RESUMO
A specific splice variant of the CD44 cell- surface protein family, CD44v6, has been shown to act as a coreceptor for the receptor tyrosine kinase c-Met on epithelial cells. Here we show that also on endothelial cells (ECs), the activity of c-Met is dependent on CD44v6. Furthermore, another receptor tyrosine kinase, VEGFR-2, is also regulated by CD44v6. The CD44v6 ectodomain and a small peptide mimicking a specific extracellular motif of CD44v6 or a CD44v6-specific antibody prevent CD44v6-mediated receptor activation. This indicates that the extracellular part of CD44v6 is required for interaction with c-Met or VEGFR-2. In the cytoplasm, signaling by activated c-Met and VEGFR-2 requires association of the CD44 carboxy-terminus with ezrin that couples CD44v6 to the cytoskeleton. CD44v6 controls EC migration, sprouting, and tubule formation induced by hepatocyte growth factor (HGF) or VEGF-A. In vivo the development of blood vessels from grafted EC spheroids and angiogenesis in tumors is impaired by CD44v6 blocking reagents, suggesting that the coreceptor function of CD44v6 for c-Met and VEGFR-2 is a promising target to block angiogenesis in pathologic conditions.
Assuntos
Receptores de Hialuronatos/metabolismo , Neovascularização Fisiológica/fisiologia , Transdução de Sinais/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/imunologia , Imunoprecipitação , Camundongos , Camundongos SCID , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/prevenção & controle , Ligação Proteica , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologiaRESUMO
In several types of cells, the activation of the receptor tyrosine kinase c-Met by its ligand hepatocyte growth factor (HGF) requires the coreceptor CD44v6. The CD44 extracellular domain is necessary for c-Met autophosphorylation, whereas the intracellular domain is required for signal transduction. We have already shown that the CD44 cytoplasmic tail recruits ezrin, radixin and moesin (ERM) proteins to the complex of CD44v6, c-Met, and HGF. We have now defined the function of the ERM proteins and the step they promote in the signaling cascade. The association of ERM proteins to the coreceptor is absolutely required to mediate the HGF-dependent activation of Ras by the guanine nucleotide exchange factor Sos. The ERM proteins need, in addition, to be linked to the actin cytoskeleton to catalyze the activation of Ras. Thus, we describe here a new function of the cytoskeleton. It is part of a "signalosome" complex that organizes the activation of Ras by Sos. So far the cytoskeleton has mainly been identified as a "responder" to signal transduction. Here, we show now that F-actin acts as an "inducer" that actively organizes the signaling cascade.
Assuntos
Actinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Glicoproteínas/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Receptores de Hialuronatos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Citoesqueleto/efeitos dos fármacos , Células HT29 , Humanos , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-met/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Proteínas Son Of Sevenless/metabolismoRESUMO
Various human cancers express elevated levels of the receptor tyrosine kinases Met or Ron and v6-containing isoforms of CD44. The activation of Met and Ron requires the presence of such CD44 v6-containing isoforms that act as coreceptors. Three amino acids within the v6 sequence were identified by mutational analysis to be essential for the coreceptor function: EWQ in the rat sequence and RWH in human. Peptides comprising these three amino acids (the smallest containing only five amino acids) efficiently act as competitors and block ligand-dependent activation of Met or Ron and subsequent cell migration.
Assuntos
Movimento Celular/fisiologia , Glicoproteínas/metabolismo , Receptores de Hialuronatos/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/genética , Células HT29 , Humanos , Receptores de Hialuronatos/genética , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-met , Ratos , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Relação Estrutura-AtividadeRESUMO
Tightly controlled concentration gradients of morphogens provide positional information and thus regulate tissue differentiation and morphogenesis in multicellular organisms. However, how such morphogenetic fields are formed and maintained remains debated. Here we show that fibroblast growth factor 8 (Fgf8) morphogen gradients in zebrafish embryos are established and maintained by two essential mechanisms. Firstly, Fgf8 is taken up into the cell by clathrin-mediated endocytosis. The speed of the uptake rate defines the range of the morphogenetic gradient of Fgf8. Secondly, our data demonstrate that after endocytosis the routing of Fgf8 from the early endosome to the late endosome shuts down signaling. Therefore, intracellular endocytic transport regulates the intensity and duration of Fgf8 signaling. We show that internalization of Fgf8 into the early endosome and subsequent transport towards the late endosome are two independent processes. Therefore, we hypothesize that Fgf8 receiving cells control both, the propagation width and the signal strength of the morphogen.
