Novel bioluminescent binding assays for interaction studies of protein/peptide hormones with their receptors

et al. 2019

The FEBS Journal

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.

Section: Introductionmentioning

confidence: 99%

Identifying the binding mechanism of LEAP2 to receptor GHSR1a

Wang

et al. 2019

The FEBS Journal

“…4C, inner panel). The NanoLuc-based binding tracers have been widely validated using different receptors in our previous studies [21–28], such as RXFP1–4, GHSR1a, leukemia inhibitory factor receptor, erythropoietin receptor, and fibroblast growth factor receptor. Using NanoLuc-PEN as a tracer, we conducted washing-based binding assays using living HEK293T cells overexpressing untagged human GPR83 (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Wang

et al. 2022

Preprint

Self 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.

“…In our previous and present studies, we validated the novel bioluminescent binding assay using a variety of protein/peptide hormones with different size and distinct receptors, including relaxin (receptor RXFP1) [ 1 ], INSL3 (receptor RXFP2) [ 5 ], chimeric relaxin family peptide R3/I5 (receptor RXFP3 and RXFP4) [ 35 ], ghrelin (receptor GSHR1a) [ 3 ], leukemia inhibitory factor (receptor LIFR/gp130) [ 4 ], erythropoietin (receptor EPOR) [ 2 ], and FGF2 (receptor FGFR1–4). Although the NanoLuc reporter is large, its attachment did not negatively affect the receptor-binding of a wide range of protein/peptide hormones, provided it was attached to an appropriate position using an appropriate linkeFor wide application of the novel binding assay, we developed two methods for convenient preparation of the bioluminescent ligands, that is, the chemical conjugation method and the genetic fusion method [ 7 ]. For the chemical conjugation method, an engineered NanoLuc reporter carrying a unique reactive moiety was chemically conjugated with a rationally designed recombinant or synthetic protein/peptide that also carries a unique reactive moiety at an appropriate position.…”

Section: Resultsmentioning

confidence: 99%

“…The NanoLuc reporter was either chemically conjugated at an appropriate position or genetically fused at one terminus of the target protein/peptide, and the resultant NanoLuc-conjugated/fused protein/peptide was a novel bioluminescent ligand, provided it retained high receptor-binding affinity. The novel bioluminescent ligands represent a novel class of non-radioactive tracers for quantitative measurements of ligand–receptor interactions [ 7 ]. However, the novel bioluminescent binding assay needs to be validated using a variety of protein/peptide hormones due to the high diversity of protein/peptide hormones and their receptors.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

PLoS ONE

Self Cite

We recently developed novel bioluminescent binding assays for several protein/peptide hormones to study their interactions with receptors using the so far brightest NanoLuc reporter. To validate the novel bioluminescent binding assay using a variety of protein/peptide hormones, in the present work we applied it to the fibroblast growth factor (FGF) family using the prototype member FGF2 as an example. A fully active recombinant FGF2 retaining a unique exposed cysteine (Cys) residue was chemically conjugated with an engineered NanoLuc carrying a unique exposed Cys residue at the C-terminus via formation of an intermolecular disulfide linkage. The NanoLuc-conjugated FGF2 (FGF2-Luc) retained high binding affinity to the overexpressed FGFR1 and the endogenous FGF receptor with the calculated dissociation constants of 161 ± 21 pM (n = 3) and 25 ± 4 pM (n = 3), respectively. In competition binding assays using FGF2-Luc as a tracer, receptor-binding potencies of wild-type or mutant FGF2s were accurately quantified. Thus, FGF2-Luc represents a novel non-radioactive tracer for the quantitative measurement of ligand–receptor interactions in the FGF family. These data suggest that the novel bioluminescent binding assay can be applied to a variety of protein/peptide hormones for ligand–receptor interaction studies.