Hypocretin stimulates [(35)S]GTP gamma S binding in Hcrtr 2-transfected cell lines and in brain homogenate

In vitro functional analyses of hypocretin/orexin receptor systems were performed using (125)I]hypocretin radioreceptor and hypocretin-stimulated (35)S]GTP gamma S binding assay in cell lines expressing human or canine (wild-type and narcoleptic-mutation) hypocretin receptor 2 (Hcrtr 2). Hypocretin-2 stimulated (35)S]GTP gamma S 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 (125)I]hypocretin or hypocretin-stimulated (35)S]GTP gamma S. In rat brain homogenates, regional specific hypocretin-stimulated (35)S]GTP gamma S binding was also observed. Hypocretin-stimulated (35)S]GTP gamma S binding, may thus be a useful functional assay for hypocretin receptors in both cell lines and brain tissue homogenates.