  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.
