  G protein-coupled receptors (GPCRs) are a large superfamily of membrane
proteins that are activated by extracellular small molecules or photons.
Neurotensin receptor 1 (NTSR1) is a GPCR that is activated by neurotensin, i.e.
a 13 amino acid peptide. Binding of neurotensin induces conformational changes
in the receptor that trigger the intracellular signaling processes. While
recent single-molecule studies have reported a dynamic monomer - dimer
equilibrium of NTSR1 in vitro, a biophysical characterization of the
oligomerization status of NTSR1 in living mammalian cells is complicated. Here
we report on the oligomerization state of the human NTSR1 tagged with mRuby3 by
dissolving the plasma membranes of living HEK293T cells into 10 nm-sized
soluble lipid nanoparticles by addition of styrene-maleic acid copolymers
(SMALPs). Single SMALPs were analyzed one after another in solution by
multi-parameter single molecule spectroscopy including brightness, fluorescence
lifetime and anisotropy for homoFRET. Brightness analysis was improved using
single SMALP detection in a confocal ABELtrap for extended observation times in
solution. A bimodal brightness distribution indicated a significant fraction of
dimeric NTSR1 in SMALPs or in the plasma membrane, respectively, before
addition of neurotensin.
