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Molecular determinants of orexin receptor-arrestin-ubiquitin complex formation
Werner C Jaeger1, Ruth M Seeber, Karin A Eidne
1Laboratory 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.
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 (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.
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 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 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.
Insights
Researchers explored orexin receptor (OX) differences, finding that OX₂ receptor stability involves more than just its C-terminus. Specific C-terminal serine/threonine clusters are key to OX₂ receptor-β-arrestin-ubiquitin complex formation, aiding drug target development.
Area of Science:
- Pharmacology
- Molecular Biology
- Neuroscience
Background:
- The orexin system is crucial for metabolic homeostasis and presents therapeutic targets for narcolepsy and metabolic syndrome.
- Orexin A and B peptides interact with orexin receptors 1 (OX₁) and 2 (OX₂), with differing affinities.
- Previous studies using bioluminescence resonance energy transfer (BRET) showed distinct orexin receptor subtype stability in forming arrestin-ubiquitin complexes.
Purpose of the Study:
- To investigate the molecular determinants responsible for the enhanced stability of orexin receptor 2 (OX₂) β-arrestin-ubiquitin complexes.
- To elucidate the role of the C-terminal tail in differentiating OX₁ and OX₂ receptor function.
Main Methods:
- Utilized bioluminescence resonance energy transfer (BRET) assays to measure complex stability.
- Employed bulk substitution and site-specific mutagenesis of C-terminal tails.
- Conducted inositol phosphate assays to assess receptor signaling.
Main Results:
- Replacing the OX₁ receptor C-terminus with that of OX₂ did not confer OX₂-like stability, suggesting intracellular domain involvement.
- Two of three putative serine/threonine clusters in the OX₂ receptor C-terminus were identified as critical for β-arrestin-ubiquitin complex formation.
Conclusions:
- This research provides fundamental insights into the molecular mechanisms governing orexin receptor-arrestin-ubiquitin complex formation.
- Understanding these functional differentiations advances the potential for developing targeted orexin receptor therapeutics.
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