Orexin receptor 2 (OX₂R) exhibits ligand-dependent spatio-temporal pharmacology through Gα_i:β-arrestin dynamics in response to Orexin-A and Orexin-B

Recent advances in our understanding of GPCR pharmacology have caused a paradigm shift in our perception of GPCR signaling. Intracellular signaling of receptors, formation of non-canonical effector complexes, β-arrestin's actions as a signal transducer, and the ability for multiple proteins to occupy the intracellular sites of the receptor simultaneously are all now widely accepted concepts. Despite this, our knowledge of Orexin Receptor 2 (OX₂R) pharmacology has remained largely unchanged. In this study, OX₂R pharmacology was investigated using various BRET techniques with a focus on spatio-temporal mechanisms. Upon stimulation with Orexin-A (OX-A) or Orexin-B (OX-B), dissociation and/or rearrangement of G_q, G_i and G_s with distinct, ligand-dependent kinetic effects was observed. Robust trafficking of G_i, but not G_q to intracellular compartments was observed with both ligands, however only G_i dissociation in response to OX-B exhibited resistance to extracellular agonist washout. Gα_i and β-arrestin2 came into closer proximity following agonist stimulation. Together, these findings indicate a series of complex signaling mechanisms that can be ligand-dependent, G protein-dependent or both. This warrants further research to determine the physiological relevance of OX₂R spatio-temporal pharmacology.