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Title: Studies of μ-opioid receptor internalisation
Author: Baptist, Myma Cynthia
ISNI:       0000 0004 2689 3898
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2009
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ELISAs were used to investigate the effects of a range of opioid ligands on the internalisation of the T7-tagged μ-opioid receptor (MOR) expressed in HEK 293 cells. The different opioid ligands were seen to vary in their abilities to induce MOR internalisation. The effect of the MOR agonists, DAMGO and morphine on MOR phosphorylation mutants (MOR-T180A and MOR-S363A) expressed in HEK 293 cells was also examined by ELISA. The ability of DAMGO to induce internalisation was significantly inhibited in the MOR-T180A in comparison to the wild type MOR. An N-terminal superecliptic pHluorin-tagged μ-opioid receptor (pHMOR) was generated to study MOR trafficking. Superecliptic pHluorin is a pH-sensitive variant of GFP which emits fluorescence at physiological extracellular pH (7.4) but not in acidic environments such as that occurring in intracellular organelles. In HEK 293 cells expressing pHMOR, fluorescence from pHluorin was only observed at the plasma membrane and this was abolished by lowering the extracellular pH to 6. Raising the intracellular pH with NH4Cl (50 mM) revealed fluorescence in intracellular compartments. The pHMOR construct was shown to be functional as indicated by the robust activation of GIRK channels as well as the rapid decrease in the level of cell surface pHMOR fluorescence following treatment with DAMGO. Total internal reflection fluorescence (TIRF) microscopy demonstrated that the surface expressed pHMORs were very dynamic and displayed characteristics which were not detected by confocal microscopy experiments such as receptor clustering following treatment with DAMGO or morphine. The agonist-activated receptors were also seen to colocalise with DsRed-tagged clathrin. The TIRF microscopy technique together with the pHluorin tagged MOR presents a very useful and promising approach to investigate dynamic changes in cell surface MOR expression in live cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available