In this study, we have compared the abilities of orexin-A and orexin-B and variants of orexin-A to activate different Ca2+ responses (influx and release) in human OX1 and OX2 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 Ca2+, suggesting similar activation of Ca2+ influx as we have previously shown for orexin-A and OX1 receptors. Amino acid-wise truncation of orexin-A reduced its ability to activate OX1 and OX2 receptors, but the response mediated by the OX2 receptor was more resistant to truncation than the response mediated by the OX1 receptor. We also performed a sequential replacement of amino acids 14 to 26 with alanine in the truncated orexin-A variant orexin-A14-33. Replacement of the same amino acids produced a fall in the potency for each receptor subtype, but the reduction was less prominent for the OX2 receptor. The most marked reduction was produced by the replacement of Leu20, Asp25, and His26 with alanine. Interestingly, extracellular Ca2+ 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 OX2 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 OX1 receptor
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