Thesis
Investigation of P2Y₁ and P2Y₁₂ receptor heterodimerisation
- Creator
- Awarding institution
- University of Strathclyde
- Date of award
- 2014
- Thesis identifier
- T13789
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- Heterodimerisation of G-Protein Coupled Receptors (GPCR) is an emerging research niche with implications for understanding signalling, pathophysiology and development of new drug targets. This research investigates P2Y₁ and P2Y₁₂ receptor heterodimerisation. Individually, these class-A GPCR are stimulated by adenine and/or uridine nucleotides in a range of tissue. They are highly expressed in platelets and dorsal root ganglion (DRG) sensory neurons, giving rise to a host of physiological functions such as platelet degranulation and nociception. A previous study in the laboratory suggested that when recombinant hP2Y₁₂ receptors are expressed in tsA201 cells they interact with the native P2Y₁ receptors to form functional heterodimers with novel pharmacological and signalling properties (Shakya-Shrestha, et al., 2010). A disadvantage of this study was that it was low throughput and only a single agonist concentration was used in patch-clamp electrophysiology recordings from single cells. The present study used Ca²⁺ imaging in a cell population to produce high throughput, multiple agonist concentration analysis. Part of the challenge to this research was to identify how P2Y₁ receptor (Gαq/11 coupled) and P2Y₁₂ receptor (Gαi/o coupled) signalling was affected by co-expression in tsA201 bioassays. By using the selective P2Y₁ receptor antagonist, MRS2179, our data demonstrates that the P2Y₁ receptor is the dominant endogenously-expressed purinergic receptor in tsA201 cells. We then showed that the selective P2Y₁₂ receptor antagonist, AR-C69931MX, significantly inhibited Ca²⁺ signalling in hP2Y₁₂-transfected, but not wild-type tsA201. Lastly, we transfected both hP2Y₁ and hP2Y₁₂ receptors into tsA201 cells (overexpressing hP2Y₁) and identified a reversal in the inhibitory actions of AR-C69931MX, compared to the hP2Y₁₂ receptor expressed bioassay. The change in pharmacology observed is consistent with P2Y₁ and P2Y₁₂ receptor heterodimerisation.
- Resource Type
- DOI
- Date Created
- 2014
- Former identifier
- 1036541
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