Understanding the pharmacological consequence of functional P2Y1- P2Y12 heterodimerisation

  • Mohammed Adnan A Safar

Student thesis: Doctoral Thesis

Abstract

G protein-coupled receptors (GPCRs) are a superfamily of transmembrane proteins that are responsible for transducing extracellular stimuli into intracellular responses. GPCRs are essential to a wide variety of distinct pathophysiologies and behaviours and represent approximately 34% of FDA-approved, human drug targets. In recent years the classical concept that GPCRs are monomeric membrane receptors has been challenged by a growing number of reports indicating that they can form dimers and higher-order functional oligomers. Furthermore, this has been demonstrated to affect receptor trafficking, ligand sensitivity, desensitization, and downstream effector response. Thus, an understanding of GPCR oligomerisation is vital to understand receptor dynamics. The P2Y1 and P2Y12 receptors belong to the class A family of GPCRs and are widely expressed throughout the body. There is growing evidence that purinergic receptors also exist as oligomers. Previously, the Kennedy lab proposed the formation of a constitutive heterodimer between coexpressed human P2Y1 and P2Y12 receptors, based on functional responses. Evidence of a physical interaction between the two receptor subtypes was, however, lacking. The aim of this project was, therefore, to use a variety of experimental techniques to determine if P2Y1 and P2Y12 receptors interact physically to form dimers and how this affected receptor function. In this study, tSA201 cells were transfected with hP2Y1 and hP2Y12 receptors containing a haemagglutinin (HA) or fluorescent protein (FP) tag. Transfection efficiency was first optimised for all receptor constructs and experimental conditions. Fluorescence microscopy then showed that both receptors localised at the cell plasma membrane and in close proximity to each other. By quantification the receptors on the cell surface, it is found that P2Y1 receptors expressed more on the cell surface in compare to P2Y12 receptors. Interestingly, hP2Y12 receptor surface expression decreased when coexpressed with the hP2Y1 receptor. The hP2Y1 and hP2Y12 receptor agonist, ADP, induced internalisation of both receptors when they were expressed on their own, but not when they were coexpressed. Physical interaction between hP2Y1 and hP2Y12 receptors was investigated using co-immunoprecipitation (co-IP), proximity ligation assay (PLA) and fluorescence lifetime imaging microscopy FoĢˆrster resonance energy transfer (FLIM-FRET). These experiments demonstrated that P2Y1 and P2Y12 receptors formed a dimer that localised to the cell plasma membrane and the distance between both receptors decreases with AR-C69931MX addition. Moreover, in this study, the P2Y1-P2Y12 dimer was found natively in BV-2 microglial cells. Also, AR-C69931MX demolished the calcium induced by ADP in BV-2 cells, which is a similar finding noticed by Kennedy lab but in there combinant system. These findings reveal P2Y1 and P2Y12 receptor heterodimerisation with implications upon receptor internalisation and signalling in recombinant and endogenous receptor cell models. The next step requires further understanding of how these events might influence the pharmacology of both receptors and their function in normal physiology and disease.
Date of Award22 Apr 2021
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SupervisorCharles Kennedy (Supervisor) & Margaret Rose Cunningham (Supervisor)

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