Assuntos
Endocitose , Endossomos/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Transdução de Sinais , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Clatrina/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Fator 8 de Crescimento de Fibroblasto/análise , Células HEK293 , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Transporte Proteico , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/análiseRESUMO
Receptor Tyrosine Kinases (RTKs) are involved in many cellular processes and play a major role in the control of cell fate. For these reasons, RTK activation is maintained under tight control. Met is an essential RTK that induces proliferation, differentiation, migration, survival and branching morphogenesis. Deregulation of Met by overexpression, amplification or lack of effective degradation leads to cancer and metastasis. We have shown that Met relies on CD44v6 for its activation and for signaling in several cancer cell lines and also in primary cells. In this paper, we show that internalization of Met is dependent on CD44v6 and the binding of Ezrin to the CD44v6 cytoplasmic domain. Both CD44v6 and Met are co-internalized upon Hepatocyte Growth Factor induction suggesting that Met-induced signaling from the endosomes relies on its collaboration with CD44v6 and the link to the cytoskeleton provided by ERM proteins.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Receptores de Hialuronatos/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Humanos , Receptores de Hialuronatos/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de SinaisRESUMO
CD44 isoforms act as coreceptors for the receptor tyrosine kinases c-Met and VEGFR-2. However, Cd44 knockout mice do not show overt phenotypes, in contrast to Met and Vegfr-2 knockout mice. We hypothesized that CD44 is being compensated for by another factor in Cd44 null mice. Using RNAi technology and blocking experiments with antibodies, peptides, and purified ectodomains, as well as overexpression studies, we identified intercellular adhesion molecule-1 (ICAM-1) as a new coreceptor for c-Met in CD44-negative tumor cells and in primary hepatocytes obtained from Cd44 null mice. Most strikingly, after partial hepatectomy, CD44v6-specific antibodies inhibited liver cell proliferation and c-Met activation in wild-type mice, whereas ICAM-1-specific antibodies interfered with liver cell proliferation and c-Met activation in Cd44 knockout mice. These data show that ICAM-1 compensates for CD44v6 as a coreceptor for c-Met in Cd44 null mice. Compensation of proteins by members of the same family has been widely proposed to explain the lack of phenotype of several knockout mice. Our experiments demonstrate the functional substitution of a protein by a heterologous one in a knockout mouse.
Assuntos
Hepatócitos/metabolismo , Receptores de Hialuronatos/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptor Cross-Talk , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Anticorpos Bloqueadores/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Teste de Complementação Genética , Fator de Crescimento de Hepatócito/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Hepatócitos/citologia , Receptores de Hialuronatos/genética , Molécula 1 de Adesão Intercelular/genética , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Cultura Primária de Células , Isoformas de Proteínas/genética , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-met/genética , RNA Interferente Pequeno/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Recent evidence has shown that the activation of receptor tyrosine kinases is not only dependent on binding of their ligands but in addition requires adhesion molecules as coreceptors. We have identified CD44v6 as a coreceptor for c-Met in several tumor and primary cells. The CD44v6 ectodomain is required for c-Met activation, whereas the cytoplasmic tail recruits ERM proteins and the cytoskeleton into a signalosome complex. Here we demonstrate that c-Met (and hepatocyte growth factor and Gab1) is haploinsufficient in a cd44-/- background, as the cd44-/-; met+/- (and cd44-/-; hgf+/- and cd44-/-; gab1+/-) mice die at birth. They have impaired synaptic transmission in the respiratory rhythm-generating network and alterations in the phrenic nerve. These results are the first genetic data showing that CD44 and c-Met collaborate in vivo and that they are involved in synaptogenesis and axon myelination in the central and peripheral nervous systems.