Abstract
Oligomerization of the human delta-opioid receptor and its regulation by ligand occupancy were explored following expression in HEK293 cells using each of co-immunoprecipitation of differentially epitope-tagged forms of the receptor, bioluminescence resonance energy transfer and time-resolved fluorescence resonance energy transfer. All of the approaches identified constitutively formed receptor oligomers, and the time-resolved fluorescence studies confirmed the presence of such homo-oligomers at the cell surface. Neither the agonist ligand [d-Ala(2),d-Leu(5)]enkephalin nor the inverse agonist ligand ICI174864 were able to modulate the oligomerization status of this receptor. Interactions between co-expressed delta-opioid receptors and beta(2)-adrenoreceptors were observed in co-immunoprecipitation studies. Such hetero-oligomers could also be detected using bioluminescence resonance energy transfer although the signal obtained was substantially smaller than for homo-oligomers of either receptor type. Signal corresponding to the delta-opioid receptor-beta(2)-adrenoreceptor hetero-oligomer was increased in the presence of agonist for either receptor. However, substantial levels of this hetero-oligomer were not detected at the cell surface using time-resolved fluorescence resonance energy transfer. These studies demonstrate that, following transient transfection of HEK293 cells, constitutively formed oligomers of the human delta-opioid receptor can be detected by a variety of approaches. However, these are not regulated by ligand occupancy. They also indicate that time-resolved fluorescence resonance energy transfer represents a means to detect such oligomers at the cell surface in populations of intact cells.
Highlights
Oligomerization of the human ␦-opioid receptor and its regulation by ligand occupancy were explored following expression in HEK293 cells using each of co-immunoprecipitation of differentially epitope-tagged forms of the receptor, bioluminescence resonance energy transfer and time-resolved fluorescence resonance energy transfer
We demonstrate that such constitutive oligomerization can be observed for this G protein-coupled receptors (GPCRs) using each of the three approaches but that ligands do not regulate these interactions appreciably
It is clear that many GPCRs have the capacity to oligomerize [1,2, 15]
Summary
All materials for tissue culture were supplied by Life Technologies Inc (Paisley, United Kingdom). [3H]DADLE (55.3 Ci/mmol) was purchased from PerkinElmer Life Sciences. [3H]Dihydroalprenolol (64 Ci/ mmol), [3H]diprenorphine (66 Ci/mmol), [3H]adenine, and [3H]cAMP were from Amersham Pharmacia Biotech. All samples were subjected to fluorescenceactivated cell sorting analysis to confirm the presence of eYFP in cells transfected with acceptor-tagged receptors. A 2-h incubation was performed at room temperature with 500,000 cells in a total volume of 100 l containing 15 nM Eu3ϩ -labeled anti-c-Myc antibody (Wallac) and 45 nM APC-labeled M5 anti-FLAGTM antibody (in house) and 50% newborn calf serum/PBS. After incubation the cells were washed twice with PBS and resuspended in 30 l of PBS before placing into wells of a 384-well microtiter plate for FRET analysis using a Victor (Wallac) configured for time-resolved fluorescence. [3H]Ligand Binding Studies (BRET)—As the BRET experiments cannot distinguish between receptors present at the cell surface and in intracellular membranes, membrane preparations were used to obtain total cell expression levels. The generation of [3H]cAMP in response to treatment of the cells with various ligands and other reagents was assessed
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