A novel BRET-based binding assay for interaction studies of relaxin family peptide receptor 3 with its ligands.
Relaxin family peptide receptor 3 (RXFP3) is an A-class G protein-coupled receptor that is implicated in the regulation of food intake and stress response upon activation by its cognate agonist relaxin-3. To study its interaction with various ligands, we developed a novel bioluminescence resonance energy transfer (BRET)-based binding assay using the brightest NanoLuc as an energy donor and a newly developed cyan-excitable orange fluorescent protein (CyOFP) as an energy acceptor. An engineered CyOFP without intrinsic cysteine residues but with an introduced cysteine at the C-terminus was overexpressed in Escherichia coli and chemically conjugated to the A-chain N-terminus of an easily labeled chimeric R3/I5 peptide via an intermolecular disulfide linkage. After the CyOFP-conjugated R3/I5 bound to a shortened human RXFP3 (removal of 33 N-terminal residues) fused with the NanoLuc reporter at the N-terminus, high BRET signals were detected. Saturation binding and real-time binding assays demonstrated that this BRET pair retained high binding affinity with fast association/dissociation. Using this BRET pair, binding potencies of various ligands with RXFP3 were conveniently measured through competition binding assays. Thus, the novel BRET-based binding assay facilitates interaction studies of RXFP3 with various ligands. The engineered CyOFP without intrinsic cysteine residues may also be applied to other BRET-based binding assays in future studies.
Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
Jia-Hui
JH
Wang
Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
Xiao-Xia
XX
Shao
Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
Meng-Jun
MJ
Hu
Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
Dian
D
Wei
Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
Ya-Li
YL
Liu
Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
Zeng-Guang
ZG
Xu
Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China. zhan-yun.guo@tongji.edu.cn.
Zhan-Yun
ZY
Guo
D001667:Binding, Competitive; D001681:Biological Assay; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements; D011485:Protein Binding; D015202:Protein Engineering; D021382:Protein Sorting Signals; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012065:Relaxin / Q000235:genetics* / Q000378:metabolism
C485182:Cyan Fluorescent Protein; C508600:IL6 protein, human; D015850:Interleukin-6; D008024:Ligands; D021382:Protein Sorting Signals; C480238:RLN3 protein, human; C410092:RXFP3 protein, human; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D012065:Relaxin; D008156:Luciferases
BRET; Binding; Interaction; RXFP3; Relaxin-3