RESUMO
Two cDNA clones homologous with human neuropeptide (NP) Y-Y1 receptor have been isolated from a mouse bone marrow cDNA library. One was thought to be the cognate of the human NPY-Y1 receptor, termed Y1 alpha receptor, and the other form, termed Y1 beta receptor, differed from the Y1 alpha receptor in the seventh transmembrane domain and C-terminal tail. Analysis of the mouse genomic DNA showed that both receptors originated from a single gene. The different peptide sequences of the Y1 beta receptor were encoded by separate exons, hence, these receptors were generated by differential RNA splicing. High affinity binding of [125I]NPY to each receptor expressed in Chinese hamster ovary (CHO) cells and sequestration of [125I]NPY after binding to each receptor were observed. In the CHO cells expressing the Y1 alpha receptor, intracellular Ca2+ increase, inhibition of forskolin-induced cAMP accumulation, and mitogen-activated protein kinase (MAPK) activation were observed by stimulation of NPY, and these responses were abolished by pretreatment with pertussis toxin. Since wortmannin completely inhibited NPY-elicited MAPK activation, we speculate that wortmannin-sensitive signaling molecule(s) such as phosphoinositide 3-kinase may lie between pertussis toxin-sensitive G-protein and MAPK. In contrast, these intracellular signals were not detected in CHO cells expressing the Y1 beta receptor. Northern blots and reverse transcriptase-polymerase chain reaction analyses indicated that the Y1 alpha receptor was highly expressed in the brain, heart, kidney, spleen, skeletal muscle, and lung, whereas the Y1 beta receptor mRNA was not detected in these tissues. However, the Y1 beta receptor was expressed in mouse embryonic developmental stage (7 and 11 days), bone marrow cells and several hematopoietic cell lines. These results suggest that the Y1 beta receptor is an embryonic and a bone marrow form of the NPY-Y1 receptor, which decreases in the expression during development and differentiation.
Assuntos
Processamento Alternativo , Expressão Gênica , Neuropeptídeo Y/metabolismo , Estrutura Secundária de Proteína , Receptores de Neuropeptídeo Y/biossíntese , Receptores de Neuropeptídeo Y/genética , Sequência de Aminoácidos , Androstadienos/farmacologia , Animais , Sequência de Bases , Medula Óssea/metabolismo , Células CHO , Cálcio/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Membrana Celular/metabolismo , Quelantes/farmacologia , Cricetinae , Primers do DNA , DNA Complementar , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Biblioteca Gênica , Humanos , Íntrons , Cinética , Camundongos , Dados de Sequência Molecular , Neuropeptídeo Y/farmacologia , Toxina Pertussis , Reação em Cadeia da Polimerase , Receptores de Neuropeptídeo Y/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfecção , Fatores de Virulência de Bordetella/farmacologia , WortmaninaRESUMO
Platelet-activating factor (PAF) is a potent lipid mediator of allergic inflammation through its interaction with eosinophils. Expression of the PAF receptor is modulated by many agents, including those responsible for cell differentiation. We report here that differentiation of a human eosinophilic leukaemia cell line, EoL-1, by sodium n-butyrate is associated with induction of PAF receptor gene expression, as indicated by: PAF receptor mRNA accumulation; increases in the binding of [3H]WEB 2086, a PAF antagonist; analysis of cell-surface expression of PAF receptor protein using a monoclonal anti-(PAF receptor) antibody; and augmentation of PAF-induced increase in the intracellular concentration of calcium. Using cDNA cloning, the receptor expressed in EoL-1 cells was identified as 'Transcript 1', one of two transcripts which was previously reported from human genomic analysis (Mutoh, Bito, Minami, Nakamura, Honda, Izumi, Nakata, Kurachi, Terano and Shimizu (1993) FEBS Lett. 322, 129-134). The PAF-induced calcium response and phosphoinositide turnover were decreased by pertussis toxin (PTX) treatment, suggesting that these signals are coupled largely with PTX-sensitive G-protein(s) in EoL-1 cells. These systems may provide a useful experimental model with which to investigate the relationship between eosinophilic differentiation and PAF receptor induction, and the role of eosinophils in allergic responses.
Assuntos
Butiratos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Expressão Gênica , Síndrome Hipereosinofílica/metabolismo , Glicoproteínas da Membrana de Plaquetas/genética , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Sequência de Aminoácidos , Animais , Azepinas/metabolismo , Sequência de Bases , Ácido Butírico , Células CHO , Clonagem Molecular , Cricetinae , DNA Complementar/química , DNA Complementar/genética , Cobaias , Humanos , Síndrome Hipereosinofílica/patologia , Inositol 1,4,5-Trifosfato/biossíntese , Dados de Sequência Molecular , Toxina Pertussis , Glicoproteínas da Membrana de Plaquetas/química , RNA Mensageiro/biossíntese , Análise de Sequência de DNA , Triazóis/metabolismo , Trítio , Células Tumorais Cultivadas , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Somatostatin has a modulatory role in regulating the membrane conductance in hippocampal neurons. To examine the signal transducing molecules involved in this process, we isolated the cDNA encoding the dominant rat hippocampal somatostatin receptor, SSTR4. Distribution of SSTR4 in the adult central nervous system was restricted to the hippocampus, cerebral cortex, striatum, hypothalamus, and thalamus, as determined by Northern blot analysis and in situ hybridization. In SSTR4-expressing Chinese hamster ovary cells, SSTR4 was functionally coupled not only to inhibition of adenylate cyclase, but also to activation of both arachidonate release and mitogen-activated protein (MAP) kinase cascade, with similar ED50 values. All of these pathways, including both MAP kinase kinase and MAP kinase activation, were completely blocked by pretreatment with pertussis toxin. On the other hand, neither inositol 1,4,5-trisphosphate synthesis nor intracellular Ca2+ mobilization was induced upon SSTR4 stimulation. These data indicate that the hippocampal functions of somatostatin might be mediated through diverse but selective second messenger systems activated via SSTR4 and reveal an unsuspected coupling of a neuronal SSTR subtype to a mitogenic signaling pathway. SSTR4, in addition, provides a useful system to study the Ca(2+)-independent, Gi-dependent (pertussis toxin-sensitive) pathway of MAP kinase activation.