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

TL;DR

This study investigates the oligomerization dynamics of human neurotensin receptor 1 in living cells and nanodiscs, revealing ligand-induced changes using single-molecule techniques in a novel nanodisc system.

Contribution

It introduces a method to analyze NTSR1 oligomerization in living cell membranes using SMALP nanodiscs and single-molecule trapping, providing new insights into receptor dynamics.

Findings

Ligand binding alters NTSR1 oligomerization state.

SMALPs effectively solubilize NTSR1 from cell membranes.

Single-molecule trapping reveals dynamic receptor behavior.

Abstract

The human neurotensin receptor 1 (NTSR1) is a G protein-coupled receptor. The receptor is activated by a small peptide ligand neurotensin. NTSR1 can be expressed in HEK cells by stable transfection. Previously we used the fluorescent protein markers mRuby3 or mNeonGreen fused to NTSR1 for EMCCD-based structured illumination microscopy (SIM) in living HEK cells. Ligand binding induced conformational changes in NTSR1 which triggered the intracellular signaling processes. Recent single-molecule studies revealed a dynamic monomer/dimer equilibrium of this receptor in artificial lipid bilayers. Here we report on the oligomerization state of human NTSR1 from living cells by trapping them into lipid nanodiscs. Briefly, SMALPs (styrene-maleic acid copolymer lipid nanoparticles) were produced directly from the plasma membranes of living HEK293T FlpIn cells. SMALPs with a diameter of 15 nm were soluble and stable. NTSR1 in SMALPs were analyzed by single-molecule intensity measurements one membrane patch at a time using a custom-built confocal anti-Brownian electrokinetic trap (ABEL trap) microscope. We found oligomerization changes before and after stimulation of the receptor with its ligand neurotensin.