RESUMO
Adenosine receptors (AdoR) are members of the G protein-coupled receptor superfamily and mediate extracellular adenosine signaling, but the mechanism of adenosine signaling is still unclear. Here we report the first characterization of an insect AdoR, encoded by the Drosophila gene CG9753. Adenosine stimulation of Chinese hamster ovary cells carrying transiently expressed CG9753 led to a dose-dependent increase of intracellular cAMP and calcium, but untransfected controls showed no such response, showing that CG9753 encodes a functional AdoR. Endogenous CG9753 transcripts were detected in the brain, imaginal discs, ring gland and salivary glands of third-instar Drosophila larvae, and CG9753 overexpression in vivo caused lethality or severe developmental anomalies. These developmental defects were reduced by adenosine depletion, consistent with the proposed function of the CG9753 product as an AdoR. Overexpression of the G protein subunit Galpha(s) or of the catalytic subunit of protein kinase A (PKA) partially mimicked and enhanced the defects caused by ectopic expression of AdoR. Our results suggest that AdoR is an essential part of the adenosine signaling pathway and Drosophila offers a unique opportunity to use genetic analysis to study conserved aspects of the adenosine signaling pathway.
Assuntos
AMP Cíclico/fisiologia , Drosophila/metabolismo , Receptores Purinérgicos P1/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Drosophila/genética , Drosophila/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Hemolinfa , Injeções , Dados de Sequência Molecular , Fenótipo , Receptores Purinérgicos P1/genética , Alinhamento de SequênciaRESUMO
GPR39, an orphan G protein-coupled receptor, has been recently identified as the receptor for the bioactive peptide obestatin. Obestatin is secreted from the stomach and acts as an anti-appetite hormone. This activity is induced whether obestatin is administered intraperitoneally or intracerebroventricularly. GPR39 is known to be expressed in the central nervous system but its precise localization is unknown. In view of the growing importance of this system, we decided to study the sites of GPR39 mRNA expression by in-situ hybridization. We find the highest levels of GPR39 mRNA in the amygdala, the hippocampus, and the auditory cortex and low levels in several other brain regions. Surprisingly, we find no expression of GPR39 in the hypothalamus, expected to be the site of the anorexigenic action of obestatin.
Assuntos
Regulação do Apetite/fisiologia , Encéfalo/metabolismo , Hormônios Peptídicos/metabolismo , Receptores Acoplados a Proteínas G/genética , Tonsila do Cerebelo/anatomia & histologia , Tonsila do Cerebelo/metabolismo , Animais , Córtex Auditivo/anatomia & histologia , Córtex Auditivo/metabolismo , Encéfalo/anatomia & histologia , Expressão Gênica/fisiologia , Hipocampo/anatomia & histologia , Hipocampo/metabolismo , Hipotálamo/anatomia & histologia , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismoRESUMO
The mas-like G protein-coupled receptors form a subfamily of G protein-coupled receptors that includes variable member numbers across different species and that have been shown to bind a wide variety of ligands from peptides to amino acid derivatives. While screening a library of peptides against different orphan G protein-coupled receptors, we found that human salusin beta activates the mouse mas-like G protein-coupled receptor, mMrgA1 with an EC(50) of about 300 nM. Salusin beta is a bioactive peptide recently discovered through bioinformatics analysis which stimulates arginine-vasopressin release from rat pituitary and causes rapid and profound hypotension and bradycardia. However, when we further analyzed the generality of the mMrgA1 activation, we found that human salusin beta does not activate corresponding human mas-like G protein-coupled receptors. Our results show that human salusin beta is a surrogate ligand of the mouse MrgA1 and raises a cautionary flag for experiments that analyze the pharmacological profiles of mas-like G protein-coupled receptors from different species.
Assuntos
Adenosina Trifosfatases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Ligantes , Camundongos , Dados de Sequência Molecular , Ratos , Receptores Acoplados a Proteínas G/genéticaRESUMO
Metastin is an antimetastatic peptide encoded by the KiSS-1 gene in cancer cells. Recent studies found that metastin is a ligand for the orphan G-protein-coupled receptor GPR54, which is highly expressed in specific brain regions such as the hypothalamus and parts of the hippocampus. This study shows that activation of GPR54 by submicromolar concentrations of metastin reversibly enhances excitatory synaptic transmission in hippocampal dentate granule cells in a mitogen-activated protein (MAP) kinase-dependent manner. Synaptic enhancement by metastin was suppressed by intracellular application of the G-protein inhibitor GDP-beta-S and the calcium chelator BAPTA. Analysis of miniature excitatory postsynaptic currents (mEPSCs) revealed an increase in the mean amplitude but no change in event frequency. This indicates that GPR54 and the mechanism responsible for the increase in EPSCs are postsynaptic. Metastin-induced synaptic potentiation was abolished by 50 microM PD98059 and 20 microM U0126, two inhibitors of the MAP kinases ERK1 and ERK2. The effect was also blocked by inhibitors of calcium/calmodulin-dependent kinases and tyrosine kinases. RT-PCR experiments showed that both KiSS-1 and GPR54 are expressed in the hippocampal dentate gyrus. Metastin is thus a novel endogenous factor that modulates synaptic excitability in the dentate gyrus through mechanisms involving MAP kinases, which in turn may be controlled upstream by calcium-activated kinases and tyrosine kinases.