Assuntos
Estatura/genética , Genes Dominantes , Mutação , Receptores da Somatotropina/genética , Sítios de Ligação , Proteínas de Transporte/sangue , Pré-Escolar , Citoplasma/metabolismo , Feminino , Heterozigoto , Hormônio do Crescimento Humano/farmacologia , Humanos , Masculino , Receptores da Somatotropina/efeitos dos fármacos , Receptores da Somatotropina/metabolismoRESUMO
The GH receptor (GHR) is a member of the cytokine receptor superfamily; its signaling involves the activation of Janus tyrosine kinases (JAK2) and Stat (signal transducers and activators of transcription) transcription factors. Using truncated and tyrosine mutants of the receptor, we show that different receptor domains are essential for the activation of Stat3 and Stat5. GH-dependent phosphorylation of JAK2, Stat3, and Stat5, as well as transactivation studies with reporter genes containing Stat3 and Stat5 DNA-binding elements, was performed in cells expressing the various GHR mutants. The membrane-proximal region of the receptor necessary for JAK2 activation is sufficient for Stat3 activation. In contrast, C-terminal tyrosine residues of GHR are absolutely required for Stat5 activation. The same residues are also involved in the regulation of JAK2 dephosphorylation, possibly through the activation of a phosphatase. Using in vitro experiments with glutathione-S-transferase fusion proteins, we demonstrate that the SH2 domain of Stat5 binds to the carboxy-terminal tyrosine-phosphorylated residues of GHR. Our results show that a cytokine receptor can mediate differently the activation of distinct Stat proteins that could be involved in cytokine-specific effects.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas do Leite , Proteínas Proto-Oncogênicas , Receptores da Somatotropina/fisiologia , Transativadores/metabolismo , Animais , Células CHO , Cricetinae , Ativação Enzimática , Glutationa Transferase/genética , Hormônio do Crescimento/farmacologia , Janus Quinase 2 , Mutagênese Sítio-Dirigida , Fosforilação , Fosfotirosina/metabolismo , Proteínas Tirosina Quinases/metabolismo , Coelhos , Receptores da Somatotropina/química , Receptores da Somatotropina/genética , Proteínas Recombinantes de Fusão , Fator de Transcrição STAT3 , Fator de Transcrição STAT5 , Transdução de Sinais , Relação Estrutura-Atividade , TransfecçãoRESUMO
A signaling pathway was delineated by which GH promotes cell survival. Experiments were performed in human leukemic cells (HL-60) and Chinese hamster ovary (CHO) cells. In HL-60 cells, GH treatment reduced starvation-induced cell death. In contrast, when HL-60 cells were treated with an anti-GH antibody, cell survival was sharply reduced. In CHO cells stably expressing either the wild-type (wtGHR) or a truncated form (delta454GHR) of the GH receptor in which GH induces a sustained activation of the receptor-associated tyrosine kinase JAK2, we found that GH stimulation inhibited programmed cell death induced by withdrawal of survival factors. This effect was enhanced in cells expressing the truncated form. In contrast, GH did not affect cell survival in CHO cells transfected with either the empty vector or a mutated GHR unable to transduce the signal (4P/AGHR). We also showed that the inhibitory action of GH on apoptosis is probably mediated via stimulation of the serine-threonine kinase Akt, as 1) GH treatment induces a prompt phosphorylation of Akt; and 2) GH effects on cell survival are abolished by transfection of an Akt mutant that exhibits dominant negative function. Experiments with pharmacological inhibitors demonstrated that GH-induced Akt phosphorylation is dependent on phosphoinositide 3-kinase activation. In contrast, we found no changes in Bcl-2 levels secondary to GHR activation.
Assuntos
Apoptose , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/fisiologia , Receptores da Somatotropina/fisiologia , Animais , Apoptose/efeitos dos fármacos , Células CHO , Cricetinae , Ativação Enzimática , Expressão Gênica , Células HL-60 , Hormônio do Crescimento Humano/farmacologia , Humanos , Janus Quinase 2 , Mutação , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptores da Somatotropina/genética , TransfecçãoRESUMO
Growth hormone receptor (GHR) signaling involves activation of the Janus Kinases (Jak) and of Stat proteins (signal transducers and activators of transcription). Growth hormone (GH) induces transcriptional activation of c-fos gene and the c-sis inducible element (SIE) of its promoter was shown to bind the Stat proteins. Using cells co-transfected with GHR and Stat 3 expression vectors, we directly demonstrate that GH induces tyrosine phosphorylation of Stat 3 and its binding to the SIE probe. We showed, using mutant forms of GHR, that only the cytoplasmic membrane proximal domain of the receptor, including a conserved proline rich region (box 1), is required for this effect.
