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.
McVey
M
M
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland.
Ramsay
D
D
Kellett
E
E
Rees
S
S
Wilson
S
S
Pope
A J
AJ
Milligan
G
G
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