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Graeme Milligan - One of the best experts on this subject based on the ideXlab platform.
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monitoring receptor oligomerization using time resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer the human δ opioid receptor displays constitutive oligomerization at the cell surface which is not regulated
Journal of Biological Chemistry, 2001Co-Authors: Mary Mcvey, Douglas Ramsay, Elaine Kellett, Stephen Rees, Shelagh Wilson, Andrew J Pope, Graeme MilliganAbstract:Abstract Oligomerization of the human δ-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-Ala2,d-Leu5]enkephalin nor the inverse agonist ligand ICI174864 were able to modulate the oligomerization status of this receptor. Interactions between co-expressed δ-opioid receptors and β2-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 δ-opioid receptor-β2-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 δ-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.
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monitoring receptor oligomerization using time resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer the human δ opioid receptor displays constitutive oligomerization at the cell surface which is not regulated
Journal of Biological Chemistry, 2001Co-Authors: Mary Mcvey, Douglas Ramsay, Elaine Kellett, Stephen Rees, Shelagh Wilson, Andrew J Pope, Graeme MilliganAbstract: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.
David B Teplow - One of the best experts on this subject based on the ideXlab platform.
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preparation of pure populations of covalently stabilized amyloid β protein Oligomers of specific sizes
Analytical Biochemistry, 2017Co-Authors: Eric Y Hayden, Joseph L Conovaloff, Ashley Mason, Gal Bitan, David B TeplowAbstract:Evidence suggests that amyloid β-protein (Aβ) Oligomers may be seminal pathogenic agents in Alzheimer's disease (AD). If so, developing oligomer-targeted therapeutics requires an understanding of oligomer structure. This has been difficult due to the instability of these non-covalently associated Aβ assemblies. We previously used rapid, zero-length, in situ chemical cross-linking to stabilize Oligomers of Aβ40. These enabled us to isolate pure, stable populations of dimers, trimers, and tetramers and to determine their structure-activity relationships. However, equivalent methods applied to Aβ42 did not produce stable Oligomers. We report here that the use of an Aβ42 homologue, [F10, Y42]Aβ42, coupled with sequential denaturation/dissociation and gel electrophoresis procedures, provides the means to produce highly pure, stable populations of Oligomers of sizes ranging from dimer through dodecamer that are suitable for structure-activity relationship determination.
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effects of the english h6r and tottori d7n familial alzheimer disease mutations on amyloid β protein assembly and toxicity
Journal of Biological Chemistry, 2010Co-Authors: Kenjiro Ono, Margaret M Condron, David B TeplowAbstract:Mutations in the amyloid beta-protein (Abeta) precursor gene cause autosomal dominant Alzheimer disease in a number of kindreds. In two such kindreds, the English and the Tottori, the mutations produce amyloid beta-proteins containing amino acid substitutions, H6R and D7N, respectively, at the peptide N terminus. To elucidate the structural and biological effects of the mutations, we began by examining monomer conformational dynamics and oligomerization. Relative to their wild type homologues, and in both the Abeta40 and Abeta42 systems, the English and Tottori substitutions accelerated the kinetics of secondary structure change from statistical coil --> alpha/beta --> beta and produced oligomer size distributions skewed to higher order. This skewing was reflected in increases in average oligomer size, as measured using electron microscopy and atomic force microscopy. Stabilization of peptide Oligomers using in situ chemical cross-linking allowed detailed study of their properties. Each substitution produced an oligomer that displayed substantial beta-strand (H6R) or alpha/beta (D7N) structure, in contrast to the predominately statistical coil structure of wild type Abeta Oligomers. Mutant Oligomers functioned as fibril seeds, and with efficiencies significantly higher than those of their wild type homologues. Importantly, the mutant forms of both native and chemically stabilized Oligomers were significantly more toxic in assays of cell physiology and death. The results show that the English and Tottori mutations alter Abeta assembly at its earliest stages, monomer folding and oligomerization, and produce Oligomers that are more toxic to cultured neuronal cells than are wild type Oligomers.
