Decoding the Signaling through Homomeric and Heteromeric Cannabinoid CB1 Receptors

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Cannabinoid CB1 receptors are one of the most abundantly expressed G-protein coupled receptors (GPCRs) in the central nervous system and play critical roles in various neurophysiological processes such as reward, emotion and motor activity. Growing evidence supports that GPCRs form heteromers that cause significant changes in their signaling. Utilizing electron microscope immunocytochemistry, CB1 receptors were found to co-localize with dopamine D2 receptors in the somata and dendrites of GABAergic neurons. Furthermore, fluorescence resonance energy transfer and co-immunoprecipitation studies have provided evidence for heteromerization of CB1 and D2 receptors in heterologous expression systems. CB1 receptors primarily signal through Pertussis toxin (PTX) sensitive Gi/o, leading to a decrease in cAMP levels. Interestingly, stimulating the CB1 receptor in a cell line co-expressing CB1 and D2 receptors was shown to increase cAMP levels, suggesting a possible Gs signaling switch through the CB1-D2 heteromer. Our preliminary data using GIRK channels as reporters of GPCR signaling in the Xenopus oocyte heterologous expression system corroborated the Gs switching behavior of CB1 receptors upon heteromerization with D2 receptors. To increase the signal to noise ratio of the CB1 receptor Gs signaling switch, we have turned to a calcium mobilization assay. Naturally, the promiscuous G alpha 16 belongs to the Gq superfamily and its activation leads to phospholipase C activation and calcium release. G alpha 16 chimeras in which 11 or 30 amino acids from the C-terminus were replaced by the corresponding sequence of Go and Gs were constructed. Calcium mobilization was monitored by GCaMP 6s in a FlexStation. We found that activation of Gi-coupled D2 receptor causes increases in intracellular calcium levels with G16/o chimeras but not with G16wt. Gs-coupled Glucagon receptor signal to both G16/s chimeras and G16wt but G16/s chimeras significantly left-shift the dose-response curve of Glucagon, indicating G16/s chimeras as good Gs signaling reporter. We are in the process of testing signaling of homomeric and heteromeric CB1 receptors through G16 chimeras in this system.