Amino Acids

, Volume 50, Issue 8, pp 1111–1119 | Cite as

Development of a novel ligand binding assay for relaxin family peptide receptor 3 and 4 using NanoLuc complementation

  • Meng-Jun Hu
  • Xiao-Xia Shao
  • Hao-Zheng Li
  • Wei-Han Nie
  • Jia-Hui Wang
  • Ya-Li Liu
  • Zeng-Guang Xu
  • Zhan-Yun Guo
Original Article


Relaxin family peptides perform a variety of biological functions by binding and activating relaxin family peptide receptor 1–4 (RXFP1–4), four A-class G protein-coupled receptors. In the present work, we developed a novel ligand binding assay for RXFP3 and RXFP4 based on NanoLuc complementation technology (NanoBiT). A synthetic ligation version of the low-affinity small complementation tag (SmBiT) was efficiently ligated to the A-chain N terminus of recombinant chimeric agonist R3/I5 using recombinant circular sortase A. After the ligation product R3/I5-SmBiT was mixed with human RXFP3 or RXFP4 genetically fused with a secretory large NanoLuc fragment (sLgBiT) at the N terminus, NanoLuc complementation was induced by high-affinity ligand–receptor binding. Binding kinetics and affinities of R3/I5-SmBiT with sLgBiT-fused RXFP3 and RXFP4 were conveniently measured according to the complementation-induced bioluminescence. Using R3/I5-SmBiT and the sLgBiT-fused receptor as a complementation pair, binding potencies of various ligands with RXFP3 and RXFP4 were quantitatively measured without the cumbersome washing step. The novel NanoBiT-based ligand binding assay is convenient for use and suitable for automation, thus will facilitate interaction studies of RXFP3 and RXFP4 with ligands in future. This assay can also be applied to some other plasma membrane receptors for pharmacological characterization of ligands in future studies.


Relaxin family Receptor Binding assay NanoLuc Complementation 



This work was supported by grants from the National Natural Science Foundation of China (31670773, 31470767).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Research involving human participants and/or animals

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

726_2018_2588_MOESM1_ESM.doc (110 kb)
Supplementary material 1 (DOC 109 kb)


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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  • Meng-Jun Hu
    • 1
  • Xiao-Xia Shao
    • 1
  • Hao-Zheng Li
    • 1
  • Wei-Han Nie
    • 1
  • Jia-Hui Wang
    • 1
  • Ya-Li Liu
    • 1
  • Zeng-Guang Xu
    • 1
  • Zhan-Yun Guo
    • 1
  1. 1.Research Center for Translational Medicine at East Hospital, School of Life Sciences and TechnologyTongji UniversityShanghaiChina

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