Differential β‐arrestin2 requirements of constitutive and agonist‐induced internalization of the CB1 cannabinoid receptor

Abstract

CB 1 cannabinoid receptor (CB 1 R) undergoes both constitutive and agonist-induced internalization, but the underlying mechanisms of these processes and the role of βarrestins in the regulation of CB 1 R function are not completely understood. In this study, we followed CB 1 R internalization using confocal microscopy and bioluminescence resonance energy transfer measurements in HeLa and Neuro-2a cells. We found that upon activation CB 1 R 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 CB 1 R. In contrast, neither β-arr2-V54D nor β-arr2-specific siRNA had a significant effect on the constitutive internalization of CB 1 R. However, both constitutive and agonist-induced internalization of CB 1 R 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 CB 1 R, 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 CB 1 R are different.

Assessment of Biased Agonism among Distinct Synthetic Cannabinoid Receptor Agonist Scaffolds

“…Distinct patterns can be observed in the resulting bias plots, allowing the SCRAs to be grouped into 6 classes (each represented in Figures 1 and 4C in a different color). Also here, 5F-APINACA (12) and EG-018 (18) (as well as EG-2201 (20) and MDMB-CHMCZCA (21), as shown in Figure S7) stand out as unique SCRAs, clearly displaying distinct shapes in the bias plot as compared to the remaining SCRAs (represented in Figure 4C in dark pink and light blue, respectively). (C8)-CP47497 (1), JWH-250 (6), BB-22 (11), and 5F-CUMYL-PINACA (17) display a pattern similar to that of (S)-5F-MDMB-PINACA (14) (depicted in green in Figure 4C), all showing a subtle preference toward β-arrestin2 signaling.…”

“…Importantly, all these observations are relative to the reference compound CP55940. Apart from the similar profiles for the carbazole-core-containing EG-018 (18), EG-2201 (20), and MDMB-CHMCZCA (21), no obvious structural features could be found that may underlie these distinct patterns. An overview of the bias plots of all SCRAs, individually plotted together with the reference compound CP55940, is given in Figure S7.…”

“…An initial panel of 19 SCRAs, encompassing a remarkable degree of chemical diversity and belonging to distinct classes, was evaluated for the ability to recruit β-arrestin2 versus mini-Gα i . As outlined further in the "Pathway Bias" section, two additional compounds, EG-2201 (20) and MDMB-CHMCZCA (21) were only included at a later stage, bringing the total panel of evaluated SCRAs to 21. An overview of the chemical structures of the selected SCRAs is given in Figure 1.…”

“…Upon binding of β-arrestin2 to CB 1 , desensitization and internalization of the receptor is induced. 21 While some studies have reported only little to no β-arrestin1 recruitment to CB 1 following receptor activation, 21 other studies indicated that β-arrestin1 provoked the activation of the extracellular signalregulated kinase pathway, thus also regulating long-term cellular events. 22,23 This was corroborated by structural studies that observed binding of β-arrestin1 to the distal part of CB 1 's C-terminus.…”

SGIP1 modulates kinetics and interactions of the cannabinoid receptor 1 and G protein‐coupled receptor kinase 3 signalosome

“…Activation of G proteins recruits GRK2/3 via interaction with G protein Gβγ dimer and fosters association between GRK2/3 and the receptor, which is consequently phosphorylated within its intracellular C terminus by the kinase (Homan & Tesmer, 2015; Nogues et al, 2017). Desensitization of CB1R expressed in Xenopus oocytes was shown to be dependent on the presence of both GRK3 and β‐arrestin2 (Daigle, Kearn & Mackie, 2008; Daigle, Kwok, et al, 2008; Gyombolai, Boros, Hunyady & Turu, 2013). Our data are in accord with these findings: the kinase activity of GRK2/3 is essential for efficient β‐arrestin2 recruitment to CB1R.…”