Or cOisogenic da an -fivefold reduction of Agrp RNA in the hypothalamus of AYla animals (Fig. 1C). We Expression of Agouti protein is normally limited to the skin where it affects pigmentation, also measured the levels of hypothalamic but ubiquitous expression causes obesity. An expressed sequence tag was identified Agrp RNA in oblob animals and found an that encodes Agouti-related protein, whose RNA is normally expressed in the hypoweightfold increase relative to coisogenic thalamus and whose levels were increased eightfold in oblob mice. Recombinant Agouticontrols. In the adrenal gland, levels of Agrp related protein was a potent, selective antagonist of Mc3r and Mc4r, melanocortin RNA in AYIa and nonmutant animals were receptor subtypes implicated in weight regulation. Ubiquitous expression of human below the level of detection, but could easily AGRP complementary DNA in transgenic mice caused obesity without altering pigmenbe detected in oblob animals. tation. Thus, Agouti-related protein is a neuropeptide implicated in the normal control ofTo determine whether AGRP antagobody weight downstream of leptin signaling.nizes melanocortin signaling, we used the baculovirus expression system to produce conditioned media containing recombinant human AGRP, and measured antag-Analysis of mouse obesity mutations has scriptase-polymerase chain reaction (RT-onist activity using a Xenopus melanohelped define regulatory circuits that gov-PCR) and Northern (RNA) hybridization phore cell line developed by Lerner and e m energy expenditure (1). In mice carry-experiments demonstrated that Agrp RNA colleagues (1 2). Melanophores provide a ing certain alleles of the Agouti coat color was expressed primarily in the adrenal gland rapid and sensitive bioassay for melanogene such as kthal yelow (A?) or YMbk and the hypothalamus (Fig. 1, B and C). To cortin agonists and antagonists because yellow (Aq), pleiotropic effects including a investigate functional overlap between Agrp pigment granule dispersion induced by ayellow coat, obesity, and increased body length are caused by ubiquitous expression a of chimeric transcripts encoding a normal A + ~o r m A B P -Agouti protein (2-4). Agouti is a paracrine ,,,,

Melanocortin receptors belong to the seven-transmembrane (TM) domain proteins that are coupled to G-proteins and signaled through intracellular cyclic adenosine monophosphate. Many structural features conserved in other G-protein coupled receptors (GPCRs) are found in the melanocortin receptors. There are five melanocortin receptor subtypes and each of the melanocortin receptor subtypes has a different pattern of tissue expression and has its own profile regarding the relative potency of different melanocortin peptides. α-, β-, and γ-MSH and ACTH are known endogenous agonist ligands for the melanocortin receptors. Agouti and AgRP are the only known naturally occurring antagonists of the melanocortin receptors. We have examined the molecular basis of all five human melanocortin receptors for different ligand binding affinity and potency using chimeric and mutated receptors. Our studies indicate that human melanocortin MC 1 receptor, human melanocortin MC 3 receptor, human melanocortin MC 4 receptor and human melanocortin MC 5 receptor utilize orthosteric sites for non selective agonists, α-MSH and NDP-β-MSH, high affinity binding and utilize allosteric sites for selective agonist or antagonist binding. Furthermore, our results indicate that molecular determinants of human melanocortin MC 2 receptor for ACTH binding and signaling are different from that of other melanocortin receptors. Many studies also indicate that agonists can induce different conformation changes of melanocortin receptors, which then lead to the activation of different signaling pathways, even when the expression level of receptor and the strength of stimulus-response coupling are the same. This finding may provide new information for the design of drugs for targeting melanocortin receptors.