Agonist-induced internalization of CB₁R is impaired by β-arr2-specific siRNA. (A) Western blot analysis shows an approximately 50% reduction in the β-arr2 protein levels of β-arr2 siRNA-transfected cells compared to control siRNA-transfected cells. n = 3 B–E, HeLa cells were transfected with Halo-CB₁R and control (B, D) or β-arr2-specific (C, E) siRNA, and analyzed with confocal microscopy after 15 min Halo-Alexa488 staining and 30 min vehicle (B, C) or WIN55 (10 μM, D, E) treatment. Vehicle treatment causes no substantial Halo-CB₁R internalization in either cell population (B, C), whereas WIN55 treatment leads to massive endocytosis of the receptor in control (D), but not in β-arr2 (E) siRNA-transfected cells. Scale bar 10 μm. (F) Quantification of the data using an intracellular to total cell fluorescence ratio shows a significant increase in intracellular receptor number upon WIN55 stimulus in control siRNA-transfected cells, and this is significantly reduced in cells transfected with β-arr2 siRNA. Data are mean + SEM, n = 6, ^*p < 0.05, ns – not significant.

BRET measurements showing the β-arr2 dependence of agonist-induced CB₁R internalization. CB₁R-Sluc was co-expressed with ICAM-YFP in HeLa cells, and BRET was measured to follow the agonist-induced removal of the receptor from the plasma membrane. (A) BRET signal decrease upon WIN55 (10 μM) stimulus can be detected in cells co-transfected with pcDNA3.1 (open circles). Co-expression of wild-type β-arr2 has no significant impact on BRET change (closed triangles). Co-expression of β-arr2-V54D substantially reduces BRET signal decrease (open triangles). Data are all mean ± SEM, n = 8. (B) BRET signal decrease upon WIN55 (10 μM) stimulus can be detected in control siRNA-transfected cells (closed triangles). The BRET signal decrease is substantially reduced in cells transfected with β-arr2 siRNA (open triangles). Data are all mean ± SEM, n = 4. Measurements were baseline-corrected to vehicle curves (indicated by horizontal dashed lines). Arrows indicate the time point of stimulation.

Constitutive internalization of CB₁R is not affected by inverse agonist treatment or dominant-negative β-arr2. Halo-CB₁R was expressed in HeLa cells alone (A, B, C) or together with wild-type β-arr2-RFP (D, E) or β-arr2-V54D-RFP (F, G), and analyzed by confocal microscopy after 15 min Halo-Alexa488 staining and 5 h 45 min incubation at 37 °C, 5% CO₂. In control cells (A), a substantial amount of Halo-CB₁R can be detected intracellularly. Internalization is enhanced in the presence of WIN55 (10 μM, B) but not affected by AM251 (30 μM, C). Constitutive internalization of the receptor can be detected in cells expressing wild-type β-arr2-RFP (D, E) and also in cells expressing β-arr2-V54D-RFP (F, G, see cell indicated with arrow). Images are representative from three independent experiments. Scale bar 10 μm.

Constitutive internalization of CB₁R is not affected by β-arr2-specific siRNA. (A and C) HeLa (A) or Neuro-2a (C) cells were transfected with Halo-CB₁R and control or β-arr2-specific siRNA, and analyzed with confocal microscopy after 15 min Halo-Alexa488 staining and 5 h 45 min incubation at 37 °C, 5% CO₂. In both control and β-arr2 siRNA-transfected cells, substantial amounts of intracellular receptors can be detected. Quantification of the data using an intracellular to total cell fluorescence ratio shows no significant difference between control or β-arr2 siRNA-transfected cells after the 6 h of incubation. B and D, In the same experiments, control or β-arr2 siRNA-transfected HeLa (B) or Neuro-2a (D) cells were incubated at 37 °C, 5% CO₂ for 5 h 15 min followed by 15 min Halo-Alexa488 staining and 30 min WIN55 (10 μM) treatment. A substantial amount of internalized receptors was detected in control, but not in β-arr2 siRNA-transfected cells. Data quantification shows that internalization upon WIN55 stimulus after 5 h 30 min is significantly reduced in β-arr2 siRNA-transfected cells. Scale bar 10 μm. Data are mean + SEM, n = 3, ^*p < 0.05.

Constitutive internalization of CB₁R is impaired by clathrin heavy chain-specific siRNA. (A) HeLa cells were transfected with Halo-CB₁R and control or clathrin heavy chain-specific siRNA (clathrin siRNA), and analyzed with confocal microscopy after 15 min Halo-Alexa488 staining and 5 h 45 min incubation at 37 °C, 5% CO₂. A substantial amount of internalized receptors was detected in control, but not in clathrin siRNA-transfected cells. Quantification of the data using an intracellular to total cell fluorescence ratio shows significant difference between control or clathrin siRNA-transfected cells after the 6 h of incubation. (B) In the same experiments, control or clathrin siRNA-transfected HeLa cells were incubated at 37 °C, 5% CO₂ for 5 h 15 min followed by 15 min Halo-Alexa488 staining and 30 min WIN55 (10 μM) treatment. A substantial amount of internalized receptors was detected in control, but not in β-arr2 siRNA-transfected cells. Data quantification shows that internalization upon WIN55 stimulus after 5 h 30 min is significantly reduced in clathrin siRNA-transfected cells. Scale bar 10 μm. Data are mean + SEM, n = 3, ^*p < 0.05.