Novel Bioluminescent Binding Assays for Ligand–Receptor Interaction Studies of the Fibroblast Growth Factor Family

Ge, Song; Shao, Xin; Wu, Qingping; Xu, Zeng‐Guang; Liu, Yali; Guo, Zhan‐Yun

doi:10.1371/journal.pone.0159263

Cited by 7 publications

(7 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, the use of radioactive materials is inconvenient for most laboratories. In recent studies, we developed novel bioluminescence ligand-receptor binding assays through chemical conjugation or genetic fusion of a small NanoLuc luciferase to the target protein/peptide hormone [14][15][16][17][18][19][20][21]. As an example, we measured the binding potencies of various ligands with the receptor GHSR1a using the NanoLuc-conjugated ghrelin (ghrelin-Luc) as a non- radioactive tracer [21].…”

Section: Introductionmentioning

confidence: 99%

Identifying the binding mechanism of LEAP2 to receptor GHSR1a

Wang

Shao

et al. 2019

The FEBS Journal

View full text Add to dashboard Cite

Liver‐expressed antimicrobial peptide 2 (LEAP2) is a highly conserved secretory peptide first isolated in 2003. However, its exact biological functions remained elusive until a recent study identified it as an endogenous antagonist for the growth hormone secretagogue receptor (GHSR1a), a G protein‐coupled receptor for which the gastric peptide ghrelin is the endogenous agonist. By tuning the ghrelin–GHSR1a system, LEAP2 has an important function in energy metabolism. In the present study, we first demonstrated that LEAP2 and ghrelin actually bound to GHSR1a in a competitive manner, rather than in a non‐competitive manner as previously reported, by binding assays and activation assays. Subsequently, we demonstrated that the antagonistic function of LEAP2 was drastically affected by the manner of its addition. LEAP2 primarily affected the maximal activation effect when added before ghrelin, whereas it primarily affected half‐maximal effective concentration when added at the same time as ghrelin. Thus, LEAP2 behaved as a competitive antagonist if added at the same time as the agonist and a non‐competitive antagonist if added before the agonist. This unusual property of LEAP2 might be caused by its slow dissociation from receptor GHSR1a. We also found that the N‐terminal fragment of LEAP2 was important for receptor binding. Our present study revealed an antagonistic mechanism for LEAP2, and will facilitate the design of novel antagonists for receptor GHSR1a in future studies.

show abstract

Section: Introductionmentioning

confidence: 99%

Identifying the binding mechanism of LEAP2 to receptor GHSR1a

Wang

Shao

et al. 2019

The FEBS Journal

View full text Add to dashboard Cite

show abstract

“…Because of its brightness and stability, Nluc has been applied in the preclinical and clinical fields in terms of screening drug targets, drug target validation, disease detection, and in vivo imaging. − With the increased sensitivity and small size, Nluc is very useful as a reporter gene for monitoring cellular biology − and can more closely mimic endogenous biology. A recent advancement enabled by Nluc is a real-time cell viability assay using a prosubstrate that is only processed to the active Nluc substrate by live cells .…”

mentioning

confidence: 99%

Highly Potent Cell-Permeable and Impermeable NanoLuc Luciferase Inhibitors

et al. 2017

View full text Add to dashboard Cite

Novel engineered NanoLuc (Nluc) luciferase being smaller, brighter, and superior to traditional firefly (Fluc) or Renilla (Rluc) provides a great opportunity for the development of numerous biological, biomedical, clinical, and food and environmental safety applications. This new platform created an urgent need for Nluc inhibitors that could allow selective bioluminescent suppression and multiplexing compatibility with existing luminescence or fluorescence assays. Starting from thienopyrrole carboxylate 1, a hit from a 42 000 PubChem compound library with a low micromolar IC against Nluc, we derivatized four different structural fragments to discover a family of potent, single digit nanomolar, cell permeable inhibitors. Further elaboration revealed a channel that allowed access to the external Nluc surface, resulting in a series of highly potent cell impermeable Nluc inhibitors with negatively charged groups likely extending to the protein surface. The permeability was evaluated by comparing EC shifts calculated from both live and lysed cells expressing Nluc cytosolically. Luminescence imaging further confirmed that cell permeable compounds inhibit both intracellular and extracellular Nluc, whereas less permeable compounds differentially inhibit extracellular Nluc and Nluc on the cell surface. The compounds displayed little to no toxicity to cells and high luciferase specificity, showing no activity against firefly luciferase or even the closely related NanoBit system. Looking forward, the structural motifs used to gain access to the Nluc surface can also be appended with other functional groups, and therefore interesting opportunities for developing assays based on relief-of-inhibition can be envisioned.

show abstract

“…The copyright holder for this preprint this version posted September 28, 2022. ; https://doi.org/10.1101/2022.09. 27.509696 doi: bioRxiv preprint consistent three-dimensional structures for mature human FAM237A (Fig. 1D).…”

Section: Introductionmentioning

confidence: 73%

“…The copyright holder for this preprint this version posted September 28, 2022. ; https://doi.org/10.1101/2022.09. 27.509696 doi: bioRxiv preprint…”

Section: Introductionmentioning

confidence: 99%

FAM237A, rather than peptide PEN and proCCK56-63, is a ligand of the orphan receptor GPR83

Wang

Shao

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

G protein-coupled receptor 83 (GPR83) is primarily expressed in the brain and is implicated in the regulation of energy metabolism and some behaviors. Recently, the PCSK1N/proSAAS-derived peptide PEN, the procholecystokinin-derived peptide proCCK56-63, and family with sequence similarity 237 member A (FAM237A) were all reported as agonists of GPR83. However, these results have not yet been reproduced by other laboratories and thus GPR83 is still officially an orphan receptor. The PEN and proCCK56-63 share sequence similarity; however, they are completely different from FAM237A, raising doubts that all of them are ligands of GPR83. To identify its actual ligand(s), in the present study we developed a NanoLuc Binary Technology (NanoBiT)-based ligand-binding assay, fluorescent ligand-based visualization, and a NanoBiT-based beta-arrestin recruitment assay for human GPR83. Using these assays, we demonstrated that mature human FAM237A could bind to GPR83 with nanomolar range affinity, which activated this receptor and induced its internalization in transfected human embryonic kidney 293T cells. However, we did not detect any interaction of PEN and proCCK56-63 with GPR83 using these assays. Thus, the results confirmed that FAM237A is an agonist of GPR83, but did not support PEN and proCCK56-63 as ligands of this receptor. Clarification of its actual endogenous agonist will pave the way for further functional studies of this brain-specific receptor. The present study also provided an efficient approach for the preparation of mature FAM237A, which would facilitate further functional studies of this difficult-to-make peptide in the future.

show abstract

Novel Bioluminescent Binding Assays for Ligand–Receptor Interaction Studies of the Fibroblast Growth Factor Family

Cited by 7 publications

References 34 publications

Identifying the binding mechanism of LEAP2 to receptor GHSR1a

Identifying the binding mechanism of LEAP2 to receptor GHSR1a

Highly Potent Cell-Permeable and Impermeable NanoLuc Luciferase Inhibitors

FAM237A, rather than peptide PEN and proCCK56-63, is a ligand of the orphan receptor GPR83

Contact Info

Product

Resources

About