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...

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The binding of glucagon to its hepatic receptor is known to result in a number of effects, including the intracellular accumulation of cAMP, the mobilization of intracellular Ca2+, and the endocytosis of glucagon and its receptor into intracellular vesicles. In this study, we begin to define the functional role of the COOH-terminal tail of the human glucagon receptor in glucagon-stimulated signal transduction and receptor internalization. We have created and expressed in Chinese hamster ovary (CHO) cells five truncation mutants in which the COOH-terminal 24, 56, 62, 67, and 73 amino acids have been removed. Cells expressing relevant truncated receptors were assayed for cell surface expression by immunofluorescence, for ligand-binding properties, for cAMP and Ca2+-mediated signal transduction properties, and for receptor endocytosis. In addition, a mutant receptor containing seven serine-to-alanine mutations in the COOH-terminal tail was studied. Our results reveal the following: 1) a region of the COOH-terminal tail that is required for proper cell surface expression, 2) the COOH-terminal 62 amino acids, which comprise the majority of the tail, are not required for ligand binding, cAMP accumulation, or Ca2+ mobilization, and 3) phosphorylation of the COOH-terminal tail is crucial for glucagon-stimulated receptor endocytosis.

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