RESUMO
EGF-CFC genes encode a novel class of extracellular, membrane-associated proteins that notably play an important role during vertebrate gastrulation. Whereas the two cysteine-rich domains that characterize these proteins, namely the extracellular EGF-like and the CFC domain, are known to be encoded by two evolutionarily conserved exons, it is generally assumed, based on weak primary sequence identity, that the remaining parts of the protein differ among vertebrates, suggesting that known members of the EGF-CFC family do not represent true orthologs. Here, by characterizing the full cDNA and genomic sequences of a new EGF-CFC gene in chick, and by comparing them with their counterparts in human (CRIPTO), mouse (cripto and cryptic), Xenopus (FRL-1) and zebrafish (one-eyed pinhead), we show that all EGF-CFC genes share an identical genomic organization over the entire coding region. Not only are the central two exons (coding for the EGF-like and CFC motifs) conserved, but also conserved are the total number of exons, their size, their intron phase and their correlation with discrete protein modules, in particular those modules that allow the EGF-CFC motif to become membrane-associated. Therefore, despite apparent divergence between their 5' and 3'-terminal exons, all known CRIPTO-related genes are structurally orthologous. We named this novel ortholog in bird, chick-cripto. We report the mRNA distribution of chick-cripto, which begins in the epiblast of the gastrula, with a pattern similar to EGF-CFC genes of other vertebrates.
Assuntos
Embrião de Galinha/metabolismo , Fator de Crescimento Epidérmico/genética , Proteínas de Homeodomínio , Glicoproteínas de Membrana , Proteínas de Neoplasias/genética , Fatores de Transcrição , Proteínas de Xenopus , Proteínas de Peixe-Zebra , Sequência de Aminoácidos , Animais , Sequência de Bases , Galinhas , Clonagem Molecular , DNA/química , DNA/genética , DNA Complementar/química , DNA Complementar/genética , Fator de Crescimento Epidérmico/química , Éxons , Proteínas Ligadas por GPI , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes/genética , Coração/embriologia , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular , Íntrons , Proteínas de Membrana , Dados de Sequência Molecular , Miocárdio/metabolismo , Proteínas de Neoplasias/química , Sistema Nervoso/embriologia , Sistema Nervoso/metabolismo , Hibridização de Ácido Nucleico/métodos , Estrutura Secundária de Proteína , RNA/genética , RNA/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Transcrição GênicaRESUMO
5-HT2B receptors, in addition to phospholipase C stimulation, are able to trigger activation of the proto-oncogene product p21ras. During mouse embryogenesis, a peak of 5-HT2B receptor expression is detected at the neurulation stage; we localized the 5-HT2B expression in neural crest cells, heart myocardium, and somites. The requirement for functional 5-HT2B receptors shortly after gastrulation, is supported by culture of embryos exposed to 5-HT2B-high affinity antagonist such as ritanserin, which induces morphological defects in the cephalic region, heart and neural tube. Functional 5-HT2B receptors are also expressed during the serotonergic differentiation of the mouse F9 teratocarcinoma-derived clonal cell line 1C11. Upon 2 days of induction by cAMP, 5-HT2B receptors become functional, and on day 4, the appearance of 5-HT2A receptors coincides with the onset of active serotonin transporter by these cells. Active serotonin uptake is modulated by serotonin suggesting autoreceptor functions for 5-HT2B receptors.
Assuntos
Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica no Desenvolvimento , Receptores de Serotonina/genética , Receptores de Serotonina/fisiologia , Serotonina/fisiologia , Animais , Diferenciação Celular , Genes ras , Camundongos , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptor 5-HT2B de SerotoninaRESUMO
The novel serotonin receptor 5-HT2B shows the highest homology to the 5-HT2 family of receptors. The pharmacological profile of membranes from 5-HT2B cDNA stably transfected LMTK- cell line, corresponds to a new 5-HT2-like receptor named 5-HT2B, although some difference exists between the mouse and rat pharmacology. A similar pharmacological profile is detected on the immortalized teratocarcinoma-derived cell line 1C11 upon 2 days of serotoninergic differenciation by cAMP. In both cell lines, the analysis 125I-DOI binding reveals the presence of a single class of sites, the affinity of which is one order of magnitude lower than the one reported for the 5-HT2A receptor. This demonstrates that the 5-HT2B receptor is functionally expressed before the complete serotoninergic differentiation of 1C11 cells. These observations are in good agreement with the presence of 5-HT2B mRNA in early mouse embryonic development. Furthermore, the major sites of 5-HT2B mRNA embryonic expression are in the heart, and in the neural fold before the closure of the neural tube. Therefore, this receptor could account at least in part for the trophic functions attributed to the 5-HT2-like receptors.