scholarly journals Heterodimerization Between the Lutropin and Follitropin Receptors is Associated With an Attenuation of Hormone-Dependent Signaling

Endocrinology ◽  
2013 ◽  
Vol 154 (10) ◽  
pp. 3925-3930 ◽  
Author(s):  
Xiuyan Feng ◽  
Meilin Zhang ◽  
Rongbin Guan ◽  
Deborah L. Segaloff
Keyword(s):  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
G Protein Coupled Receptors ◽  
Fsh Receptor ◽  
Bioluminescence Resonance Energy Transfer ◽  
Protein Protein Interactions ◽  
Lh Receptor ◽  
G Protein Coupled ◽  
Stimulation Of

The LH receptor (LHR) and FSH receptor (FSHR) are each G protein-coupled receptors that play critical roles in reproductive endocrinology. Each of these receptors has previously been shown to self-associate into homodimers and oligomers shortly after their biosynthesis. As shown herein using bioluminescence resonance energy transfer to detect protein-protein interactions, our data show that the LHR and FSHR, when coexpressed in the same cells, specifically heterodimerize with each other. Further experiments confirm that at least a portion of the cellular LHR/FSHR heterodimers are present on the cell surface and are functional. We then sought to ascertain what effects, if any, heterodimerization between the LHR and FSHR might have on signaling. It was observed that when the LHR was expressed under conditions promoting the heterodimerization with FSHR, LH or human chorionic gonadotropin (hCG) stimulation of Gs was attenuated. Conversely, when the FSHR was expressed under conditions promoting heterodimerization with the LHR, FSH-stimulated Gs activation was attenuated. These results demonstrate that the coexpression of the LHR and FSHR enables heterodimerizaton between the 2 gonadotropin receptors and results in an attenuation of signaling through each receptor.

scholarly journals Bioluminescence Resonance Energy Transfer as a Method to Study Protein-Protein Interactions: Application to G Protein Coupled Receptor Biology

Molecules ◽  
2019 ◽  
Vol 24 (3) ◽  
pp. 537 ◽  
Author(s):  
Chayma El Khamlichi ◽  
Flora Reverchon-Assadi ◽  
Nadège Hervouet-Coste ◽  
Lauren Blot ◽  
Eric Reiter ◽  
...  
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Receptor Biology ◽  
G Protein Coupled

The bioluminescence resonance energy transfer (BRET) approach involves resonance energy transfer between a light-emitting enzyme and fluorescent acceptors. The major advantage of this technique over biochemical methods is that protein-protein interactions (PPI) can be monitored without disrupting the natural environment, frequently altered by detergents and membrane preparations. Thus, it is considered as one of the most versatile technique for studying molecular interactions in living cells at “physiological” expression levels. BRET analysis has been applied to study many transmembrane receptor classes including G-protein coupled receptors (GPCR). It is well established that these receptors may function as dimeric/oligomeric forms and interact with multiple effectors to transduce the signal. Therefore, they are considered as attractive targets to identify PPI modulators. In this review, we present an overview of the different BRET systems developed up to now and their relevance to identify inhibitors/modulators of protein–protein interaction. Then, we introduce the different classes of agents that have been recently developed to target PPI, and provide some examples illustrating the use of BRET-based assays to identify and characterize innovative PPI modulators in the field of GPCRs biology. Finally, we discuss the main advantages and the limits of BRET approach to characterize PPI modulators.

scholarly journals Demonstration of Improvements to the Bioluminescence Resonance Energy Transfer (BRET) Technology for the Monitoring of G Protein–Coupled Receptors in Live Cells

2008 ◽  
Vol 13 (9) ◽  
pp. 888-898 ◽  
Author(s):  
Martina Kocan ◽  
Heng B. See ◽  
Ruth M. Seeber ◽  
Karin A. Eidne ◽  
Kevin D.G. Pfleger
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
G Protein Coupled Receptors ◽  
Live Cells ◽  
Bioluminescence Resonance Energy Transfer ◽  
G Protein Coupled

The bioluminescence resonance energy transfer (BRET) technique has become extremely popular for studying protein-protein interactions in living cells and real time. Of particular interest is the ability to monitor interactions between G protein–coupled receptors, such as the thyrotropin-releasing hormone receptor (TRHR), and proteins critical for regulating their function, such as β-arrestin. Using TRHR/β-arrestin interactions, we have demonstrated improvements to all 3 generations of BRET (BRET1, BRET2, and eBRET) by using the novel forms of luciferase, Rluc2 and Rluc8, developed by the Gambhir laboratory. Furthermore, for the 1st time it was possible to use the BRET2 system to detect ligand-induced G protein–coupled receptor/β-arrestin interactions over prolonged periods (on the scale of hours rather than seconds) with a very stable signal. As demonstrated by our Z′-factor data, these luciferases increase the sensitivity of BRET to such an extent that they substantially increase the potential applicability of this technology for effective drug discovery high-throughput screening. ( Journal of Biomolecular Screening 2008:888-898)

scholarly journals Homo- and hetero-oligomeric interactions between G-protein-coupled receptors in living cells monitored by two variants of bioluminescence resonance energy transfer (BRET): hetero-oligomers between receptor subtypes form more efficiently than between less closely related sequences

2002 ◽  
Vol 365 (2) ◽  
pp. 429-440 ◽  
Author(s):  
Douglas RAMSAY ◽  
Elaine KELLETT ◽  
Mary McVEY ◽  
Stephen REES ◽  
Graeme MILLIGAN
Keyword(s):  
Opioid Receptor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
G Protein Coupled Receptors ◽  
Receptor Subtypes ◽  
Bioluminescence Resonance Energy Transfer ◽  
Oligomer Formation ◽  
G Protein Coupled ◽  
Energy Acceptor

Homo- and hetero-oligomerization of G-protein-coupled receptors (GPCRs) were examined in HEK-293 cells using two variants of bioluminescence resonance energy transfer (BRET). BRET2 (a variant of BRET) offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared with traditional BRET. Previously recorded homo-oligomerization of the human δ-opioid receptor was confirmed using BRET2. Homo-oligomerization of the κ-opioid receptor was observed using both BRET techniques. Both homo- and hetero-oligomers, containing both δ- and κ-opioid receptors, were unaffected by the presence of receptor ligands. BRET detection of opioid receptor homo- and hetero-oligomers required expression of 50000–100000 copies of the receptor energy acceptor construct per cell. The effectiveness of δ—κ-opioid receptor hetero-oligomer formation was as great as for homomeric interactions. The capacity of the two opioid receptors to form oligomeric complexes with the β2-adrenoceptor was also assessed. Although such interactions were detected, at least 250000 copies per cell of the energy acceptor were required. Requirement for high levels of receptor expression was equally pronounced in attempts to measure hetero-oligomer formation between the κ-opioid receptor and the thyrotropin-releasing hormone receptor-1. These studies indicate that constitutively formed homo- and hetero-oligomers of opioid receptor subtypes can be detected in living cells containing less than 100000 copies of the receptors. However, although hetero-oligomeric interactions between certain less closely related GPCRs can be detected, they appear to be of lower affinity than homo- or hetero-oligomers containing closely related sequences. Interactions recorded between certain GPCR family members in heterologous expression systems are likely to be artefacts of extreme levels of overexpression.

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