RESUMO
Human melanin-concentrating hormone (hMCH) is a potent but nonselective agonist at human melanin-concentrating hormone receptors 1 and 2 (hMCH-1R and hMCH-2R, respectively). To determine the structural features of this neuropeptide which are necessary for efficient binding to and activation of the receptors, Ala-substituted, open-chain, and truncated analogues were synthesized and tested in the binding assays in CHO cells expressing hMCH-1R and hMCH-2R, and in functional assays measuring the level of intracellular calcium mobilization in human HEK-293 cells expressing these receptors. A compound consisting merely of the cyclic core of hMCH with the Arg attached to the N-terminus of the disulfide ring was found to activate both hMCH-1R and hMCH-2R about as effectively as full-length hMCH. Thus, the sequence Arg-cyclo(S-S)(Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys) appears to constitute the "active core" that is necessary for agonist potency at hMCH-1R and hMCH-2R. A potent and approximately 4-fold more selective agonist at hMCH-1R than at hMCH-2R is also reported.
Assuntos
Hormônios Hipotalâmicos/química , Hormônios Hipotalâmicos/fisiologia , Melaninas/química , Melaninas/fisiologia , Fragmentos de Peptídeos/fisiologia , Hormônios Hipofisários/química , Hormônios Hipofisários/fisiologia , Receptores do Hormônio Hipofisário/metabolismo , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Humanos , Hormônios Hipotalâmicos/metabolismo , Isomerismo , Melaninas/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/fisiologia , Hormônios Hipofisários/metabolismo , Conformação Proteica , Receptores Acoplados a Proteínas G , Receptores do Hormônio Hipofisário/agonistasRESUMO
Melanin-concentrating hormone (MCH) is a 19-aa cyclic neuropeptide originally isolated from chum salmon pituitaries. Besides its effects on the aggregation of melanophores in fish several lines of evidence suggest that in mammals MCH functions as a regulator of energy homeostasis. Recently, several groups reported the identification of an orphan G protein-coupled receptor as a receptor for MCH (MCH-1R). We hereby report the identification of a second human MCH receptor termed MCH-2R, which shares about 38% amino acid identity with MCH-1R. MCH-2R displayed high-affinity MCH binding, resulting in inositol phosphate turnover and release of intracellular calcium in mammalian cells. In contrast to MCH-1R, MCH-2R signaling is not sensitive to pertussis toxin and MCH-2R cannot reduce forskolin-stimulated cAMP production, suggesting an exclusive G(alpha)q coupling of the MCH-2R in cell-based systems. Northern blot and in situ hybridization analysis of human and monkey tissue shows that expression of MCH-2R mRNA is restricted to several regions of the brain, including the arcuate nucleus and the ventral medial hypothalamus, areas implicated in regulation of body weight. In addition, the human MCH-2R gene was mapped to the long arm of chromosome 6 at band 6q16.2-16.3, a region reported to be associated with cytogenetic abnormalities of obese patients. The characterization of a second mammalian G protein-coupled receptor for MCH potentially indicates that the control of energy homeostasis in mammals by the MCH neuropeptide system may be more complex than initially anticipated.
Assuntos
Encéfalo/metabolismo , Cromossomos Humanos Par 22 , Receptores do Hormônio Hipofisário/genética , Receptores do Hormônio Hipofisário/metabolismo , Receptores do Hormônio Hipofisário/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Células COS , Membrana Celular/fisiologia , Chlorocebus aethiops , Mapeamento Cromossômico , Cricetinae , Feminino , Humanos , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Oncorhynchus keta , Especificidade de Órgãos , Hipófise/química , Hipófise/fisiologia , Ensaio Radioligante , Receptores Acoplados a Proteínas G , Receptores do Hormônio Hipofisário/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TransfecçãoAssuntos
Carcinógenos/farmacologia , DNA/efeitos da radiação , Proteínas/efeitos da radiação , RNA/efeitos da radiação , 2-Acetilaminofluoreno/farmacologia , Acetoxiacetilaminofluoreno/farmacologia , Albuminas , Alquilantes/farmacologia , Centrifugação Isopícnica , Fenômenos Químicos , Química , Cromatografia em Gel , DNA/isolamento & purificação , Metanossulfonato de Etila/farmacologia , Hidroxiacetilaminofluoreno/farmacologia , Técnicas In Vitro , Mecloretamina/farmacologia , Metanossulfonato de Metila/farmacologia , Muramidase , Raios UltravioletaRESUMO
Significantly enhanced attachment to Ehrlich ascites and Escherichia coli cells was observed for radioactive DNA and RNA in the presence of chemical mutagens and ultimate carcinogens. In some instances, formation of nucleic acid-protein adducts by these compounds further (or similarly) enhanced the binding. DNA irradiated with ultraviolet light in the presence of a protein bound more efficiently than either an unirradiated mixture of these two macromolecules or DNA irradiated alone. The spectrum of compounds tested and found active in this system includes alkylating agents, aromatic amines, and carcinogenic metals. Precarcinogens and nonultimate carcinogenic chemicals, as well as tumor-promoting agents, did not increase the binding. However, addition of extracts from mouse or rat livers activated precarcinogenic and proximate carcinogenic chemicals and resulted in enhanced cellular attachment of indicator nucleic acids in their presence. Possible usefulness of this test system for fast and efficient screening for environmental carcinogens and mutagens, as well as possible relevance of the observed phenomena to in vivo effects of chemical and physical carcinogens, is considered.
