A homogeneous single-label quenching resonance energy transfer assay for a δ-opioid receptor–ligand using intact cells

The Analyst ◽  
2013 ◽  
Vol 138 (17) ◽  
pp. 4907 ◽  
Author(s):  
Kari Kopra ◽  
Shweta ◽  
Eija Martikkala ◽  
Pekka Hänninen ◽  
Ulla Petäjä-Repo ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Opioid Receptor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Intact Cells ◽  
Receptor Ligand ◽  
Δ Opioid Receptor

scholarly journals Heterodimerization of Mu Opioid Receptor Protomer with Dopamine D2 Receptor Modulates Agonist-Induced Internalization of Mu Opioid Receptor

Biomolecules ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 368 ◽  
Author(s):  
Vasudevan ◽  
Borroto-Escuela ◽  
Huysentruyt ◽  
Fuxe ◽  
Saini ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Opioid Receptor ◽  
Dopamine D2 Receptor ◽  
D2 Receptor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Mu Opioid Receptor ◽  
Potential Threat ◽  
Mu Opioid ◽  
Dopamine D2

The interplay between the dopamine (DA) and opioid systems in the brain is known to modulate the additive effects of substances of abuse. On one hand, opioids serve mankind by their analgesic properties, which are mediated via the mu opioid receptor (MOR), a Class A G protein-coupled receptor (GPCR), but on the other hand, they pose a potential threat by causing undesired side effects such as tolerance and dependence, for which the exact molecular mechanism is still unknown. Using human embryonic kidney 293T (HEK 293T) and HeLa cells transfected with MOR and the dopamine D2 receptor (D2R), we demonstrate that these receptors heterodimerize, using an array of biochemical and biophysical techniques such as coimmunoprecipitation (co-IP), bioluminescence resonance energy transfer (BRET1), Fӧrster resonance energy transfer (FRET), and functional complementation of a split luciferase. Furthermore, live cell imaging revealed that D2LR, when coexpressed with MOR, slowed down internalization of MOR, following activation with the MOR agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO).

scholarly journals Bioluminescence resonance energy transfer identify scaffold protein CNK1 interactions in intact cells

FEBS Letters ◽  
2004 ◽  
Vol 579 (3) ◽  
pp. 648-654 ◽  
Author(s):  
Marco A. Lopez-Ilasaca ◽  
Julio C. Bernabe-Ortiz ◽  
Soon-Young Na ◽  
Victor J. Dzau ◽  
Ramnik J. Xavier
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Scaffold Protein ◽  
Bioluminescence Resonance Energy Transfer ◽  
Intact Cells

Fluorescence resonance energy transfer (FRET) microscopy in living cells as a novel tool for the study of cytokine action

2005 ◽  
Vol 72 (S1) ◽  
pp. 14-19 ◽  
Author(s):  
Arieh Gertler ◽  
Eva Biener ◽  
Krishnan V. Ramanujan ◽  
Jean Djiane ◽  
Brian Herman
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Direct Interaction ◽  
Receptor Internalization ◽  
Placental Lactogen ◽  
Hormonal Stimulation ◽  
Intact Cells ◽  
Fluorescence Resonance

Fluorescence resonance energy transfer (FRET) microscopy was used to study interactions between proteins in intact cells. We showed that growth hormone (GH) causes transient homodimerization of GH receptors tagged with yellow or cyan fluorescent proteins. The peak of FRET signaling occurred 2 to 4 min after hormonal stimulation and was followed by a decrease in FRET signal. Repeating those experiments in cells pretreated with the inhibitor of internalization methyl-β-cyclodextrin, or in potassium-depleted cells showed no difference in the kinetics of FRET signaling as compared with the non-treated cells, indicating that the decrease in FRET signal does not result from receptor internalization by the pathways inhibited by methyl-β-cyclodextrin or potassium depleted but might occur by other pathways of internalization. Using a similar methodology, we also demonstrated that ovine placental lactogen (oPL) causes transient heterodimerization of GH and prolactin (PRL) receptors 2·5 to 3 min after oPL application. On the other hand, oGH or oPRL had no effect at all, further substantiating the finding the oPL, which lacks a specific receptor, acts in homologous systems by heterodimerization of GH and PRL receptors. We also demonstrated that both PRL and leptin (LEP) are capable of transactivation of the oncogenic receptors erbB2 and erbB3. Upon PRL or LEP stimulation of HEK-293T cells transfected with LEP or PRL receptors and erbB2 or erbB3, erbB proteins are first phosphorylated and then activate MAPK (erk1/erk2). However, the FRET experiments failed to document any evidence of a direct interaction between erbB2 and the PRL or LEP receptors, suggesting that erbB activation probably occurs via activated JAK2, translocated from the respective receptors to erbB2.

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