Investigation On The Molecular Mechanisms For The Regulation Of Cannabinoid Receptor-G-protein Coupling And Their Signal Transduction Pathways

Cannabis is an ancient medicinal plant, its first use of records can be traced back 5000 years ago. The latest study found that cannabinoids work through binding and activation of two types of cannabinoid receptors CB1 and CB2 in specific cell surface. CB1 receptors are among the most abundant G protein-coupled receptors in the central nervous system where they mediate the majority of the psychotropic and behavioral effects of cannabis. In comparison, CB2 receptors are expressed in peripheral immune tissues suggesting a role in immune response. Therefore, further investigation of cannabinoid receptors structure and function relationship and their signal transduction pathways could further clarify the molecular mechanisms of related diseases, provide a better theoretical basis and broader thought to develop more effective and innovative therapeutic medicine.7TM receptor coupling selectivity to G-proteins is controlled by a large contact area that involves several portions of the receptor and each subunit of the G protein. However, fidelity structure determinants discriminate between different G-proteins are not well understood. The CB1 receptor is primarily known to be functionally coupled to the Gi-mediated pathway, through which it negatively regulates cyclic AMP production. However, several lines of evidence suggest that CB1 receptors can also stimulate the formation of cAMP through coupling to Gs. In the present study, we demonstrate that the CB1 receptor is capable of dually coupling to Gs-mediated cAMP accumulation and Gi-induced activation of ERK1/2 and Ca2+mobilization. In comparison, the cannabinoid CB2 receptor selectively couples to Gi and mediates an inhibitory effect on cAMP production. Through the analysis of CB1/CB2 chimeric receptors, the second intracellular loop (ICL2) of the CB1 receptor was primarily responsible for mediating selective coupling to Gs and Gi, whereas the C-terminal region of the receptor plays an important role in defining the effectiveness of G protein activation. Furthermore, the results obtained from mutagenesis indicate that mutation of Leu-222 in ICL2 to either Ala or Pro resulted in a switch in G protein coupling from Gs to Gi. Moreover, mutants with replacement of Leu-222 with either Ile or Val led to. balanced coupling of the receptor with Gs and Gi. We also noted that the three positive charged amino acids (His-219,Arg-220 and Arg-226) presented at the ICL2 are associated with selective coupling to Gs and essential for CB1 keeping in constraint state. Data from theoretical modeling of the GPCR-Ga complex reveal that different mutations of Leu-222 could lead to distinct local conformation, which constitutes the molecular basis of selective coupling of CB1 receptor to different G-proteins. Taken together, these results first demonstrate that L222, within a highly conserved DRY(X)6L motif in CB1 receptor, plays a critical role in mediating selective coupling to Gs and Gi, two contrary proteins with opposing effects in regulation of cAMP, and this mechanism likely plays an important role in mediating the specificity of other members of the GPCR family. Our studies provide new insight into the mechanisms governing the coupling of the CB1 receptor to G proteins and cannabinoid-induced tolerance and biphasic effects.In contrast to CB1, the CB2 only couple to Gi and inhibit adenylyl cyclase activity. In an attempt to convert the CB2 into a Gs-linked adenylate cyclase activating receptor we proceeded to make global domain replacement of the intracellular face of the CB2 with the corresponding regions of the CB1. The ICL2 exchange resulted in impaired cAMP inhibition and had significantly basal cAMP accumulation in the absence of agonist. Furthermore, results from multi-domains chimeras indicate replacement both of ICL2 and Cter resulted in a chimera capable of induce intracellular cAMP upon agonist treatment, and this effect was synergistically enhanced in the presence of forskolin. Moreover, we also mutated the pro-139 in CB2 receptor corresponding to L222 in CB1 receptor into alanine and leucine, and found that the P139L abrogated the Gi coupling while the P139A could still couple to Gi but with moderate impairment in inhibition of cAMP accumulation. Taken together, these results demonstrated that the ICL2 can alter the binding affinities of G proteins to which a receptor is coupled, but interaction among multi-loops is thoroughly involved in fully determining G-protein selectivity. Moreover, these results also initially suggested that the residue located at DRY(X)6L motif serves as a key site responsible for selective coupling to Gi in class A GPCRs. Desensitization and receptor trafficking tightly control the temporal and spatial regulation of GPCR signaling. We examined agonist-induced internalization and sorting mechanisms using cannabinoid CB2 receptors fused to green fluorescent protein (EGFP). The cannabinoid agonist WIN55,212-2 can induce CB2 receptor internalization in dose-dependent and time-course manner. CB2 receptors internalized initially via clathrin-coated pits but did not require activated Gi-protein. Furthermore, agonist induced internalization was not blocked by AM630 and required ubiquitin-proteasome pathway involved in this process. Upon short time agonist challenge, internalized CB2 receptors were mainly sorted to recycling endosome and then slowly recycled to the cell surface. Lastly, we also characterized the structure determinants involved in both receptor internalization and G-protein activation, and results indicating that multi-region of the carboxy terminus of the receptor was necessary for internalization as well as G-protein coupling. Collectively, this study show that CB2 receptor trafficking is dynamically regulated by cannabimimetic drugs.