Abstract
Relaxin is a prototype of the relaxin family peptide hormones and plays important biological functions by binding and activating the G protein-coupled receptor RXFP1. To study their interactions, in the present work, we applied the newly developed bioluminescent ligand–receptor binding assay to the relaxin-RXFP1 system. First, a fully active easily labeled relaxin, in which three Lys residues of human relaxin-2 were replaced by Arg, was prepared through overexpression of a single-chain precursor in Pichia pastoris and in vitro enzymatic maturation. Thereafter, the B-chain N-terminus of the easily labeled relaxin was chemically cross-linked with a C-terminal cysteine residue of an engineered NanoLuc through a disulfide linkage. Receptor-binding assays demonstrated that the NanoLuc-conjugated relaxin retained high binding affinity with the receptor RXFP1 (K d = 1.11 ± 0.08 nM, n = 3) and was able to sensitively monitor binding of a variety of ligands with RXFP1. Using the novel bioluminescent binding assay, we demonstrated that three highly conserved B-chain Arg residues of relaxin-3 had distinct contributions to binding of the receptor RXFP1. In summary, our present work provides a novel bioluminescent ligand–receptor binding assay for the relaxin-RXFP1 system to facilitate their interaction studies, such as characterization of relaxin analogues or screening novel agonists or antagonists of RXFP1.
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Acknowledgments
We thank Promega Corporation for providing the plasmids encoding NanoLuc. This work was supported by the National Natural Science Foundation of China (31470767, 31270824) and the Fundamental Research Funds for the Central Universities (2000219098).
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Handling Editor: J. D. Wade.
Q.-P. Wu and L. Zhang contributed equally to this work.
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Wu, QP., Zhang, L., Shao, XX. et al. Application of the novel bioluminescent ligand–receptor binding assay to relaxin-RXFP1 system for interaction studies. Amino Acids 48, 1099–1107 (2016). https://doi.org/10.1007/s00726-015-2146-3
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DOI: https://doi.org/10.1007/s00726-015-2146-3