DOWNSTREAM TRANSLATION INITIATION GENERATES POST-TRANSCRIPTIONAL TYPE-1 CANNABINOID RECEPTOR N-TERMINAL VARIANTS

The type-1 cannabinoid receptor (CB1) is a G-protein-coupled receptor (GPCR) involved in multiple brain processes. In neurons, CB1 is predominantly localised at the presynaptic membrane, where it detects endocannabinoids released from the postsynaptic neuron to suppress neurotransmitter release. Additionally, CB1 is also present in mitochondrial membranes, where it regulates cellular respiration and energy production. Interestingly, deletion of the first 22 amino acids selectively disrupts mitochondrial targeting, while preserving synaptic signalling. However, the mechanisms that control CB1 sorting to the presynapse and mitochondria remain unknown. Our results indicate that post-transcriptional mechanisms generate CB1 N‑terminal variants, providing a potential molecular basis for the different subcellular localisations of the receptor. Specifically, by disrupting the annotated start codon in CB1 constructs, together with additional manipulations of the upstream Kozak sequence and downstream start codons, we demonstrated that CB1 translation can initiate downstream at alternative methionines within its N‑terminal domain, producing truncated receptor variants. At the endogenous level, the usage of alternative downstream translation initiation sites (TIS) was confirmed by ribosome profiling. Additionally, we found that the N-terminal tail of full-length CB1 is cleaved by a disintegrin and metalloprotease-containing protein 10 (ADAM10), suggesting a two-step regulatory mechanism controlling CB1’s N‑terminal domain. Further understanding the mechanisms that regulate the expression of these N-terminal variants and defining their specific functional impacts will provide new insights into the molecular determinants of CB1’s diverse functions, help identify more selective drug targets, and ultimately support the development of safer cannabinoid-based therapeutic approaches.