Differential β-arrestin2 requirements for 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 β-arrestins 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.

Mutation in the V2 vasopressin receptor gene, AVPR2, causes nephrogenic syndrome of inappropriate diuresis

“…The dissimilarity in the agonist induced and constitutive internalization observed in this study in case of I130N-V2R is not unique among GPCRs, since β-arrestin-dependent, agonist-induced endocytosis and β-arrestin-independent constitutive internalization was introduced earlier (22)(23)(24). It is also notable, that basal internalization is not obviously a consequence of constitutive activity (25).…”

Mutations in the ‘DRY’ motif of the CB1 cannabinoid receptor result in biased receptor variants

“…4A), raising the question whether the reduced E max value of CB 1 R-AAY in this assay reflects a true loss of agonist-induced G-protein activation, or it is caused merely by the absence of basal activity, while WIN55-induced G-protein activation remains unaffected. However, repeating these experiments in HeLa cells, where basal activity of CB 1 R is minimal (Gyombolai et al 2013), also showed substantially impaired WIN55-induced G-protein activation of CB 1 R-AAY (Supplementary Figure 1, see section on supplementary data given at the end of this article), suggesting that this mutation reduces not only the basal but also the WIN55-induced G o protein activation of CB 1 R. In contrast, CB 1 R-DAA proved to be G-protein-biased, as its b-arr recruitment in response to agonist stimulus was practically absent, but was still able to activate G-proteins, although at a lower level (w70% of the WT CB 1 R). According to our data, plasma membrane expression of this mutant is ca.…”

“…Moreover, CB 1 R shows basal G-protein activation and constitutive internalization under diverse cellular conditions (Leterrier et al 2006, McDonald et al 2007, Turu et al 2007. Like most other 7TMRs, CB 1 R also recruits b-arr following activation, which leads to the desensitization and internalization of the receptor (Kouznetsova et al 2002, Daigle et al 2008, Gyombolai et al 2013. The binding between b-arrs and CB 1 R is relatively weak, and the affinity of the receptor for b-arr2 (b-arr2) is substantially higher than that for b-arr1 (b-arr1) (Gyombolai et al 2013).…”

“…Like most other 7TMRs, CB 1 R also recruits b-arr following activation, which leads to the desensitization and internalization of the receptor (Kouznetsova et al 2002, Daigle et al 2008, Gyombolai et al 2013. The binding between b-arrs and CB 1 R is relatively weak, and the affinity of the receptor for b-arr2 (b-arr2) is substantially higher than that for b-arr1 (b-arr1) (Gyombolai et al 2013). Furthermore, b-arr1 recruitment of CB 1 R appears to be agonist-dependent (Laprairie et al 2014, Flores-Otero et al 2014.…”

Involvement of clathrin and -arrestins in Aggregatibacter actinomycetemcomitans endocytosis of human vascular endothelial cells

“…As typical of G proteins coupled receptors (GPCRs), PAFR activation by PC could be regulated by β-arrestins (McLaughlin et al, 2006;Eckels et al, 2009). β-Arrestins could bind to activated GPCRs and clathrin to promote the clathrin-mediated endocytosis and transcytosis (Shenoy and Lefkowitz, 2011;Gyombolai et al, 2013). The role of β-arrestins in inflammation process has been well known by regulation of chemokine responsiveness (Vroon et al, 2006;Cattaruzza et al, 2013;Porter et al, 2010).…”