RESUMO
Site-directed mutation of serine 110 (Ser(3.35)) and serine 114 (Ser(3.39)) in the human melatonin MT(1) receptor to alanine residues reduced ligand binding affinities of seven known melatonin receptor agonists and partial agonists by 3- to 15-fold. These mutants also displayed a relative reduction in their affinities for melatonin-mediated functional responses of 30- and 14-fold, respectively. In contrast to the observed effects of the agonists and partial agonists, the melatonin receptor antagonist luzindole was found to bind to mutants Ser(3.35)Ala and Ser(3.39)Ala with affinities equivalent to that determined for the wild-type melatonin MT(1) receptor. Luzindole was subsequently confirmed as an antagonist of melatonin-mediated functional responses for both mutant receptors. These studies have identified that in the human melatonin MT(1) receptor, Ser(3.35) and Ser(3.39), in transmembrane domain 3, are critical for the formation of the high-affinity ligand binding site for agonists and partial agonists but not for the antagonist luzindole.
Assuntos
Receptores de Superfície Celular/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Serina/metabolismo , Acetamidas/farmacologia , Amidas/farmacologia , Substituição de Aminoácidos , Animais , Sítios de Ligação/genética , Ligação Competitiva/efeitos dos fármacos , Células COS , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Indóis/farmacologia , Melatonina/análogos & derivados , Melatonina/farmacologia , Mutagênese Sítio-Dirigida , Naftalenos/farmacologia , Ligação Proteica/efeitos dos fármacos , Ensaio Radioligante , Receptores de Superfície Celular/agonistas , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Melatonina , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/genética , Relação Estrutura-Atividade , Transfecção , Triptaminas/farmacologiaRESUMO
Melatonin receptors bind and become activated by melatonin. The melatonin-related receptor, despite sharing considerable amino acid sequence identity with melatonin receptors, does not bind melatonin and is currently an orphan G protein-coupled receptor. To investigate the structure and function of both receptors, we engineered a series of 14 chimeric receptor constructs, allowing us to determine the relative contribution of each transmembrane domain to ligand binding and receptor function. Results identified that when sequences encoding transmembrane domains 1, 2, 3, 5, or 7 of the melatonin mt(1) receptor were replaced by the corresponding domains of the melatonin-related receptor, the resultant chimeric receptors all displayed specific 2-[(125)I]iodomelatonin binding. Replacement of sequences incorporating transmembrane domains 4 or 6, however, resulted in chimeric receptors that displayed no detectable 2-[(125)I]iodomelatonin binding. The subsequent testing of a "reverse" chimeric receptor in which sequences encoding transmembrane domains 4 and 6 of the melatonin-related receptor were replaced by the corresponding melatonin mt(1) receptor sequences identified specific 2-[(125)I]iodomelatonin binding and melatonin-mediated modulation of cyclic AMP levels. To further investigate these findings, site-directed mutagenesis was performed on residues within transmembrane domain 6 of the melatonin mt(1) receptor. This identified Gly(258) (Gly(6.55)) as a critical residue required for high affinity ligand binding and receptor function.
Assuntos
Receptores de Superfície Celular/genética , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/genética , Animais , Ligação Competitiva , Células COS , Clonagem Molecular , AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/genética , Humanos , Radioisótopos do Iodo , Melatonina/metabolismo , Melatonina/farmacologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Ligação Proteica , Engenharia de Proteínas , Estrutura Secundária de Proteína , Receptores de Melatonina , Proteínas Recombinantes de Fusão/química , Alinhamento de Sequência , TransfecçãoRESUMO
A cyclic AMP response element (CRE)-luciferase reporter gene assay was used to characterise the functional responses of human melatonin mt(1) and human melatonin MT(2) receptors, stably expressed in the human embryonic kidney cell line HEK293, to a series of six naphthalenic analogues of melatonin. By comparison to the observed melatonin-mediated inhibition of stimulated luciferase levels the naphthalenic series was identified as comprising agonists, partial agonists and one antagonist of melatonin mt(1) and melatonin MT(2) receptor function. Three of the agonist/partial agonist members of this series were also identified as displaying a functional selectivity for the melatonin MT(2) receptor. Competitive displacement of 2-[125I]iodomelatonin binding to the ovine pars tuberalis melatonin ML(1) receptor demonstrated a close correlation to the observed functional luciferase responses of the human melatonin mt(1) receptor. We conclude that the CRE-luciferase reporter gene assay provides an effective functional screening method for the pharmacological characterisation of human melatonin receptor subtypes.