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effects of the english h6r and tottori d7n familial alzheimer disease mutations on amyloid β protein assembly and toxicity
Journal of Biological Chemistry, 2010Co-Authors: Kenjiro Ono, Margaret M Condron, David B TeplowAbstract:Mutations in the amyloid β-protein (Aβ) precursor gene cause autosomal dominant Alzheimer disease in a number of kindreds. In two such kindreds, the English and the Tottori, the mutations produce amyloid β-proteins containing amino acid substitutions, H6R and D7N, respectively, at the peptide N terminus. To elucidate the structural and biological effects of the mutations, we began by examining monomer conformational dynamics and oligomerization. Relative to their wild type homologues, and in both the Aβ40 and Aβ42 systems, the English and Tottori substitutions accelerated the kinetics of secondary structure change from statistical coil → α/β → β and produced oligomer size distributions skewed to higher order. This skewing was reflected in increases in average oligomer size, as measured using electron microscopy and atomic force microscopy. Stabilization of peptide Oligomers using in situ chemical cross-linking allowed detailed study of their properties. Each substitution produced an oligomer that displayed substantial β-strand (H6R) or α/β (D7N) structure, in contrast to the predominately statistical coil structure of wild type Aβ Oligomers. Mutant Oligomers functioned as fibril seeds, and with efficiencies significantly higher than those of their wild type homologues. Importantly, the mutant forms of both native and chemically stabilized Oligomers were significantly more toxic in assays of cell physiology and death. The results show that the English and Tottori mutations alter Aβ assembly at its earliest stages, monomer folding and oligomerization, and produce Oligomers that are more toxic to cultured neuronal cells than are wild type Oligomers.
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elucidation of amyloid β protein oligomerization mechanisms discrete molecular dynamics study
Journal of the American Chemical Society, 2010Co-Authors: Brigita Urbanc, Gal Bitan, M Betnel, Luis Cruz, David B TeplowAbstract:Oligomers of amyloid beta-protein (Abeta) play a central role in the pathology of Alzheimer's disease. Of the two predominant Abeta alloforms, Abeta(1-40) and Abeta(1-42), Abeta(1-42) is more strongly implicated in the disease. We elucidated the structural characteristics of Oligomers of Abeta(1-40) and Abeta(1-42) and their Arctic mutants, [E22G]Abeta(1-40) and [E22G]Abeta(1-42). We simulated oligomer formation using discrete molecular dynamics (DMD) with a four-bead protein model, backbone hydrogen bonding, and residue-specific interactions due to effective hydropathy and charge. For all four peptides under study, we derived the characteristic oligomer size distributions that were in agreement with prior experimental findings. Unlike Abeta(1-40), Abeta(1-42) had a high propensity to form paranuclei (pentameric or hexameric) structures that could self-associate into higher-order Oligomers. Neither of the Arctic mutants formed higher-order Oligomers, but [E22G]Abeta(1-40) formed paranuclei with a similar propensity to that of Abeta(1-42). Whereas the best agreement with the experimental data was obtained when the charged residues were modeled as solely hydrophilic, further assembly from spherical Oligomers into elongated protofibrils was induced by nonzero electrostatic interactions among the charged residues. Structural analysis revealed that the C-terminal region played a dominant role in Abeta(1-42) oligomer formation whereas Abeta(1-40) oligomerization was primarily driven by intermolecular interactions among the central hydrophobic regions. The N-terminal region A2-F4 played a prominent role in Abeta(1-40) oligomerization but did not contribute to the oligomerization of Abeta(1-42) or the Arctic mutants. The oligomer structure of both Arctic peptides resembled Abeta(1-42) more than Abeta(1-40), consistent with their potentially more toxic nature.
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elucidation of amyloid β protein oligomerization mechanisms discrete molecular dynamics study
Journal of the American Chemical Society, 2010Co-Authors: Brigita Urbanc, Gal Bitan, M Betnel, Luis Cruz, David B TeplowAbstract:Oligomers of amyloid β-protein (Aβ) play a central role in the pathology of Alzheimer’s disease. Of the two predominant Aβ alloforms, Aβ1−40 and Aβ1−42, Aβ1−42 is more strongly implicated in the disease. We elucidated the structural characteristics of Oligomers of Aβ1−40 and Aβ1−42 and their Arctic mutants, [E22G]Aβ1−40 and [E22G]Aβ1−42. We simulated oligomer formation using discrete molecular dynamics (DMD) with a four-bead protein model, backbone hydrogen bonding, and residue-specific interactions due to effective hydropathy and charge. For all four peptides under study, we derived the characteristic oligomer size distributions that were in agreement with prior experimental findings. Unlike Aβ1−40, Aβ1−42 had a high propensity to form paranuclei (pentameric or hexameric) structures that could self-associate into higher-order Oligomers. Neither of the Arctic mutants formed higher-order Oligomers, but [E22G]Aβ1−40 formed paranuclei with a similar propensity to that of Aβ1−42. Whereas the best agreement wi...
