title: Quantitative measurement of cell membrane receptor internalization by the nanoluciferase reporter: Using the G protein-coupled receptor RXFP3 as a model.
abstract: Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the brightest bioluminescence to date. In the present work, we developed NanoLuc as a sensitive bioluminescent reporter to measure quantitatively the internalization of cell membrane receptors, based on the pH dependence of the reporter activity. The G protein-coupled receptor RXFP3, the cognate receptor of relaxin-3/INSL7, was used as a model receptor. We first generated stable HEK293T cells that inducibly coexpressed a C-terminally NanoLuc-tagged human RXFP3 and a C-terminally enhanced green fluorescent protein (EGFP)-tagged human RXFP3. The C-terminal EGFP-tag and NanoLuc-tag had no detrimental effects on the ligand-binding potency and intracellular trafficking of RXFP3. Based on the fluorescence of the tagged EGFP reporter, the ligand-induced RXFP3 internalization was visualized directly under a fluorescence microscope. Based on the bioluminescence of the tagged NanoLuc reporter, the ligand-induced RXFP3 internalization was measured quantitatively by a convenient bioluminescent assay. Coexpression of an EGFP-tagged inactive [E141R]RXFP3 had no detrimental effect on the ligand-binding potency and ligand-induced internalization of the NanoLuc-tagged wild-type RXFP3, suggesting that the mutant RXFP3 and wild-type RXFP3 worked independently. The present bioluminescent internalization assay could be extended to other G protein-coupled receptors and other cell membrane receptors to study ligand-receptor and receptor-receptor interactions.
authors.affiliation: Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, China; Central Laboratory, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, China.
authors.forename: Ge
authors.initials: G
authors.lastname: Song
authors.affiliation: Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, China.
authors.forename: Xiao-Xia
authors.initials: XX
authors.lastname: Shao
authors.affiliation: Central Laboratory, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China.
authors.forename: Ya-Li
authors.initials: YL
authors.lastname: Liu
authors.affiliation: Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, China; Central Laboratory, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China. Electronic address: zhan-yun.guo@tongji.edu.cn.
authors.forename: Zhan-Yun
authors.initials: ZY
authors.lastname: Guo
mesh_terms: D001681:Biological Assay; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012065:Relaxin / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling
chemical_list: D008024:Ligands; C480238:RLN3 protein, human; C410092:RXFP3 protein, human; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012065:Relaxin; D008156:Luciferases
keywords: Bioluminescence; GPCR; Internalization; RXFP3; Receptor; Relaxin-3