Tyramine, ␤-phenylethylamine, tryptamine, and octopamine are biogenic amines present in trace levels in mammalian nervous systems. Although some ''trace amines'' have clearly defined roles as neurotransmitters in invertebrates, the extent to which they function as true neurotransmitters in vertebrates has remained speculative. Using a degenerate PCR approach, we have identified 15 G protein-coupled receptors (GPCR) from human and rodent tissues. Together with the orphan receptor PNR, these receptors form a subfamily of rhodopsin GPCRs distinct from, but related to the classical biogenic amine receptors. We have demonstrated that two of these receptors bind and͞or are activated by trace amines. The cloning of mammalian GPCRs for trace amines supports a role for trace amines as neurotransmitters in vertebrates. Three of the four human receptors from this family are present in the amygdala, possibly linking trace amine receptors to affective disorders. The identification of this family of receptors should rekindle the investigation of the roles of trace amines in mammalian nervous systems and may potentially lead to the development of novel therapeutics for a variety of indications.

The principal inhibitory neurotransmitter GABA (gamma-aminobutyric acid) exerts its effects through two ligand-gated channels, GABA(A) and GABA(C) receptors, and a third receptor, GABA(B) , which acts through G proteins to regulate potassium and calcium channels. Cells heterologously expressing the cloned DNA encoding the GABA(B)R1 protein exhibit high-affinity antagonist-binding sites, but they produce little of the functional activity expected from studies of endogenous GABA(B) receptors in the brain. Here we describe a new member of the GABA(B) polypeptide family, GABA(B)R2, that shows sequence homology to GABA(B)R1. Neither GABA(B)R1 nor GABA(B)R2, when expressed individually, activates GIRK-type potassium channels; however, the combination of GABA(B)R1 and GABA(B)R2 confers robust stimulation of channel activity. Both genes are co-expressed in individual neurons, and both proteins co-localize in transfected cells. Moreover, immunoprecipitation experiments indicate that the two polypeptides associate with each other, probably as heterodimers. Several G-protein-coupled receptors (GPCRs) exist as high-molecular-weight species, consistent with the formation of dimers by these receptors, but the relevance of these species for the functioning of GPCRs has not been established. We have now shown that co-expression of two GPCR structures, GABA(B)R1 and GABA(B)R2, belonging to the same subfamily is essential for signal transduction by GABA(B) receptors.

The central nervous system octapeptide, neuropeptide FF (NPFF), is believed to play a role in pain modulation and opiate tolerance. Two G protein-coupled receptors, NPFF1 and NPFF2, were isolated from human and rat central nervous system tissues. NPFF specifically bound to NPFF1 (K d ‫؍‬ 1.13 nM) and NPFF2 (K d ‫؍‬ 0.37 nM), and both receptors were activated by NPFF in a variety of heterologous expression systems. The localization of mRNA and binding sites of these receptors in the dorsal horn of the spinal cord, the lateral hypothalamus, the spinal trigeminal nuclei, and the thalamic nuclei supports a role for NPFF in pain modulation. Among the receptors with the highest amino acid sequence homology to NPFF1 and NPFF2 are members of the orexin, NPY, and cholecystokinin families, which have been implicated in feeding. These similarities together with the finding that BIBP3226, an anorexigenic Y1 receptor ligand, also binds to NPFF1 suggest a potential role for NPFF1 in feeding. The identification of NPFF1 and NPFF2 will help delineate their roles in these and other physiological functions.

