We report the isolation of a gene encoding a novel member of the family of melanocortin receptors. The mouse melanocortin-5 receptor (mMC5R) responds to melanocortins with an increase in intracellular cyclic 3',5'-adenosine monophosphate (cAMP) concentrations. Stimulation of the mMC5R by the melanocortins revealed a hierarchy of potency in which alpha-melanocyte stimulating hormone (alpha-MSH) > beta-melanocyte stimulating hormone (beta-MSH) > adrenocorticotropic hormone (ACTH) > gamma- melanocyte stimulating hormone (gamma-MSH). Further structure-activity studies indicated that amino- and carboxyl-terminal portions of alpha-MSH appear to be key determinants in the activation of mMC5R whereas the melanocortin core heptapeptide sequence is devoid of pharmacological activity. Northern blot analysis demonstrated the expression of mMC5R mRNA in mouse skeletal muscle, lung, spleen, and brain.

using four heat-flux plates and four temperature probes. Evapotranspiration and CO 2 flux were measured using the eddy-covariance method with an Applied Technologies sonic anemometer and LI-COR 6262 infrared gas analyser mounted on 2-m towers 25 . The mean and standard error for energy flux, gross primary production and evapotranspiration at sites 3 and 4 were calculated on the basis of 30-min averages. CO 2 fluxes at sites 11, 17 and 21 were determined using eddycovariance methods and 2.5-m towers 26 . Mean values and standard errors at these sites were calculated using the daily mean CO 2 fluxes. The daily methane fluxes were integrated over the thaw period to obtain annual emission. Winter methane fluxes were assumed to be zero. CH 4 flux was measured during the thaw season, Jun-Aug, at 27 MNT and MAT sites along the Dalton Highway in 1996 using a static chamber method 30 . Air samples were taken over periods of 30-45 min and were analysed on a gas chromatograph equipped with a flame ionization detector.

To elucidate the molecular basis for the interaction of ligands with the human melanocortin-4 receptor (hMC4R), agonist structure-activity studies and receptor point mutagenesis were performed. Structure-activity studies of [Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) identified D-Phe7-Arg8-Trp9 as the minimal NDP-MSH fragment that possesses full agonist efficacy at the hMC4R. In an effort to identify receptor residues that might interact with amino acids in this tripeptide sequence 24 hMC4R transmembrane (TM) residues were mutated (the rationale for choosing specific receptor residues for mutation is outlined in the Results section). Mutation of TM3 residues D122 and D126 and TM6 residues F261 and H264 decreased the binding affinity of NDP-MSH 5-fold or greater, thereby identifying these receptor residues as sites potentially involved in the sought after ligand-receptor interactions. By examination of the binding affinities and potencies of substituted NDP-MSH peptides at receptor mutants, evidence was found that core melanocortin peptide residue Arg8 interacts at a molecular level with hMC4R TM3 residue D122. TM3 mutations were also observed to decrease the binding of hMC4R antagonists. Notably, mutation of TM3 residue D126 to alanine decreased the binding affinity of AGRP (87-132), a C-terminal derivative of the endogenous melanocortin antagonist, 8-fold, and simultaneous mutations D122A/D126A completely abolished AGRP (87-132) binding. In addition, mutation of TM3 residue D122 or D126 decreased the binding affinity of hMC4R antagonist SHU 9119. These results provide further insight into the molecular determinants of hMC4R ligand binding.

Peptide alpha amidation is required to produce some hormones, such as gastrin, from their glycine-extended precursors. This terminal posttranslational processing reaction is thought to be essential for the biological activation of many peptide hormones; only amidated gastrin exerts a physiological effect that results in gastric acid secretion. However, both amidated gastrin and glycine-extended gastrin stimulate proliferation of exocrine pancreatic cell line AR4-2J through selective receptors for the substrate and the product, respectively, of peptide alpha amidation. Thus, the amidation reaction may function as a determinant of the specific biological actions of products derived from prohormones.

The melanocortin-1 receptor (MC1R) is a seven-transmembrane (TM) G-protein-coupled receptor whose natural ligands are the melanocortin peptides, adrenocorticotropic hormone, and ␣-, ␤-, and ␥-melanocyte stimulating hormone (MSH). To test a previously constructed three-dimensional model of the molecular interaction between the long-acting, superpotent , and simultaneous mutation of multiple TM4, -5, and -6 tyrosine and phenylalanine residues suggests that aromatic-aromatic ligand-receptor interactions also participate in binding these melanocortins to the MC1R. These experiments appear to have identified some of the critical receptor residues involved in the ligand-receptor interactions between these melanocortins and the hMC1R.The term melanocortin refers to several of the post-translational products of the pro-opiomelanocortin gene, adrenocorticotropic hormone (ACTH), 1 and ␣-, ␤-, and ␥-MSH. Besides the well known effects of these peptides on adrenal cortical steroidogenesis (ACTH) and pigmentation (␣-MSH and ACTH), melanocortins have also been implicated in a myriad of physiological processes including learning and memory, blood pressure control, immune modulation, and weight homeostasis among others (1-3).All melanocortin peptides contain a core or "message" HisPhe-Arg-Trp sequence and, with the exception of ␥-MSH, also share a common heptapeptide sequence Met-Glu-His-Phe-ArgTrp-Gly (Fig. 1) MT-II)) (4, 5). Both NDP-MSH and MT-II have extremely prolonged activity (days) as compared with ␣-MSH (hours) in the classical frog and lizard skin darkening bioassays (4, 5).There is some evidence to suggest that the basis for this residual activity lies, at least in part, on the slow dissociation rates of these ␣-MSH analogs (6). This unique property of prolonged activity makes an understanding of the molecular interactions of these two compounds with its target receptors of particular interest.To date five melanocortin receptor subtypes with different patterns of tissue expression in the central nervous system and peripheral body have been cloned. Designated melanocortin receptor (MCR) 1-5 for the order in which they were isolated, all five receptor subtypes belong to the family of seven transmembrane G-protein-coupled receptors and upon stimulation all subtypes activate adenylate cyclase and elevate intracellular 3Ј,5Ј-adenosine monophosphate. A unified nomenclature has been set forth in which the melanocyte melanocortin receptor, previously referred to as the ␣-MSH receptor, has been designated the melanocortin-1 receptor (MC1R). This newer terminology was adopted since studies with the cloned melanocortin receptor subtypes have demonstrated that with the exception of the melanocortin-2 receptor (the adrenocortical ACTH receptor) which is only activated by ACTH, all other subtypes are promiscuous in their activation by the melanocortins.Each of the five MCR subtypes has its own signature profile of melanocortin activation. The human MC1R (hMC1R) is activated equally well by both ACTH and ␣-MSH while ␤-MSH is appro...