In vitro functional analyses of hypocretin/orexin receptor systems were performed using [I-125]hypocretin radioreceptor and hypocretin-stimulated [S-35]GTPgammaS binding assay in cell lines expressing human or canine (wild-type and narcoleptic-mutation) hypocretin receptor 2 (Hcrtr 2). Hypocretin-2 stimulated [S-35]GTPgammaS binding in human and canine Hcrtr 2 expressing cell lines, while cell lines expressing the mutated canine Hcrtr 2 did not exhibit specific binding for [I-125]hypocretin or hypocretin-stimulated [S-35]GTPgammaS. In rat brain homogenates, regional specific hypocretin-stimulated [S-35]GTPgammaS binding was also observed. Hypocretin-stimulated [S-35]GTPgammaS binding, may thus be a useful functional assay for hypocretin receptors in both cell lines and brain tissue homogenates. (C) 2002 Elsevier Science (USA). All rights reserved.