Quantitative measurement of cell membrane receptor internalization by the nanoluciferase reporter: Using the G protein-coupled receptor RXFP3 as a model

摘要: Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the brightest bioluminescence to date. In present work, we NanoLuc as sensitive bioluminescent measure quantitatively internalization of cell membrane receptors, based on pH dependence activity. The G protein-coupled receptor RXFP3, cognate relaxin-3/INSL7, was used model receptor. We first generated stable HEK293T cells that inducibly coexpressed C-terminally NanoLuc-tagged human RXFP3 and enhanced green fluorescent protein (EGFP)-tagged RXFP3. C-terminal EGFP-tag NanoLuc-tag had no detrimental effects ligand-binding potency intracellular trafficking Based fluorescence tagged EGFP reporter, ligand-induced visualized directly under microscope. measured by convenient assay. Coexpression an EGFP-tagged inactive [E141R]RXFP3 effect wild-type suggesting mutant worked independently. assay could be extended other receptors study ligand-receptor receptor-receptor interactions.