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Distinct recognition of OX1 and OX2 receptors by orexin peptides
Sylwia Ammoun1, Tomas Holmqvist, Ramin Shariatmadari
1Department of Neuroscience, Physiology, Uppsala University, BMC, P.O. Box 572, SE-75123 Uppsala, Sweden.
Abstract:
In this study, we have compared the abilities of orexin-A and orexin-B and variants of orexin-A to activate different Ca(2+) responses (influx and release) in human OX(1) and OX(2) receptor- expressing Chinese hamster ovary cells. Responses mediated by activation of both receptor subtypes with either orexin-A or -B were primarily dependent on extracellular Ca(2+), suggesting similar activation of Ca(2+) influx as we have previously shown for orexin-A and OX(1) receptors. Amino acid-wise truncation of orexin-A reduced its ability to activate OX(1) and OX(2) receptors, but the response mediated by the OX(2) receptor was more resistant to truncation than the response mediated by the OX(1) receptor. We also performed a sequential replacement of amino acids 14 to 26 with alanine in the truncated orexin-A variant orexin-A(14-33). Replacement of the same amino acids produced a fall in the potency for each receptor subtype, but the reduction was less prominent for the OX(2) receptor. The most marked reduction was produced by the replacement of Leu20, Asp25, and His26 with alanine. Interestingly, extracellular Ca(2+) dependence of responses to some of the mutated peptides was different from those of orexin-A and -B. The mutagenesis also suggests that although the determinants required from orexin-A for binding to and activation of the receptor are highly conserved between the orexin receptor subtypes, the OX(2) receptor requires fewer determinants. This might in part explain why orexin-B has the affinity and potency equal to orexin-A for this subtype, although it has 10- to 100-fold lower affinity and potency for the OX(1) receptor.
Insights
Orexin-A and orexin-B activate calcium responses through OX1 and OX2 receptors. Orexin receptor 2 is more resistant to orexin-A truncation and requires fewer binding determinants than OX1 receptor.
Area of Science:
- Neuroscience
- Molecular Pharmacology
Background:
- Orexins (orexin-A and orexin-B) are neuropeptides involved in regulating sleep-wake cycles and appetite.
- They exert their functions through two G protein-coupled receptors: OX1 and OX2 receptors.
Purpose of the Study:
- To compare the abilities of orexin-A, orexin-B, and orexin-A variants to activate calcium responses in OX1 and OX2 receptors.
- To investigate the role of specific amino acid residues in orexin-A for receptor activation and binding.
Main Methods:
- Utilized Chinese hamster ovary (CHO) cells expressing human OX1 or OX2 receptors.
- Measured calcium influx and release responses upon stimulation with orexins and their variants.
- Performed amino acid truncation and alanine-scanning mutagenesis on orexin-A.
Main Results:
- Both orexin-A and orexin-B activated OX1 and OX2 receptors, primarily via extracellular calcium influx.
- Truncation of orexin-A reduced its potency, with OX2 receptors showing greater resistance than OX1 receptors.
- Mutagenesis identified Leu20, Asp25, and His26 in orexin-A as critical for OX1 receptor activation.
- OX2 receptors demonstrated higher tolerance to mutations and required fewer determinants for binding and activation compared to OX1 receptors.
- This explains orexin-B's equal affinity and potency for OX2 receptors, contrasting with its lower affinity for OX1 receptors.
Conclusions:
- Orexin receptor subtypes exhibit differential sensitivity to ligand structure and binding determinants.
- The OX2 receptor's unique properties contribute to orexin-B's comparable efficacy with orexin-A at this receptor.
- Findings provide insights into orexin receptor pharmacology and ligand design.
