RESUMEN
The biogenesis of exosomes, small secreted vesicles involved in signalling processes, remains incompletely understood. Here, we report evidence that the syndecan heparan sulphate proteoglycans and their cytoplasmic adaptor syntenin control the formation of exosomes. Syntenin interacts directly with ALIX through LYPX(n)L motifs, similarly to retroviral proteins, and supports the intraluminal budding of endosomal membranes. Syntenin exosomes depend on the availability of heparan sulphate, syndecans, ALIX and ESCRTs, and impact on the trafficking and confinement of FGF signals. This study identifies a key role for syndecan-syntenin-ALIX in membrane transport and signalling processes.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Exosomas/metabolismo , Transducción de Señal , Sindecanos/metabolismo , Sinteninas/metabolismo , Animales , Sitios de Unión , Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Ratones , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Interferencia de ARN , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Sindecanos/genética , Sinteninas/genética , Factores de Tiempo , TransfecciónRESUMEN
Syndecans are heparan sulfate proteoglycans that modulate the activity of several growth factors and cell adhesion molecules. PDZ domains in the adaptor protein syntenin interact with syndecans and with the phosphoinositide PIP(2), which is involved in the regulation of the actin cytoskeleton and membrane trafficking. Here, we show that the syntenin PDZ domain-PIP(2) interaction controls Arf6-mediated syndecan recycling through endosomal compartments. FGF receptor accompanies syndecan along the syntenin-mediated recycling pathway, in a heparan sulfate- and FGF-dependent manner. Syndecans that cannot recycle via this pathway become trapped intracellularly and inhibit cell spreading. This syntenin-mediated syndecan recycling pathway may regulate the surface availability of a number of cell adhesion and signaling molecules.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteoglicanos/metabolismo , Factor 6 de Ribosilación del ADP , Adhesión Celular/fisiología , Línea Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Endosomas/metabolismo , Endosomas/ultraestructura , Humanos , Modelos Biológicos , Fosfatidilinositol 4,5-Difosfato/química , Sindecano-2 , Sindecanos , SinteninasRESUMEN
Mammalian heparanase, strongly implicated in the regulation of cell growth, migration, and differentiation, plays a crucial role in inflammation, angiogenesis, and metastasis. There is thus a clear need for understanding how heparanase activity is regulated. Cells can generate an active form of the enzyme from a larger inactive precursor protein by a process of secretion-recapture, internalization, and proteolytic processing in late endosomes/lysosomes. Cell surface heparan sulfate proteoglycans are the sole known components with a role in this trafficking of the heparanase precursor. Here, we provide evidence that heparan sulfate proteoglycans are not strictly required for this process. More importantly, by heparanase transfection, binding, and uptake experiments and by using a combination of specific inhibitors and receptor-defective cells, we have identified low density lipoprotein receptor-related proteins and mannose 6-phosphate receptors as key elements of the receptor system that mediates the capture of secreted heparanase precursor and its trafficking to the intracellular site of processing/activation.
Asunto(s)
Glucuronidasa/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Precursores de Proteínas/metabolismo , Receptor IGF Tipo 2/metabolismo , Animales , Células CHO , Línea Celular , Línea Celular Transformada , Cricetinae , Cricetulus , Endosomas/metabolismo , Fibroblastos/metabolismo , Glicosilación , Humanos , Lisosomas/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Glypican (GPC)-3 inhibits cell proliferation and regulates cell survival during development. This action is demonstrated by GPC3 loss-of-function mutations in humans and mice. Here, we show that the GPC3 core protein is processed by a furinlike convertase. This processing is essential for GPC3 modulating Wnt signaling and cell survival in vitro and for supporting embryonic cell movements in zebrafish. The processed GPC3 core protein is necessary and sufficient for the cell-specific induction of apoptosis, but in vitro effects on canonical and noncanonical Wnt signaling additionally require substitution of the core protein with heparan sulfate. Wnt 5A physically associates only with processed GPC3, and only a form of GPC3 that can be processed by a convertase is able to rescue epiboly and convergence/extension movements in GPC3 morphant embryos. Our data imply that the Simpson-Golabi-Behmel syndrome may in part result from a loss of GPC3 controls on Wnt signaling, and suggest that this function requires the cooperation of both the protein and the heparan sulfate moieties of the proteoglycan.