Biophysical insights into orexin receptor-β-arrestin complexes and their role in cellular signalling

[Truncated abstract] The orexin endocrine system has been associated with a range of physiological functions including regulation of sleep and wake states, energy metabolism, addictive behaviour and modulation of stress hormones. The orexin system is characterized by two primarily hypothalamic endogenous peptide agonists that specifically stimulate two G protein-coupled receptors, orexin receptor 1 (OxR1) and orexin receptor 2 (OxR2) present in tissues of the central nervous system, and also in the periphery. Activation of these receptors primarily stimulates the Gq/phospholipase C/protein kinase C signalling pathway, a process that is dampened by specific recruitment and binding of β- arrestin proteins. Furthermore, β-arrestins may confer a wide range of regulatory and signalling functions to activated receptor complexes in addition to inhibiting G protein-mediated signalling, including internalization, G proteinindependent signalling and degradation of the activated receptor complex. It has been previously detected that these receptors display subtype-specific differences in β-arrestin recruitment that is only observed over extended periods of up to several hours of agonist treatment utilizing extended bioluminescence resonance energy transfer (eBRET) techniques. In addition, these receptors displayed altered abilities to recycle rapidly upon activation. This work aimed to explore and contrast key structural and functional properties of human orexin receptor-β-arrestin association and complex formation upon heterologous expression in a cell line. Key structural determinants in the C-terminus of OxR2 were found to alter the ability of OxR2 to bind with β-arrestin. It was found that although primary structure is largely implicated in the ability of β-arrestins to bind some GPCRs, it appears that secondary structure may be equally as important...