Assuntos
Membrana Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Receptores da Somatotropina/metabolismo , Transativadores/metabolismo , Ativação Transcricional/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Linhagem Celular , Cricetinae , Citoplasma/metabolismo , DNA/metabolismo , Sondas de DNA , Proteínas de Ligação a DNA/genética , Hormônio do Crescimento/farmacologia , Cinética , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Receptores da Somatotropina/genética , Fator de Transcrição STAT3 , Deleção de Sequência , Transdução de Sinais/fisiologia , Transativadores/genética , TransfecçãoRESUMO
A novel form of congenital growth hormone insensitivity syndrome (GHIS), which lacks the classic phenotype associated with this condition, is described. Dominant inheritance is shown to result from a heterozygous 876-1 G to C transversion of the 3' splice acceptor site preceding exon 9 in the growth hormone receptor (GHR) gene. The result of this mutation is a severely truncated cytoplasmic domain of the GHR, which is incapable of transmitting a signal. The mutant receptor is shown to form a heterodimer with the wild-type GHR, the activity of which is inhibited in a dominant-negative manner.
Assuntos
Estatura/genética , Genes Dominantes , Mutação/genética , Receptores da Somatotropina/genética , Sequência de Bases , Pré-Escolar , Feminino , Transtornos do Crescimento/genética , Humanos , Recém-Nascido , Masculino , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
The peptide hormones, prolactin (PRL) and growth hormone (GH), are known to regulate numerous target tissues. Among such targets are cells of the immune system, including T cells, B cells, macrophages and natural killer cells. We have cloned a panel of PRL- and GH-inducible T cell genes for use in studies to understand how these hormones through the expression of these genes modulate the biology of immune function cells. This article focuses on the signalling pathways emanating from the PRL receptor (PRL-R) and GH receptor (GH-R), and the expression of PRL-inducible target genes.
Assuntos
Linfócitos B/imunologia , Regulação da Expressão Gênica/imunologia , Hormônio do Crescimento/imunologia , Células Matadoras Naturais/imunologia , Macrófagos/imunologia , Prolactina/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Clonagem Molecular , HumanosRESUMO
Structure of growth-hormone receptor and the class I type of cytokine receptors: common structural features; cytokine-receptor isoforms; oligomerization of receptor components initiates cytokine signalling. Role of the Jak kinases in mediating specific functions of growth-hormone receptor and cytokine receptors. Role of signal transducer and activator of transcription (Stat) proteins in growth hormone and cytokine functions. Other pathways activated by cytokine receptors: the mitogen-activated protein kinase pathway; insulin-receptor substrates 1 and 2 and phosphatidylinositol-3-kinase pathways; the Src pathways and other tyrosine kinase pathways; the phospholipase C/protein kinase C/Ca2+ pathways. Regulation of growth-hormone receptor and cytokine receptor signaling: binding sites, internalization and ubiquitination; phosphatases and Janus kinase/Stat inhibitors. Conclusions and future prospects.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Citocinas/metabolismo , Receptores da Somatotropina/metabolismo , Transativadores/metabolismo , Regulação da Expressão Gênica , Modelos Biológicos , Transdução de SinaisRESUMO
The cloning of receptor targets procedure, used so far to identify proteins associated with tyrosine kinase receptors was modified to clone SH2 proteins able to bind to the growth hormone receptor (GHR). The cytoplasmic region of GHR, a member of the cytokine receptor superfamily does not contain tyrosine kinase activity. It was thus phosphorylated in bacteria by the Elk tyrosine kinase and radiolabeled to screen a mouse expression library. With this probe, we identified Shc and the p85 subunit of phosphatidylinositol 3-kinase as direct targets of the receptor. The other proteins identified, Csk, Shb, Grb4, and Grb10 are new potential transducers for cytokine receptors. We show in Huh-7 hepatoma cells that Grb10 and GHR associate under GH stimulation. Co-transfections in 293 cells further show that Grb10 interacts with both the GHR and Jak2. Functional tests demonstrate that Grb10 inhibits transcription of two reporter genes containing, respectively, the serum response element of c-fos and the GH response element 2 of the Spi2.1 gene, whereas it has no effect on a reporter gene containing only Stat5 binding elements. Our results suggest that Grb10 is a new target for a member of the cytokine receptor family that down-regulates some GH signaling pathways downstream of Jak2 and independently of Stat5.