Fig. 7: Bystander BRET shows the bivalent ligands’ ability to induce cointernalization of D3R-NTSR1.
Bystander BRET between D3R-Rluc or NTSR1-Rluc and rGFP-CAAX as membrane marker or rGFP-FYVE as endosome marker is used to monitor receptor trafficking. a, b Time course of receptor internalisation. Stimulation of NTSR1 with NT(8-13) results in time-dependent sequestration and internalisation as detected by a decreased BRET signal between Rluc and rGFP-CAAX (n = 4) and an increased BRET signal between Rluc and rGFP-FYVE (n = 3). In cells devoid of β-arrestin1 and β-arrestin2, a a small fraction of NTSR1 (n = 3) is sequestered from the cell membrane, b but does not translocate into FYVE-labelled endosomes (n = 3). Internalisation of the D3R is not observed (n = 4). Data are displayed as individual results from biologically independent experiments. c, d Stimulating D3R-Rluc-NTSR1 coexpressing cells with the bivalent ligands results in D3R trafficking from the membrane to endosomes. NT(8-13) is able to induce a small increase in the endosomal compartment. Concentration-response curves for D3R internalisation reveal a bell-shaped profile for bivalent ligands 1d and 2d but not for monovalent NT(8-13). Data show mean ± s.e.m. of c n = 6 and d n = 5 independent experiments. .
