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 Ser352 and residue cluster S335S336T338T340 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.
Keywords: β-arrestin, cannabinoid, clathrin, internalization, mutagenesis function approaches, recycling.
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