Heteromultimerization of Cannabinoid CB1 Receptor and Orexin OX1 Receptor Generates a Unique Complex in Which Both Protomers Are Regulated by Orexin A

Agonist-induced internalization was observed for both inducible and constitutively expressed forms of the cannabinoid CB1 receptor. These were also internalized by the peptide orexin A, which has no direct affinity for the cannabinoid CB1 receptor, but only when the orexin OX1 receptor was co-expressed along with the cannabinoid CB1 receptor. This effect of orexin A was concentration-dependent and blocked by OX1 receptor antagonists. Moreover, the ability of orexin A to internalize the CB1 receptor was also blocked by CB1 receptor antagonists. Remarkably, orexin A was substantially more potent in producing internalization of the CB1 receptor than in causing internalization of the bulk OX1 receptor population, and this was true in cells in which the CB1 receptor was maintained at a constant level, whereas levels of OX1 could be varied and vice versa. Both co-immunoprecipitation and cell surface, homogenous time-resolved fluorescence resonance energy transfer based on covalent labeling of N-terminal SNAP and CLIP tags present in the extracellular N-terminal domain of the receptors confirmed the capacity of these two receptors to heteromultimerize. These studies confirm the capacity of the CB1 and OX1 receptors to interact directly and demonstrate that this complex has unique regulatory characteristics. The higher potency of the agonist orexin A to regulate the CB1-OX1 heteromer compared with the OX1-OX1 homomer present in the same cells and the effects of CB1 receptor antagonists on the function of orexin A suggest an interplay between these two systems that may modulate appetite, feeding, and wakefulness.