Mary Mcvey - One of the best experts on this subject based on the ideXlab platform.
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monitoring receptor oligomerization using time resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer the human δ opioid receptor displays constitutive oligomerization at the cell surface which is not regulated
Journal of Biological Chemistry, 2001Co-Authors: Mary Mcvey, Douglas Ramsay, Elaine Kellett, Stephen Rees, Shelagh Wilson, Andrew J Pope, Graeme MilliganAbstract:Abstract Oligomerization of the human δ-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-Ala2,d-Leu5]enkephalin nor the inverse agonist ligand ICI174864 were able to modulate the oligomerization status of this receptor. Interactions between co-expressed δ-opioid receptors and β2-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 δ-opioid receptor-β2-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 δ-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.
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monitoring receptor oligomerization using time resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer the human δ opioid receptor displays constitutive oligomerization at the cell surface which is not regulated
Journal of Biological Chemistry, 2001Co-Authors: Mary Mcvey, Douglas Ramsay, Elaine Kellett, Stephen Rees, Shelagh Wilson, Andrew J Pope, Graeme MilliganAbstract: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.
Stephen M Strittmatter - One of the best experts on this subject based on the ideXlab platform.
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amyloid β induced signaling by cellular prion protein and fyn kinase in alzheimer disease
Prion, 2013Co-Authors: Stephen M StrittmatterAbstract:Alzheimer disease (AD) is the most prevalent cause of dementia. Amyloid-β (Aβ) Oligomers are potent synaptotoxins thought to mediate AD-related phenotypes. Cellular prion protein (PrPC) has been identified as a high-affinity receptor for Aβ Oligomers. Herein, we review the functional consequences of Aβ oligomer binding to PrPC on the neuronal surface. We highlight recent evidence that Fyn kinase mediates signal transduction downstream of the PrPC-Aβ oligomer complex. These studies suggest that PrPC has a central role in AD pathogenesis and may provide a target for therapeutic intervention in AD.
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cellular prion protein mediates impairment of synaptic plasticity by amyloid β Oligomers
Nature, 2009Co-Authors: Juha Lauren, David A Gimbel, Haakon B Nygaard, John Gilbert, Stephen M StrittmatterAbstract:A pathological hallmark of Alzheimer's disease is an accumulation of insoluble plaque containing the amyloid-beta peptide of 40-42 amino acid residues. Prefibrillar, soluble Oligomers of amyloid-beta have been recognized to be early and key intermediates in Alzheimer's-disease-related synaptic dysfunction. At nanomolar concentrations, soluble amyloid-beta Oligomers block hippocampal long-term potentiation, cause dendritic spine retraction from pyramidal cells and impair rodent spatial memory. Soluble amyloid-beta Oligomers have been prepared from chemical syntheses, transfected cell culture supernatants, transgenic mouse brain and human Alzheimer's disease brain. Together, these data imply a high-affinity cell-surface receptor for soluble amyloid-beta Oligomers on neurons-one that is central to the pathophysiological process in Alzheimer's disease. Here we identify the cellular prion protein (PrP(C)) as an amyloid-beta-oligomer receptor by expression cloning. Amyloid-beta Oligomers bind with nanomolar affinity to PrP(C), but the interaction does not require the infectious PrP(Sc) conformation. Synaptic responsiveness in hippocampal slices from young adult PrP null mice is normal, but the amyloid-beta oligomer blockade of long-term potentiation is absent. Anti-PrP antibodies prevent amyloid-beta-oligomer binding to PrP(C) and rescue synaptic plasticity in hippocampal slices from oligomeric amyloid-beta. Thus, PrP(C) is a mediator of amyloid-beta-oligomer-induced synaptic dysfunction, and PrP(C)-specific pharmaceuticals may have therapeutic potential for Alzheimer's disease.
