are investigated. Here, work is presented investigating the signalling capabilities of the Adenosine A2A receptor (A2AR), the Calcitonin receptor-like receptor (CLR), the Calcitonin receptor (CTR), corticotrophin-releasing factor receptor type 2 (CRFR2) and the growth hormone-releasing hormone receptor (GHRHR). For the A2AR, agonist-mediated cAMP production and β-arrestin recruitment is studied, as well as receptor internalisation. In particular, the effects of small molecule inhibitors of internalisation are investigated with regards to cAMP production, attempting to decipher the importance of receptor internalisation in A2AR-mediated signalling. More specifically, the A2AR is demonstrated to signal in a sustained manner, following agonist stimulation, whilst revealing an inability to recruit β-arrestins or undergo agonist-induced internalisation. Furthermore, the study was expanded to include other Adenosine receptors: the A1R, A2BR and A3R, as well as the β2 adrenoceptor, to allow for comparison between different class A GPCRs. Work pertaining to the CLR begins with an investigation into interactions between a subset of class B GPCRs and RAMPs at the cell surface, with RAMP-receptor interactions revealed to be cell type-dependent in some instances, before subsequently verifying interactions via bioluminescent resonance energy transfer (BRET) microscopy. Subsequently, the effect(s) of RAMP1 mutations upon agonist-mediated signalling is investigated in terms of the abilities of mutant RAMP1-CLR heterodimers to mediate cAMP production and β-arrestin recruitment, in various cell types. Such work validates the effects of RAMP1 mutations against previously published reports, whilst laying a framework upon which RAMP-CLR dynamics will be investigated via single molecule total internal reflection fluorescence (TIRF) microscopy, as part of a collaborative effort. Indeed, RAMP1 W74K and W84A appear to affect CLR-mediated signalling in CHO-K1, COS-7 and HEK293 cell backgrounds, as well as influencing the diffusion pattern of CLR molecules. Finally, a characterisation of the effects of mutations within various class B1 GPCRs upon signalling is presented, as part of a collaborative project, quantifying the abilities of CTR, CRFR2 and GHRHR mutants to mediate cAMP production, intracellular Ca2+ mobilisation and β-arrestin recruitment, relative to the wild type cognate receptor. In particular, the majority of CRFR2 mutants revealed either a diminished or similar signalling capability, relative to their wild type counterpart, whereas GHRHR V225I had no impact upon agonist-mediated signalling but CTR A429S acquired the ability to recruit β-arrestin2.

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4 May 2022 (No publication date)
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Theses / dissertations
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University of Cambridge
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Ladds, G