Distinct Recognition of OX1 and OX2Receptors by Orexin Peptides

• Published in: The Journal of pharmacology and experimental therapeutics 2003-05, Vol.305 (2), p.507
• Main Authors: Sylwia Ammoun, Tomas Holmqvist, Ramin Shariatmadari, Hendrica B. Oonk, Michel Detheux, Marc Parmentier, Karl E. O. Ã kerman, Jyrki P. Kukkonen
• Format: Article
• Language: eng ; jpn
• ISSN: 0022-3565; 1521-0103
• DOI: 10.1124/jpet.102.048025

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.