Cannabinoid Receptor Signaling is Dependent on Sub-Cellular Location

G protein-coupled receptors (GPCRs) are membrane bound signaling molecules that regulate many aspects of human physiology. Recent advances have demonstrated that GPCR signaling can occur both at the cell surface and internal cellular membranes. Our findings suggest that cannabinoid receptor 1 (CB1) signaling is highly dependent on its subcellular location. We find that intracellular CB1 receptors predominantly couple to Gi while plasma membrane receptors couple to Gs. Here we show subcellular location of CB1, and its signaling, is contingent on the choice of promoters and receptor tags. Heterologous expression with a strong promoter or N-terminal tag resulted in CB1 predominantly localizing to the plasma membrane and signaling through Gs. Conversely, CB1 driven by low expressing promoters and lacking N-terminal genetic tags largely localized to internal membranes and signals via Gi. Lastly, we demonstrate that genetically encodable non-canonical amino acids (ncAA) offer a solution to the problem of non-native N-terminal tags disrupting CB1 signaling. We identified sites in CB1R and CB2R which can be tagged with fluorophores without disrupting CB signaling or trafficking using (trans-cyclooctene attached to lysine (TCO*A)) and copper-free click chemistry to attach fluorophores in live cells. Together, our data demonstrate the origin of location bias in cannabinoid signaling which can be experimentally controlled and tracked in living cells through promoters and novel CBR tagging strategies.