Towards monitoring conformational changes of the GPCR neurotensin receptor 1 by single-molecule FRET

Abstract

Neurotensin receptor 1 (NTSR1) is a G protein-coupled receptor that is important for signaling in the brain and the gut. Its agonist ligand neurotensin (NTS), a 13-amino-acid peptide, binds with nanomolar affinity from the extracellular side to NTSR1 and induces conformational changes that trigger intracellular signaling processes. Our goal is to monitor the conformational dynamics of single fluorescently labeled NTSR1. For this, we fused the fluorescent protein mNeonGreen to the C terminus of NTSR1, purified the receptor fusion protein from membranes, and reconstituted NTSR1 into liposomes with polar lipids. Using single-molecule anisotropy measurements, NTSR1 was found to be monomeric in liposomes, with a small fraction being dimeric and oligomeric, showing homoFRET. Similar results were obtained for NTSR1 in detergent solution. Furthermore, we demonstrated agonist binding to NTSR1 by time-resolved single-molecule Förster resonance energy transfer (smFRET), using neurotensin labeled with the fluorophore ATTO594.

NTSR1 glycosylation and MMP dependent cleavage generate three distinct forms of the protein

“…Fig. 5), we excluded the possibility of the NTSR1-high form to be a dimer or polymer form of the protein [30][31][32][33] . As most GPCRs are known to be glycosylated 25,34 and as NTSR1 contains three putative N-glycosylation sites in its N-terminal end (Fig.…”

Monitoring oligomerization dynamics of individual human neurotensin receptors 1 in living cells and in SMALP nanodiscs

“…In contrast, the fluorescent protein mNeonGreen exhibits a similarly high absorbance coefficient (maximum =116,000 M -1 •cm -1 at 505 nm; www.fpbase.org) combined with a much higher QY=0.8 24 . Therefore, we expected that mNeonGreen as the marker fused to the C-terminus of the NTSR1, i.e., NTSR1-mNG, should provide an optimal signal-to-noise ratio for imaging and for single-molecule detection 3 .…”

“…GPCRs are membrane proteins that can recognize extracellular ligands, respond to mechanical stimuli, light, or pheromones, and convert them into intracellular signals. Understanding the molecular mechanisms of these receptors builds the basis for therapeutic drug development, as one-third of all drugs used today are targeting GPCRs 2,3 . Only 30 of the 750 identified GPCRs are currently targets for clinical drugs 4,5 .…”

“…Recent single-molecule studies revealed a dynamic monomer/dimer equilibrium of the purified receptor in artificial lipid bilayers 3,6,[21][22][23] . For comparison with the native lipid environment, we started to investigate the oligomerization state of NTSR1 in living human cells.…”