title: Allosteric regulation by oleamide of the binding properties of 5-hydroxytryptamine7 receptors.
abstract: Oleamide belongs to a family of amidated lipids with diverse biological activities, including sleep induction and signaling modulation of several 5-hydroxytryptamine (5-HT) receptor subtypes, including 5-HT1A, 5-HT2A/2C, and 5-HT7. The 5-HT7 receptor, predominantly localized in the hypothalamus, hippocampus, and frontal cortex, stimulates cyclic AMP formation and is thought to be involved in the regulation of sleep-wake cycles. Recently, it was proposed that oleamide acts at an allosteric site on the 5-HT7 receptor to regulate cyclic AMP formation. We have further investigated the interaction between oleamide and 5-HT7 receptors by performing radioligand binding assays with HeLa cells transfected with the 5-HT7 receptor. Methiothepin, clozapine, and 5-HT all displaced specific [3H]5-HT (100 nM) binding, with pK(D) values of 7.55, 7.85, and 8.39, respectively. Oleamide also displaced [3H]5-HT binding, but the maximum inhibition was only 40% of the binding. Taking allosteric (see below) cooperativity into account, a K(D) of 2.69 nM was calculated for oleamide. In saturation binding experiments, oleamide caused a 3-fold decrease in the affinity of [3H]5-HT for the 5-HT7 receptor, without affecting the number of binding sites. A Schild analysis showed that the induced shift in affinity of [3H]5-HT reached a plateau, unlike that of a competitive inhibitor, illustrating the allosteric nature of the interaction between oleamide and the 5-HT7 receptor. Oleic acid, the product of oleamide hydrolysis, had a similar effect on [3H]5-HT binding, whereas structural analogs of oleamide, trans-9,10-octadecenamide, cis-8,9-octadecenamide, and erucamide, did not alter [3H]5-HT binding significantly. The findings support the hypothesis that oleamide acts via an allosteric site on the 5-HT7 receptor regulating receptor affinity.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: P B
authors.initials: PB
authors.lastname: Hedlund
authors.forename: M J
authors.initials: MJ
authors.lastname: Carson
authors.forename: J G
authors.initials: JG
authors.lastname: Sutcliffe
authors.forename: E A
authors.initials: EA
authors.lastname: Thomas
mesh_terms: D000494:Allosteric Regulation; D006367:HeLa Cells; D006801:Humans; D006993:Hypnotics and Sedatives / Q000737:chemistry / Q000494:pharmacology; D009829:Oleic Acids / Q000737:chemistry / Q000494:pharmacology*; D011985:Receptors, Serotonin / Q000187:drug effects* / Q000378:metabolism; D014162:Transfection
chemical_list: D006993:Hypnotics and Sedatives; D009829:Oleic Acids; D011985:Receptors, Serotonin; C104425:serotonin 7 receptor; C029407:oleylamide