Molecular determinants of orexin receptor-arrestinubiquitin complex formation

Background and Purpose: The orexin system regulates a multitude of key physiological processes, particularly involving maintenance of metabolic homeostasis. Consequently, there is considerable potential for pharmaceutical development for the treatment of disorders from narcolepsy to metabolic syndrome. It acts through the hormonal activity of two endogenous peptides, orexin A binding to orexin receptors 1 and 2 (OX(1) and OX(2)) with similar affinity, and orexin B binding to OX(2) with higher affinity than OX(1) receptors. We have previously revealed data differentiating orexin receptor subtypes with respect to their relative stability in forming orexin receptor-arrestin-ubiquitin complexes measured by BRET. Recycling and cellular signalling distinctions were also observed. Here, we have investigated, using BRET, the molecular determinants involved in providing OX(2) receptors with greater β-arrestin-ubiquitin complex stability. Experimental Approach: The contribution of the C-terminal tail of the OX receptors was investigated by bulk substitution and site-specific mutagenesis using BRET and inositol phosphate assays. Key Results: Replacement of the OX(1) receptor C-terminus with that of the OX(2) receptor did not result in the expected gain of function, indicating a role for intracellular domain configuration in addition to primary structure. Furthermore, two out of the three putative serine/threonine clusters in the C-terminus were found to be involved in OX(2) receptor-β-arrestin-ubiquitin complex formation. Conclusions and Implications: This study provides fundamental insights into the molecular elements that influence receptor-arrestin-ubiquitin complex formation. Understanding how and why the orexin receptors can be functionally differentiated brings us closer to exploiting these receptors as drug targets. Linked Articles: This article is part of a themed section on Orexin Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-2