An investigation of homo and heterodimerization of the human delta opioid receptor

McVey, Mary (2001) An investigation of homo and heterodimerization of the human delta opioid receptor. PhD thesis, University of Glasgow.

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Abstract

Homo and heterodimerization of G-protein coupled receptors (GPCRs) is a concept which has gained credibility as another mechanism by which GPCR signalling can increase in complexity. The generation of Flag and c-myc N-terminally tagged forms of the human deltaOR, with and without GFP fused to the C-terminus, has allowed identification of each receptor form after transient transfection in HEK293 ceils. Stable cell lines expressing Flag-deltaOR or Flag-deltaOR-GFP were generated in HEK293cells. High affinity [3H]antagonist binding and agonist stimulated inhibition of adenylyl cyclase activity was observed. Agonist stimulated internalization of Flag-deltaOR-GFP was followed in live cells with a of 1/2< 10min. Constitutively formed (mouse) deltaOR homodimers were identified using co-immunprecipitation techniques (Cvejic and Devi 1997). The human deltaOR is demonstrated here to also form constitutive homodimers using co-immunoprecipitation techniques. Constitutive heterodimerization of human deltaOR with the deltaOR, IP prostanoid receptor, IP prostaniod receptor-GFP, beta1AR-GFP and beta2AR-GFP was also observed. Further investigations of the lysis procedures and antibodies used for immunoprecipitation indicated that the heterodimers were not a reflection of the experimental conditions used. Fluorescently labelled antibodies were used to specifically label N-terminally tagged GPCRs expressed at the cell-surface. The spectral overlap property of the fluorescent labels chosen allowed fluorescence resonance energy transfer (FRET) to be used to determine homo and heterodimerization of antibody-bound GPCRs. The fluorescent donor molecule, Europium, also has the property of long-lived fluorescence after excitation. Thus allowing the FRET to be time-resolved (TR-FRET) increasing the sensitivity of the developed assay. Constitutively formed cell-surface deltaOR homodimers were identified using TR- FRET. The presence of agonist was unable to modulate this interaction. Heterodimerization between the deltaOR and P2AR-GFP, which had been observed using co-immunoprecipitation techniques, was not evident using the cell-surface TR-FRET in intact cells. The presence of agonist for each receptor within the dimer or the presence of both agonists together did not generate any heterodimerization between the deltaOR and P2AR-GFP receptors. No significant level of heterodimers between the deltaOR and deltaOR was observed using TR-FRET. Optimisation of the TR-FRET assay has allowed the assay to be performed in a homogeneous format although this is less sensitive than the heterogeneous assay described.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Graeme Milligan
Keywords: Molecular biology, Biochemistry
Date of Award: 2001
Depositing User: Enlighten Team
Unique ID: glathesis:2001-72381
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 24 May 2019 15:12
Last Modified: 24 May 2019 15:12
URI: http://theses.gla.ac.uk/id/eprint/72381

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