RESUMO
IGF peptides belong to a complex system that is known to play a major role in the control of growth and development in mammals. Even if studies performed in nonmammalian species tend to demonstrate an important function of these molecules, use of heterologous ligands, especially in fish, partly limit our knowledge of the physiological role(s) of IGFs. We report in this study the cloning, production, and characterization in an evolved fish, the turbot Psetta maxima, of mature IGF-I and IGF-II. The deduced 68-amino-acid IGF-I and 70-amino-acid IGF-II show 75% and 74% sequence identity with their mammalian counterparts, respectively, confirming the high sequence homology observed in other species. The development of a simple and efficient system for the production and purification of both IGF-I and IGF-II in Escherichia coli was used to investigate the in vitro regulation of GH release in the turbot. Our results demonstrated for the first time in a Euteleost species that both peptides specifically inhibited GH release. Both hormones were equally potent in inhibiting GH release from dispersed pituitary cells, with maximal inhibitory effects of 92% and 91% at 1 nM doses after 12 days of culture, respectively. The biologically active recombinant turbot IGFs that we obtained will allow us to further investigate potential and perhaps the specific role(s) of these hormones in turbot as, in contrast with mammals, growth in fish is potentially continued during "adult" life.
Assuntos
Clonagem Molecular , Linguados/genética , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/farmacologia , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Membrana Celular/metabolismo , Células Cultivadas , Meios de Cultura Livres de Soro , DNA Complementar/química , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Feminino , Linguados/metabolismo , Hormônio do Crescimento/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/química , Fator de Crescimento Insulin-Like II/química , Cinética , Dados de Sequência Molecular , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Placenta/metabolismo , Gravidez , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
The insulin receptor (IR) and the structurally related insulin-like growth factor type 1 receptor (IGF-1R) belong to the tyrosine kinase (TK) receptor family. In this study, we have carried out the molecular characterization of both receptors from turbot (Psetta maxima), an evoluted teleost flatfish. The cDNA encoding the precursors of IGF-1R and the nearly entire IR (only the first 16 amino acids of the alpha subunit are missing when compared to the published human sequence) were cloned from an embryonic cDNA library. The deduced polypeptides contain all the topological features characterizing the insulin/IGF-1 receptor family. They are highly conserved compared to their mammalian counterparts, particularly within domains involved in the catalytic activity and in the transduction pathways. Nevertheless, some differences in the primary sequences, especially in the carboxy-terminal domain of the precursors, may affect the functions fulfilled by these receptors. As in mammals, the long IGF-1R 5'-untranslated sequence contains open reading frames and potential Sp1 binding sites. Northern blot analyses have revealed a major IR transcript of 11 kb, which is approximately the size of IGF-1R transcript (Eliès, G., Groigno, L., Wolff, J., Boeuf, G., Boujard, D., 1996. Characterization of the insulin-like growth factor type 1 receptor messenger in two teleost species. Mol. Cell. Endocrinol. 124, 131-140.). If IR and IGF-1R transcripts are detected by RT-PCR at all developmental stages and in all tissues examined, in situ hybridization studies have shown that these mRNA can be visualized as ubiquitous signals only in young larvae, whereas IGF-1R and IR expression appears weaker during later development and in adult tissues.
Assuntos
RNA Mensageiro/metabolismo , Receptor IGF Tipo 1/genética , Receptor de Insulina/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Clonagem Molecular , Embrião não Mamífero , Linguados , Biblioteca Gênica , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , RNA Mensageiro/análise , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de SequênciaRESUMO
The insulin-like growth factor type 1 receptor (IGF-1R) is a tyrosine kinase which plays essential role in the regulation of growth and development. In this study, we have cloned cDNAs encoding the tyrosine kinase domain of the IGF-1R from two species of fish. The turbot and trout nucleotide sequences share 82% identity. Moreover, the deduced polypeptides are also highly conserved (> 90% identity) compared with the IGF-1R sequences described in other vertebrates, particularly within domains involved in the catalytic activity and in the transduction pathway. Northern blot analyses have revealed a unique 13-kb mRNA transcript. Using an RT-PCR approach, we have also shown that the polyadenylation status seems to vary according to the developmental stage in turbot: polyadenylated in oocytes and in the first larval stages, the mRNA becomes undetectable in the polyadenylated fraction in later stages or in adult somatic tissues. These results suggest that IGF-1R mRNA undergoes complex post-transcriptional regulation.
