A dynamic and screening-compatible nanoluciferase-based complementation assay enables profiling of individual GPCR–G protein interactions

Laschet, Céline; Dupuis, Nadine; Hanson, Julien

doi:10.1074/jbc.ra118.006231

Cited by 54 publications

(58 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…35 Increasing studies using NanoBiT have been published, while an issue was raised about the reliability of the measurement of interaction dynamics with this method and similar fragment complementation techniques. 56 The high sensitivity and precision of detection were revealed in our study, particularly by the two-step competition curves of exenatide. The measured K d for the receptor NTD in fusion with LgBiT or SmBiT was quite compatible with that estimated from the native NTD of GLP-1R using the classical biophysical method.…”

Section: Discussionmentioning

confidence: 53%

“…NanoLuc Binary Technology (NanoBiT), based on its enhanced luminescence detection, provides a highly sensitive and convenient method for the investigation of protein‐protein interactions . Increasing studies using NanoBiT have been published, while an issue was raised about the reliability of the measurement of interaction dynamics with this method and similar fragment complementation techniques . The high sensitivity and precision of detection were revealed in our study, particularly by the two‐step competition curves of exenatide.…”

Section: Discussionmentioning

confidence: 71%

See 1 more Smart Citation

Dimerization/oligomerization of the extracellular domain of the GLP‐1 receptor and the negative cooperativity in its ligand binding revealed by the improved NanoBiT

Song

Shen

et al. 2020

FASEB j.

View full text Add to dashboard Cite

The glucagon‐like peptide‐1 receptor (GLP‐1R), a family B G‐protein coupled receptor (GPCR), regulates the insulin secretion following stimulation by ligands. The transmembrane domain (TM) mediates GLP‐1R homodimerization, which modulates its ligand binding and signaling. We investigated the possible involvement of the N‐terminal extracellular domain (NTD) in dimerization/oligomerization and dimer‐associated ligand binding by NanoLuc Binary Technology (NanoBiT). With improved NanoBiT detection using a decreasing substrate concentration, the negative cooperativity of ligand binding to the NTD was confirmed by accelerated dissociation and Scatchard analysis. The dimerization/oligomerization of the isolated NTD was observed by NanoBiT and validated by analytical ultracentrifugation, deriving the comparable dimerization affinity (~105 M−1). The NTD was also involved in the dimerization/oligomerization of the full‐length GLP‐1R with mutated TM4 at the cellular level. In an analysis of the parameters of the NTD binding, the Kd for the probe GLP‐1 (7‐36, A8G) was similar (6‐8 μM) in both the 1:1 binding model and the receptor dimerization model. Compared with GLP‐1 and dulaglutide, exenatide showed two‐site binding with Ki values of 1.4 pM and 8.7 nM. Our study indicates the involvement of NTD in the GLP‐1R dimerization/oligomerization and suggests that further investigations on the role in other family B GPCRs are needed.

show abstract

Section: Discussionmentioning

confidence: 53%

Section: Discussionmentioning

confidence: 71%

Dimerization/oligomerization of the extracellular domain of the GLP‐1 receptor and the negative cooperativity in its ligand binding revealed by the improved NanoBiT

Song

Shen

et al. 2020

FASEB j.

View full text Add to dashboard Cite

show abstract

“…S1). In contrast, a recent publication showed that NanoBiT was successful for a direct recruitment assay with the D2R fused to the small bit (28). We do not know why these split fragments work with the direct recruitment assay but not the membrane recruitment assay, but we speculated that this might result from unfavorable orientation of the attachments of the large and small fragments.…”

Section: Discussionmentioning

confidence: 81%

“…Complementation-based luciferase split assays have been widely used in the last decade to detect proteinprotein interaction in multiple biological systems; NanoLuc has advantages in both decreased size and increased brightness (15). Several NanoBiT-based GPCR complementation methods have been developed, including direct β-arrestin recruitment, direct G protein interaction and internalization, all of which have the small fragment fused to the C terminus of the receptor (27)(28)(29). Curiously, we found that the NanoBiT system, when adapted to the β-arrestin membrane recruitment assay, failed to show a response with any of the three receptors tested, including the D2R (Fig.…”

