Involvement of β-arrestin-2 and clathrin in agonist-mediated internalization of the human cannabinoid CB2 receptor.

The CB2 cannabinoid receptor is a promising therapeutic target for the treatment of inflammatory diseases, neuropathic pain, liver diseases, cancer and cardiovascular diseases. Obtaining detailed information on the internalization and trafficking of the human CB2 receptor in response to agonist will have a significant impact on drug discovery. Visualization and quantitative detection of EGFP-tagged CB2 receptor showed that, upon WIN55,212-2 stimulation, the CB2 receptor was rapidly internalized in a dose- and time-dependent manner from the cell membrane into the cytoplasm. Pretreatment with hypertonic sucrose, MDC clathrin inhibitor, or siRNA-mediated knock-down of clathrin heavy chain led to significant inhibition of agonist-induced CB2 internalization. Using the RNA interference method, we showed that knockdown of β-arrestin2 expression significantly impaired receptor internalisation. Further investigation demonstrated that the internalized CB2 receptors were co-localized with the early endosome probe and were recycled to the cell surface after the removal of agonist, but treatment with specific cell-permeable proteasome inhibitor MG132 a inhibited the recycling of internalized CB2 receptor, suggesting that the proteasome-mediated degradation pathway may be involved in CB2 internalization. Moreover, the single residue Ser(352) and residue cluster S(335)S(336)T(338)T(340) at the C-terminal tail are shown to be essential for receptor phosphorylation and β-arrestin2 association. These data provide new insights into the mechanisms regulating agonist-mediated internalization and trafficking of the human CB2 receptor.