title: Molecular determinants of orexin receptor-arrestin-ubiquitin complex formation.
abstract: OBJECTIVE
  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 (OX₁ and OX₂) with similar affinity, and orexin B binding to OX₂ with higher affinity than OX₁ 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 OX₂ receptors with greater β-arrestin-ubiquitin complex stability.
  
  
  METHODS
  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.
  
  
  RESULTS
  Replacement of the OX₁ receptor C-terminus with that of the OX₂ 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 OX₂ receptor-β-arrestin-ubiquitin complex formation.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratory for Molecular Endocrinology-G Protein-Coupled Receptors, Western Australian Institute for Medical Research (WAIMR) and Centre for Medical Research, The University of Western Australia, Perth, WA, Australia.
authors.forename: Werner C
authors.initials: WC
authors.lastname: Jaeger
authors.forename: Ruth M
authors.initials: RM
authors.lastname: Seeber
authors.forename: Karin A
authors.initials: KA
authors.lastname: Eidne
authors.forename: Kevin D G
authors.initials: KD
authors.lastname: Pfleger
mesh_terms: D000595:Amino Acid Sequence; D019393:Arrestin / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D018698:Glutamic Acid / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009479:Neuropeptides; D064446:Orexin Receptors / Q000235:genetics / Q000378:metabolism*; D000068797:Orexins; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D013912:Threonine / Q000737:chemistry / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*
chemical_list: D019393:Arrestin; D018076:DNA, Complementary; C000596229:HCRT protein, human; D007295:Inositol Phosphates; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides; D064446:Orexin Receptors; D000068797:Orexins; D025801:Ubiquitin; D013912:Threonine; D018698:Glutamic Acid; D012694:Serine
keywords: BRET; GPCR; arrestin; hypocretin; orexin; ubiquitin