Using the novel HiBiT tag to label cell surface relaxin receptors for BRET proximity analysis

Abstract

Relaxin family peptide 1 (RXFP1) is the receptor for relaxin a peptide hormone with important therapeutic potential. Like many G protein‐coupled receptors (GPCRs), RXFP1 has been reported to form homodimers. Given the complex activation mechanism of RXFP1 by relaxin, we wondered whether homodimerization may be explicitly required for receptor activation, and therefore sought to determine if there is any relaxin‐dependent change in RXFP1 proximity at the cell surface. Bioluminescence resonance energy transfer (BRET) between recombinantly tagged receptors is often used in GPCR proximity studies. RXFP1 targets poorly to the cell surface when overexpressed in cell lines, with the majority of the receptor proteins sequestered within the cell. Thus, any relaxin‐induced changes in RXFP1 proximity at the cell surface may be obscured by BRET signal originating from intracellular compartments. We therefore, utilized the newly developed split luciferase system called HiBiT to specifically label the extracellular terminus of cell surface RXFP1 receptors in combination with mCitrine‐tagged receptors, using the GABA B heterodimer as a positive control. This demonstrated that the BRET signal detected from RXFP1‐RXFP1 proximity at the cell surface does not appear to be due to stable physical interactions. The fact that there is also no relaxin‐mediated change in RXFP1‐RXFP1 proximity at the cell surface further supports these conclusions. This work provides a basis by which cell surface GPCR proximity and expression levels can be specifically studied using a facile and homogeneous labeling technique such as HiBiT.

Unravelling cytosolic delivery of cell penetrating peptides with a quantitative endosomal escape assay

“…The split luciferase assay comprises of two subunits: large BiT protein (LgBiT, 17.8 kDa) and a high affinity complementary peptide (HiBiT, 1.3 kDa) 18 . This assay is useful for measuring intracellular protein interactions (e.g., GPCR homodimerisation) 19 , protein dynamics 20 and cellular internalisation (investigating the role of PIP2) 21 . We express LgBiT as a fusion protein with actin, which localises the LgBiT in the cytosol.…”

Strategies for Site‐Specific Labeling of Receptor Proteins on the Surfaces of Living Cells by Using Genetically Encoded Peptide Tags

“…[81,117] Recently, surface-specific labeling by the HiBiT-LgBiT system was used to study the potential of the relaxin family receptor-1 (RXFP 1 ) to undergo ligand-induced homodimerization. [118] The assay was based on the distancedependent bioluminescence resonance energy transfer (BRET) between a luciferase-and mCitrine-tagged RXFP 1 . Pairs comprised of the constitutively active Nluc and mCitrine reporters showed BRET responses indicative of ligand-independent formation of RXFP 1 dimers.…”

“…Applied to the monitoring of internalization of the adenosine A1 receptor (A 1 AR) and β 2 adrenergic receptor (β 2 AR) in live cells, a decrease of the luminescent signal is observed when internalized receptors are excluded from interactions with the cell‐impermeable LgBiT [81,117] . Recently, surface‐specific labeling by the HiBiT‐LgBiT system was used to study the potential of the relaxin family receptor‐1 (RXFP 1 ) to undergo ligand‐induced homodimerization [118] . The assay was based on the distance‐dependent bioluminescence resonance energy transfer (BRET) between a luciferase‐ and mCitrine‐tagged RXFP 1 .…”

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

“…The numerous studies have demonstrated nanoBRET to utilize a broad range of fluorophores—from different fluorescent proteins (CyOFP, red-shifted CyOFP1, mNeonGreen, mScarlet, mCitrine, TurboFP635, mOrange, TagRFP, etc.) [ 185 , 186 , 187 , 188 , 189 , 190 ] to fluorescent dyes (TAMRA, BODIPY, 4-nitro-7-aminobenzofurazan (NBD), Alexa Fluor TM , etc.) [ 191 , 192 , 193 , 194 , 195 ].…”

“…Comprehensive reviews on NanoBRET methodology and applications have been published recently [ 197 , 198 ]. NanoBRET is successfully used for studying protein–protein interactions [ 184 , 199 ], or investigations on receptor activation [ 190 , 199 , 200 ], oligomerization [ 201 ], conformational states [ 186 ], proximities [ 189 , 202 ], and receptor–ligand interactions [ 200 , 201 , 203 ]. NanoBRET technology has been also applied for in vivo imaging.…”