Thesis
P2Y1-P2Y12 receptor heterodimers : novel insights from microglial cell signalling and computational approaches
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2025
- Thesis identifier
- T17295
- Person Identifier (Local)
- 201788476
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- P2Y1 and P2Y12 receptors are class A G protein-coupled receptors and previous studies in the Kennedy lab suggested that they may physically interact to form functional heterodimers or possibly higher order oligomers. Little is known, however, about their properties. The aim of this project, therefore, was to characterise the pharmacological and signalling properties of the putative heterodimer and to investigate the possible molecular nature of the interaction. First, Ca2+ imaging was used as a bioassay to characterise the pharmacological properties of native P2Y receptors in BV-2 microglial cells. The P2Y1 and P2Y12 receptor agonist, adenosine-5'-diphosphate (ADP) and the selective P2Y1 receptor agonist, MRS2365, evoked concentration-dependent mobilisation of intracellular Ca2+ stores, which was abolished by the P2Y1 receptor antagonist, MRS2179. In contrast, P2Y12 receptor antagonists, including ARC69931MX and AZD1283, had no effect against MRS2365, but partially reduced the response to ADP. Canonically, P2Y1 receptors elicit Ca2+ mobilisation via the Gq/11 G proteins, whereas P2Y12 receptors couple to Gi/o. Pretreatment with the Gi/o inhibitor, pertussis toxin, had no effect on the action of MRS2365, but partially inhibited the response to ADP. The inhibitory action of MRS2179 against both agonists was unaffected by pertussis toxin, but that of AZD1283 against ADP was abolished. Thus, the Ca2+ mobilisation evoked by ADP in BV-2 cells involves both pertussis toxin-sensitive and -insensitive mechanisms. The nature of the physical interaction of the receptors was then investigated using the protein structure prediction tool, AlphaFold2. Initial benchmarking studies modelled monomeric β2-adrenoceptors, opioid and CB1 cannabinoid receptors accurately and confidently. Next, AlphaFold2-Multimer modelled monomeric β2-adrenoceptors bound to Gs, and dimeric GABAB receptors at the Venus flytrap lobes with high confidence, but it was less successful at modelling the GABAB receptor at the transmembrane regions and opioid receptor homo- and heterodimers. Finally, monomeric P2Y1 as well as monomeric and homodimeric P2Y12 receptors were modelled with high confidence, as were the interactions between the monomeric receptors and G proteins, but the P2Y1-P2Y12 receptor heterodimer was modelled with lower confidence, both in the absence and presence of G proteins. These data are consistent with my hypothesis that P2Y1-P2Y12 receptor heterodimers contribute to ADP-induced release of Ca2+ stores in BV-2 cells. They revealed that pertussis toxin-sensitive G proteins contribute to this response, whilst the modelling studies provided insight into the structure of monomeric and homodimeric P2Y1 and P2Y12 receptors and produced a potential model for the P2Y1-P2Y12 receptor heterodimer. Thus, these studies form a basis for future studies to investigate the physiological relevance of P2Y1-P2Y12 receptor heterodimers.
- Advisor / supervisor
- Cunningham, Margaret Rose
- Kennedy, Charles
- Resource Type
- DOI
- Date Created
- 2024
- Embargo Note
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