Characterization Of Agonist-Mediated Signaling And Internalization Of Human Cannabinoid Receptor CB2

G protein-coupled receptors (GPCRs) are a class of integral membrane proteins that contain seven membrane-spanning helices and represent one of the largest and most diverse membrane protein superfamily in mammals, involved in signal transduction across membranes. Dysregulation of GPCR signaling has been linked to several pathologies such as depression, schizophrenia, insomnia, Alzheimer’s, Parkinson’s disease, cardio-cerebral vascular diseases, inflammation, color blindness and asthma and so on. GPCRs are important drug targeted for the pharmaceutical industry, and more than20%of currently top-selling drugs are GPCR related. Therefore, the study on the relationship between structure and function of CPCRs, as well as their complex signaling pathways and signal transduction mechanisms, will boost drug market undoubtfully. CB2is one of the GPCRs members. CB2receptors, expressed in all immune cells, was found to play a variety role in regulation of immune cell migration and proliferation. Therefore, a better understanding of CB2mediated endocytosis and its signal transduction mechanism will be extremely important in elucidating the importance of the immunomodulatory role of CB2and play a decisive role in developing anti-inflammation drugs.Our research indicated that the CB2expressed in HEK293cells rapidly internalized via clathrin upon exposure to agonist WIN55,212-2,and mainly regulated by arrestin3.Inhibition of clathrin function by pretreatment with sucrose or clathrin inhibitor-MβDC or down-regulation of clathrin expression by transfection with clathrin siRNA resulted in a significant inhibition of CB2endocytosis, indicating that the endocytosis of CB2was clathrin vesicle-dependent. Over-expression of dynamin dominant negative mutant K44A also inhibited the endocytosis of CB2, suggesting the dynamin is also involved in the regulation of endocytosis of CB2. Moreover, internalized CB2receptor was co-localized with endosome marker-Alexa Fluor594-labeled transferrin but not lysosomal marker-lysotracker DND-99. Internalized CB2receptors were partly recycled to the membrane while pretreatment with lysosomal inhibitor-MG132dramatically inhibited CB2endocytosis, indicating that the internalized CB2receptor were partly trafficked back to the cell membrane via endosomal pathway, and some may enter the ubiquitin degradation pathway.Pretreatment with Pertussis toxin PTX significantly inhibited WIN55,212-2mediated inhibition of forskolin-induced intracellular cAMP levels, inhibition of WIN55,212-2-mediated ERK1/2downstream signaling, but not the CB2endocytosis, suggesting the involvement of Gi protein in CB2downstream signals, but not in endocytosis.Studies from CB2chimeras with replacement of the intracellular face of the CB2with the corresponding regions of the CB1and several site-directed mutants in the ICL2revealed that the ICL2of CB1confers the CB2chimera dually couple to Gi and Gs. Further studies showed that mutation of Pro-139into Leu can stimulate cAMP production, and activate ERK1/2signaling through the cAMP/PKA pathway and Gi-dependent pathway, indicating that Pro-139, within a highly conserved DRY(X)5PP motif in CB2receptor, plays a critical role in mediating selective coupling to G proteins. We also studied the mutants within the highly conserved DRY(X)5PP motif by substitution of Leu135into Ala, He and Val, and found that the size of amino acids and the length of side chain in Leu135are very closely related to receptor folding, self-activation and G-protein coupling.Using deletion and site-directed mutagenesis strategies, we found that the carboxy-terminal played an important role in the regulation of internalization and desensitization of CB2receptor. Either deletion of CB2carboxy-terminal E341-C360or substitution of S335, S336, T338and T340to alanine (SSTT/A mutation) significantly impacts the endocytosis and desensitization of CB2receptor. Decreasing binding of arrestin3to Cter-1and SSTT/A mutation was observed, the Cter-1deletion mutant and SSTT/A mutation almost completely lost its binding affinity to arrestin3, and Cter-1deletion mutant and SSTT/A mutant have sustained ERK1/2signal than the wild type. These data supports a key role for the phosphoryolation and desensitization of the serine and threonine residues within the distal carboxyl terminus of the receptor.In conclusion, our study clarified the detailed mechanism of CB2receptor internalization and unveiled the critical role of the intracellular loop and the amino acid residues in the regulation of CB2-G-protein coupling, and the effects of amino acids size on receptor folding, and the involvement of carboxyl terminus in the internalization, desensitization and arrestin3binding process, and found that the participation of G-protein pathway in the phosphorylation of ERK1/2. We hope that the follow-up experiments will further detail the CB2specific signaling pathways from activation to production of ERK1/2.