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

Anika Westphal; Hendrik Sielaff; Stefanie Reuter; Thomas Heitkamp; Ralf Mrowka; Michael Börsch

doi:10.1117/12.2506797

22 February 2019 Ligand-induced oligomerization of the human GPCR neurotensin receptor 1 monitored in living HEK293T cells

Anika Westphal, Hendrik Sielaff, Stefanie Reuter, Thomas Heitkamp, Ralf Mrowka, Michael Börsch

Author Affiliations +

Proceedings Volume 10882, Multiphoton Microscopy in the Biomedical Sciences XIX; 108820W (2019) https://doi.org/10.1117/12.2506797
Event: SPIE BiOS, 2019, San Francisco, California, United States

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. Förster resonance energy transfer between identical fluorophores detected by fluorescence anisotropy), by colocalization microscopy and by time-lapse imaging using structured illumination microscopy (SIM).

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Anika Westphal, Hendrik Sielaff, Stefanie Reuter, Thomas Heitkamp, Ralf Mrowka, and Michael Börsch "Ligand-induced oligomerization of the human GPCR neurotensin receptor 1 monitored in living HEK293T cells", Proc. SPIE 10882, Multiphoton Microscopy in the Biomedical Sciences XIX, 108820W (22 February 2019); https://doi.org/10.1117/12.2506797

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