Approximately 50 % of current pharmaceutical drugs target the G protein-coupled receptor (GPCR) family, rendering an understanding of the mechanisms behind their actions critical. Dopamine is a key neurotransmitter involved in motor function and behaviour and has been associated with disorders such as Parkinson's disease and schizophrenia. Within the present study, a Fluorescence Resonance Energy Transfer (FRET)-based conformational change sensor was created to study dopamine D2S receptor responses to ligands to gain an understanding of the mechanisms underlying ligand-induced activation. Chimaeric receptors were created with a tetracysteine sequence FlAsH binding motif at varying positions within intracellular loop 3 and CFP fused to the C-terrninal where FlAsH and CFP formed a convenient FRET pair. The constructs were transiently transfected into HEK293 cells and characterised using confocal and FRET microscopy. The 353-CFP-D2s receptor was well expressed at the cell surface and produced agonist-induced FRET changes and therefore was used to create a stably expressing HEK293 cell line. Radioligand binding and eSS]GTPyS binding assays showed that the 353-CFP-D2s receptor had a pharmacological profile similar to the native D2S receptor. On application of agonist (NP A, dopamine, m-tyramine, p- tyramine and (- )-3-PPP) to the 353-CFP-D2s receptor, a concentration dependent decrease in FRET was detected which was completely reversed when agonist was removed. The extent of FRET response was found to broadly correlate with the extent of G protein activation. The forward rate constant of the FRET change was also found to alter in an agonist-concentration dependent manner while the reverse rate was concentration independent. Throughout the project, the control and optimisation of experimental variables was found to be crucial for successful use of this FRET -based system. In conclusion, this sensor and the use of this technique could be very useful to aid understanding of activation of this important GPCR. 111.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:558734 |
| Date | January 2012 |
| Creators | Fotheringham, Helen L. |
| Publisher | University of Reading |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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