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Differential β-arrestin2 requirements for constitutive and agonist-induced internalization of the CB1 cannabinoid receptor.

By October 31, 2013No Comments

pm2Differential β-arrestin2 requirements for constitutive and agonist-induced internalization of the CB1 cannabinoid receptor.

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Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary. pal.gyombolai@eok.sote.hu

Abstract

CB1 cannabinoid receptor (CB1R) undergoes both constitutive and agonist-induced internalization, but the underlying mechanisms of these processes and the role of β-arrestins in the regulation of CB1R function are not completely understood. In this study, we followed CB1R internalization using confocal microscopy and bioluminescence resonance energy transfer measurements in HeLa and Neuro-2a cells. We found that upon activation CB1R binds β-arrestin2 (β-arr2), but not β-arrestin1. Furthermore, both the expression of dominant-negative β-arr2 (β-arr2-V54D) and siRNA-mediated knock-down of β-arr2 impaired the agonist-induced internalization of CB1R. In contrast, neither β-arr2-V54D nor β-arr2-specific siRNA had a significant effect on the constitutive internalization of CB1R. However, both constitutive and agonist-induced internalization of CB1R were impaired by siRNA-mediated depletion of clathrin heavy chain. We conclude that although clathrin is required for both constitutive and agonist-stimulated internalization of CB1R, β-arr2 binding is only required for agonist-induced internalization of the receptor suggesting that the molecular mechanisms underlying constitutive and agonist-induced internalization of CB1R are different.
Copyright © 2013. Published by Elsevier Ireland Ltd.
PMID:

 

23541635

 

[PubMed – in process]

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Fig. 1.

BRET measurements showing the recruitment of β-arr1 and β-arr2 to CB1R, AT1R and β2AR upon agonist stimulus. (A) CB1R-mVenus was co-expressed with β-arr1-Rluc or β-arr2-Rluc in HeLa cells, and BRET was measured upon WIN55 (10 μM) stimulus. Measurements were baseline-corrected to vehicle curves (indicated by horizontal dashed line). Arrow indicates the time point of stimulation. Data are mean ± SEM. (B–D) BRET titration curves showing the relative affinities of AT1R (B), β2AR (C) and CB1R (D) to β-arrestin isoforms. HeLa cells were transfected with constant amounts of β-arr1-Rluc or β-arr2-Rluc, and varying amounts of the mVenus-tagged receptor, yielding different acceptor/donor ratios. Average BRET change between 3 and 5 min after agonist stimulus (100 nM angiotensin II (B), 1 μM isoproterenol (C) or 10 μM WIN55 (D)) was plotted against mVenus/Rluc intensity ratios measured at the beginning of each experiment. Data resulting from at least 3 independent experiments were fitted using non-linear regression with a one-site binding equation.

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Fig. 2.

Confocal microscopy analysis indicates class A β-arrestin binding pattern of CB1R. β-arr1-GFP (A–D) or β-arr2-GFP (E–H) and CB1R-mCherry were co-expressed in HeLa cells and analyzed by confocal microscopy. Under control conditions, β-arr1-GFP shows diffuse cytoplasmic and nuclear localization (A). After 20 min of WIN55 (10 μM) stimulus, no change in β-arr1-GFP distribution can be detected (C). β-arr2-GFP shows diffuse cytoplasmic localization in control cells (E, inset). After 20 min of WIN55 (10 μM) stimulus, β-arr2-GFP can be detected in punctuate structures, however only in the close proximity of the plasma membrane (G, inset, arrows indicate β-arr2-GFP puncta). A large proportion of CB1R is constitutively intracellular, reflecting to spontaneous endocytosis of the receptor (B, D, F, H). Images are representative from three independent experiments. Scale bar 10 μm.

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Fig. 3.

Agonist-induced internalization of CB1R is impaired by dominant-negative β-arr2. Halo-CB1R was expressed in HeLa cells alone (A–C) or together with wild-type β-arr2-RFP (D–G) or β-arr2-V54D-RFP (H–K), and analyzed with confocal microscopy after 15 min Halo-Alexa488 staining and 30 min vehicle (A, D, E, H, I) WIN55 (10 μM, B, F, G, J, K) or WIN55 + AM251 (10 μM + 30 μM, respectively, C) treatment. Vehicle treatment causes no substantial Halo-CB1R internalization (A, D, H) in either cell population, whereas WIN55 treatment leads to massive endocytosis of the receptor in control (B) or β-arr2-RFP- (F, G), but not in β-arr2-V54D-RFP- (J, K) expressing cells. WIN55-induced internalization is blocked by co-treatment with AM251 (C). Images are representative from three independent experiments. Scale bar 10 μm.

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Fig. 4.

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.

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Fig. 5.

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.

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Fig. 6.

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.

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Fig. 7.

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.

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Fig. 8.

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.

Corresponding author contact information
Corresponding author. Address: Department of Physiology, Faculty of Medicine, Semmelweis University, H-1444 Budapest, P.O. Box 259, Hungary. Tel.: +36 1 266 9180; fax: +36 1 266 6504.

Copyright © 2013 Published by Elsevier Ireland Ltd.

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