Biochemical and molecular characterisation of the proposed P2Y₁-P2Y₁₂ receptor heterodimer in tsA201 cells

  • Samuel Davis

Student thesis: Master's Thesis

Abstract

The P2Y receptor family represents a wide-ranging set of G-protein-coupled receptors that respond to purinergic ligands, and are involved in many physiological processes. From signalling assays in tsA201 cells, the Kennedy lab has previously proposed the formation of a constitutive heterodimer between co-expressed human P2Y₁ and P2Y₁₂ receptors; this could represent a target for the control of pain sensing neurones. Therefore, it was aimed in this project to characterise if the receptors physically interacted using coimmunoprecipitation,and also to investigate the endogenous expression of other potentially interacting hP2Y receptors in the tsA201 cell line. In this study, tsA201 cells were determined to express mRNA for hP2Y₁,₂,₄,₆,₁₂,₁₄ receptors using reverse transcriptase PCR and confirmatory Sanger sequencing. Furthermore, the cells demonstrated Ca2⁺ responses to all of the natural hP2Y receptor agonists tested, the order of potency being ADP > ATP > UTP > UDP. When co-expressing hP2Y₁ and hP2Y₁₂ receptors oppositely tagged with a HA or fluorescent protein (FP), immunoprecipitating for the HA tag and blotting for the FP showed a visible high molecular weight protein complex of ~130 to >250 KDa for either combination of receptors. This implied that the FP tagged receptor had co-immunoprecipitated with the other HA tagged receptor as this protein was also visible when blotting for the HA tag, signifying the presence of both tagged receptors in the complex.These results were overall suggestive of functional hP2Y₁,₂,₁₂ receptor expression in the tsA201 cell line, and possibly also hP2Y₄,₆,₁₄ receptors. The high molecular weight protein complex detected could represent a constitutive hP2Y₁ and hP2Y₁₂ receptor heterodimer as proposed. Whilst it was not possible to specifically either confirm or refute the physical formation of the heterodimer with the data acquired, it was potentially supportive, and furthermore suggested other future paths of investigation.
Date of Award28 Apr 2017
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SupervisorCharles Kennedy (Supervisor) & Margaret Rose Cunningham (Supervisor)

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