Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer. The human delta -opioid receptor displays constitutive oligomerization at the cell surface, which is not regulated by receptor occupancy.
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.
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland.
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McVey
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Ramsay
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Kellett
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Rees
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Wilson
A J
AJ
Pope
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Milligan
D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000737:chemistry; D004745:Enkephalins / Q000737:chemistry; D000939:Epitopes; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D008163:Luminescent Measurements; D009292:Narcotic Antagonists / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D017465:Receptors, Opioid, delta / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014162:Transfection
D004745:Enkephalins; D000939:Epitopes; D008024:Ligands; D009292:Narcotic Antagonists; D018343:Receptors, Adrenergic, beta-2; D017465:Receptors, Opioid, delta; D004743:Enkephalin, Leucine; C040643:N,N-diallyl-tyrosyl-alpha-aminoisobutyric acid-phenylalanyl-leucine