Ligand-induced oligomerization of the human GPCR neurotensin receptor 1 monitored in living HEK293T cells

TL;DR

This study investigates how the human neurotensin receptor 1 (NTSR1), a GPCR, oligomerizes in living cells upon ligand binding, using advanced fluorescence microscopy techniques to monitor receptor states in real-time.

Contribution

It provides the first in vivo evidence of ligand-induced oligomerization of NTSR1 in the plasma membrane of living HEK293T cells.

Findings

Ligand binding promotes NTSR1 oligomerization in living cells.

Multiple fluorescence techniques confirm receptor oligomerization.

Oligomerization dynamics can be monitored in real-time.

Abstract

The human neurotensin receptor 1 (NTSR1) is a G protein-coupled receptor that can be expressed in HEK293T cells by stable transfection. Its ligand is a 13-amino-acid peptide that binds with nanomolar affinity from the extracellular side to NTSR1. Ligand binding induces conformational changes that trigger the intracellular signaling processes. Recent single-molecule studies revealed a dynamic monomer - dimer equilibrium of the receptor in vitro. Here we report on the oligomerization state of the human NTSR1 in the plasma membrane of HEK293T cells in vivo. We fused different fluorescent marker proteins mRuby3 or mNeonGreen to the C-terminus of NTSR1 and mutated a tetracysteine motif into intracellular loop 3 (ICL3) for subsequent FlAsH labeling. Oligomerization of NTSR1 was monitored before and after stimulation of the receptor with its ligand by FLIM and homoFRET microscopy (i.e. Forster resonance energy transfer between identical fluorophores detected by fluorescence anisotropy), by colocalization microscopy and by time-lapse imaging using structured illumination microscopy (SIM).