ARTICLESMelanin-concentrating hormone (MCH), a cyclic 19-aminoacid polypeptide, is produced predominantly by neurons in the lateral hypothalamus and zona incerta which project broadly throughout the brain 1 . Several lines of evidence implicate MCH as an important mediator in the regulation of energy balance and body weight. Central MCH administration stimulates food intake while fasting results in an increase in MCH expression 2 . Mice lacking the gene encoding MCH are lean, hypophagic and maintain elevated metabolic rates 3 . In contrast, mice overexpressing the MCH gene are susceptible to obesity and insulin resistance 4 . Although these findings support a rationale for MCH antagonists in the treatment of obesity, it is unclear if sufficient MCH tone exists to produce a robust and sustained loss of body weight after chronic MCH-receptor blockade. Moreover, because genetic manipulation of the MCH gene also affects the expression of neuropeptide E-I and neuropeptide G-E, which are processed from the same prehormone precursor as MCH, the observed phenotypes may be influenced by changes in the levels of these less characterized peptides 5 . The effects of MCH are mediated through receptors in the rhodopsin superfamily of G protein-coupled receptors (GPCRs). MCH1 receptor (MCH1-R) has been isolated from rodents and humans 6,7 , whereas MCH2-R has thus far been identified only in humans 8,9 . To assess the role of MCH1-R, we identified a selective, high-affinity MCH1-R antagonist and evaluated it in several animal models. We report here that the acute administration of a MCH1-R antagonist attenuated central MCH-stimulated food intake, and reduced consumption of palatable food. Moreover, chronic administration of this antagonist produced a robust and sustained decrease in body weight in rats with diet-induced obesity. As the distribution of MCH1-R binding sites in the central nervous system (CNS) is suggestive of a role for MCH in the regulation of mood and stress, we tested the MCH1-R antagonist in several animal models of depression and anxiety. Pharmacological blockade of the MCH1-R produced a profile similar to clinically used antidepressants and anxiolytics, suggesting that the MCH1-R might be a novel target for the treatment of depression and anxiety. SNAP-7941 is a selective, high-affinity MCH1-R antagonistScreening of our GPCR-biased compound collection against the human MCH1-R in a functional assay measuring intracellular Ca 2+ mobilization resulted in the discovery of a highpotency antagonist, SNAP-7941,4-tetrahydro-5-pyrimidinecarboxylate hydrochloride) (Fig. 1a). SNAP-7941 is a competitive antagonist of MCH in a [3 H]phosphoinositide accumulation assay in a mammalian cell line expressing the human MCH1-R (Fig. 1b). The Schild regression from these data estimated a pA 2 of 9.24 with a slope of 0.98 (r 2 = 0.94) for SNAP-7941 (Fig. 1b, inset), which predicts a K b of 0.57 nM. This compound was greater than 1,000-fold selective for MCH1-R compared with the human MCH2-R, as well as GPCRs associated with food...

Trace amines have been implicated in a number of neuropsychiatric disorders including depression and schizophrenia. Although long known to modulate neurotransmission indirectly through the release of catecholamines, the identification of the Trace Amine 1 receptor (TA1) offers a mechanism by which trace amines can influence synaptic activity directly. TA1 binds and is activated by trace amines such as b-phenylethylamine and tyramine. Our pharmacological characterization of mouse TA1 showed that, as in rat and primate, amphetamine is an agonist at this receptor but with surprisingly high potency. Without selective ligands for TA1 that do not also possess catecholamine-releasing properties, however, it has not been possible to study its physiological role in the central nervous system. To that end, a line of mice lacking the TA1 receptor was generated to characterize its contribution to the regulation of behavior. Compared with wild-type littermates, TA1 knockout (KO) mice displayed a deficit in prepulse inhibition. Knockout animals, in which the TA1-agonist influence of amphetamine was absent, showed enhanced sensitivity to the psychomotor-stimulating effect of this drug, which was temporally correlated with significantly larger increases in the release of both dopamine and norepinephrine in the dorsal striatum and associated with a 262% increase in the proportion of striatal high-affinity D2 receptors. TA1 therefore appears to play a modulatory role in catecholaminergic function and represents a potentially novel mechanism for the treatment of neuropsychiatric disorders. Furthermore, the TA1 KO mouse may provide a useful model for the development of treatments for some positive symptoms of schizophrenia.