  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).
