title: Application of the novel bioluminescent ligand-receptor binding assay to relaxin-RXFP1 system for interaction studies.
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.
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
authors.forename: Qing-Ping
authors.initials: QP
authors.lastname: Wu
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
authors.forename: Xiao-Xia
authors.initials: XX
authors.lastname: Shao
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
authors.forename: Jia-Hui
authors.initials: JH
authors.lastname: Wang
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
authors.forename: Zeng-Guang
authors.initials: ZG
authors.lastname: Xu
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China. yaliliuliu@163.com.
authors.forename: Ya-Li
authors.initials: YL
authors.lastname: Liu
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China. zhan-yun.guo@tongji.edu.cn.
authors.forename: Zhan-Yun
authors.initials: ZY
authors.lastname: Guo
mesh_terms: D000595:Amino Acid Sequence; D001681:Biological Assay; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012065:Relaxin / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D008024:Ligands; C417543:RXFP1 protein, human; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; D011994:Recombinant Proteins; D012065:Relaxin; D008156:Luciferases
keywords: Binding; Bioluminescence; Ligand; RXFP1; Receptor; Relaxin