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cellular prion protein mediates impairment of synaptic plasticity by amyloid β Oligomers
Nature, 2009Co-Authors: Juha Lauren, David A Gimbel, Haakon B Nygaard, John Gilbert, Stephen M StrittmatterAbstract:The hypothesis that soluble amyloid-β peptide oligomer plays a central role in Alzheimer's disease is well established, yet no mechanistic basis for Aβ oligomer effects on neurons has been described. Several lines of evidence point to the existence of a high-affinity cell-surface receptor for soluble Aβ Oligomers on neurons as central to Alzheimer's disease pathology and now cellular prion protein PrPC has been identified as a candidate for that role. PrP, a plasma membrane glycoprotein associated with lipid rafts, binds Aβ Oligomers selectively with high affinity and mediates the deleterious effects of the peptide. These data raise the possibility that PrPC-specific drugs might have therapeutic potential in Alzheimer's, and point to an unexpected link between infectious prion diseases and Alzheimer's disease. This paper shows that the effects of amyloid-beta Oligomers on long-term potentiation are mediated by the cellular prion protein — notorious for its involvement in diseases such as bovine spongiform encephalopathy, new variant Creutzfeldt–Jakob disease and kuru. A pathological hallmark of Alzheimer’s disease is an accumulation of insoluble plaque containing the amyloid-β peptide of 40–42 amino acid residues1. Prefibrillar, soluble Oligomers of amyloid-β have been recognized to be early and key intermediates in Alzheimer’s-disease-related synaptic dysfunction2,3,4,5,6,7,8,9. At nanomolar concentrations, soluble amyloid-β Oligomers block hippocampal long-term potentiation7, cause dendritic spine retraction from pyramidal cells5,8 and impair rodent spatial memory2. Soluble amyloid-β Oligomers have been prepared from chemical syntheses, transfected cell culture supernatants, transgenic mouse brain and human Alzheimer’s disease brain2,4,7,9. Together, these data imply a high-affinity cell-surface receptor for soluble amyloid-β Oligomers on neurons—one that is central to the pathophysiological process in Alzheimer’s disease. Here we identify the cellular prion protein (PrPC) as an amyloid-β-oligomer receptor by expression cloning. Amyloid-β Oligomers bind with nanomolar affinity to PrPC, but the interaction does not require the infectious PrPSc conformation. Synaptic responsiveness in hippocampal slices from young adult PrP null mice is normal, but the amyloid-β oligomer blockade of long-term potentiation is absent. Anti-PrP antibodies prevent amyloid-β-oligomer binding to PrPC and rescue synaptic plasticity in hippocampal slices from oligomeric amyloid-β. Thus, PrPC is a mediator of amyloid-β-oligomer-induced synaptic dysfunction, and PrPC-specific pharmaceuticals may have therapeutic potential for Alzheimer’s disease.
Douglas Ramsay - One of the best experts on this subject based on the ideXlab platform.
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monitoring receptor oligomerization using time resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer the human δ opioid receptor displays constitutive oligomerization at the cell surface which is not regulated
Journal of Biological Chemistry, 2001Co-Authors: Mary Mcvey, Douglas Ramsay, Elaine Kellett, Stephen Rees, Shelagh Wilson, Andrew J Pope, Graeme MilliganAbstract:Abstract Oligomerization of the human δ-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-Ala2,d-Leu5]enkephalin nor the inverse agonist ligand ICI174864 were able to modulate the oligomerization status of this receptor. Interactions between co-expressed δ-opioid receptors and β2-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 δ-opioid receptor-β2-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 δ-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.
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monitoring receptor oligomerization using time resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer the human δ opioid receptor displays constitutive oligomerization at the cell surface which is not regulated
Journal of Biological Chemistry, 2001Co-Authors: Mary Mcvey, Douglas Ramsay, Elaine Kellett, Stephen Rees, Shelagh Wilson, Andrew J Pope, Graeme MilliganAbstract: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.
