Figure 1
6TM-MOR interacts with β2-AR upon heterologous expression.
(a) 6TM-MOR (anti-FLAG, red) was located mainly intracellularly (ER marker anti-calnexin, green, left panel) when expressed alone (top row of each panel), but translocated to the plasma membrane (anti-Na+/K+-ATPase, green, right panel) when co-expressed with β2-AR (bottom row of each panel) in HEK293 cells. (b) β2-AR and 6TM-MOR co-internalized in response to 10 μM isoproterenol stimulation (ISO). EEA1 was used as an early endosomes marker (red, left panel) and 6TM-MOR was visualized with anti-FLAG (red, right panel). For (a) and (b) DNA was counterstained with Hoechst 33342 (blue). Scale bar: 10 μm. (c) Myc-tagged 6TM-MOR, but not 7TM-MOR, was immunoprecipitated with β2-AR from HEK293 cell lysates stably expressing FLAG-tagged β2-ARs. IP: precipitating antibody; WB: probing antibody; EV: empty vector. (d) Residues mediating the interaction between 6TM-MOR and β2-AR were identified along the putative surface of heterodimerization between helices H5 and H6 of 6TM-MOR and β2-AR. Residues predicted to mediate the interaction are indicated. When 6TM-MOR was co-expressed with the β2-AR mutant consisting of four alanine substitutions of the residues indicated in red, 6TM-MOR localized to intracellular compartments (Supplementary Fig. S1).