Section: Discussionmentioning

confidence: 99%

A novel luminescence-based β-arrestin membrane recruitment assay for unmodified GPCRs

Pedersen

Pham²,

Mancebo³

et al. 2020

Preprint

View full text Add to dashboard Cite

G protein-coupled receptors (GPCRs) signal through activation of G proteins and subsequent modulation of downstream effectors. More recently, G protein-independent signaling via the arrestin pathway has also been implicated in important physiological functions. This has led to great interest in the identification of biased ligands that favor either the G protein or arrestin-signaling pathways. Currently available screening techniques that measure arrestin recruitment have required C-terminal receptor modifications that can in principle alter protein interactions and thus signaling. Here, we have developed a novel luminescence-based assay to measure arrestin recruitment to any unmodified receptor.NanoLuc, an engineered luciferase from ophlorus gracilirostris (deep sea shrimp), is smaller and brighter than other well-established luciferases. Recently, several publications have explored functional NanoLuc split sites for use in complementation assays. Here, we have identified a novel split site and have fused the N-terminal fragment to a membrane tether and the C-terminal fragment to the N-terminus of either β-arrestin 1 or 2. Upon receptor activation, arrestin is recruited to the plasma membrane in an agonist concentrationdependent manner and the two NanoLuc fragments complement to reconstitute functional luciferase, which allows quantification of recruitment with a single luminescence signal. Our assay avoids potential artifacts related to C-terminal receptor modification. The split NanoLuc arrestin recruitment assay has promise as a new generic assay for measuring arrestin recruitment to diverse GPCR types in heterologous or native cells.

show abstract

“…The complemented HiBiT-LgBiT protein showed a similar luminescence output to the full length NanoLuc, making it an ideal system to study proteins expressed at endogenous levels (Schwinn et al, 2018). NanoBiT has been used to monitor protein-protein interactions, including GPCR oligomerization (Botta et al, 2019), and the recruitment of G proteins and β arrestin to GPCRs (Hisano et al, 2018;Laschet et al, 2018;Storme et al, 2018), with the rapid complementation and maturation rate of the split NanoLuc luciferase enabling kinetic measurements. These studies made use of GPCRs with NanoBiT fused to their C-terminal domains.…”

Section: Introductionmentioning

confidence: 99%

Monitoring ligand-induced changes in receptor conformation with NanoBiT conjugated nanobodies

Soave

Heukers

Kellam

et al. 2020

Preprint

View full text Add to dashboard Cite

max 150 words)Camelid single-domain antibody fragments (nanobodies) offer the specificity of an antibody in a single 15kDa immunoglobulin domain. Their small size allows for easy genetic manipulation of the nanobody sequence to incorporate protein tags, facilitating their use as biochemical probes. The nanobody VUN400, which recognises the second extracellular loop of the human CXCR4 chemokine receptor, was used as a probe to monitor specific CXCR4 conformations. VUN400 was fused via its C-terminus to the 11-amino acid HiBiT tag (VUN400-HiBiT) which complements to LgBiT protein, forming a full length functional NanoLuc luciferase. Here, complemented luminescence was used to detect VUN400-HiBiT binding to CXCR4 receptors expressed in living HEK293 cells. VUN400-HiBiT binding to CXCR4 could be prevented by orthosteric and allosteric ligands, allowing VUN400-HiBiT to be used as a probe to detect specific conformations of CXCR4. These data demonstrate that the high specificity offered by extracellular-targeted nanobodies can be utilised to probe receptor pharmacology.

show abstract

A dynamic and screening-compatible nanoluciferase-based complementation assay enables profiling of individual GPCR–G protein interactions

Cited by 54 publications

References 53 publications

Dimerization/oligomerization of the extracellular domain of the GLP‐1 receptor and the negative cooperativity in its ligand binding revealed by the improved NanoBiT

Dimerization/oligomerization of the extracellular domain of the GLP‐1 receptor and the negative cooperativity in its ligand binding revealed by the improved NanoBiT

A novel luminescence-based β-arrestin membrane recruitment assay for unmodified GPCRs

Monitoring ligand-induced changes in receptor conformation with NanoBiT conjugated nanobodies

Contact Info

Product

Resources

About