Endocrine Abstracts

Introduction: β-Arrestins are cytosolic proteins, which play key roles in the desensitization, internalization and signaling of G protein-coupled receptors. Currently, little is known about the role of β-arrestins in the internalization of the CB1 cannabinoid receptor (CB1R). Therefore, the interaction between β-arrestins and CB1R, and its role in receptor internalization were studied.

Materials and methods: The β-arrestin binding of CB1R was studied by confocal microscopy and bioluminescence resonance energy transfer (BRET). To follow CB1R internalization, plasma membrane CB1 receptors were selectively stained using Halo-labeling technique. Internalization of the receptors was also monitored by a BRET assay through measuring non-specific BRET between CB1R and a plasma membrane marker protein (ICAM).

Results: We found that upon activation CB1R binds transiently to β-arrestin2 (β-arr2), but not β-arrestin1. Dominant negative β-arr2 (β-arr2-V54D) or knock-down of β-arr2 by siRNA inhibited the agonist-induced internalization of CB1Rs. Similar inhibitory effects on agonist-induced CB1R internalization were also demonstrated using BRET measurements. In contrast, neither β-arr2-V54D nor β-arr2-siRNA had a significant effect on the constitutive internalization of CB1R.

Conclusions: We conclude that upon activation, CB1R binds β-arr2 in a transient manner (class A GPCR), and this binding is required for the agonist-induced internalization of the receptor. In contrast, constitutive CB1R internalization is independent of the β-arr2 binding of the receptor, suggesting that the molecular mechanisms underlying these two processes are different.

Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Funding: This work was supported, however funding details unavailable.