Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 9 de 9
Filtrar
1.
Cell ; 187(14): 3726-3740.e43, 2024 Jul 11.
Artículo en Inglés | MEDLINE | ID: mdl-38861993

RESUMEN

Many growth factors and cytokines signal by binding to the extracellular domains of their receptors and driving association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affect signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo-designed fibroblast growth factor receptor (FGFR)-binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and mitogen-activated protein kinase (MAPK) pathway activation. The high specificity of the designed agonists reveals distinct roles for two FGFR splice variants in driving arterial endothelium and perivascular cell fates during early vascular development. Our designed modular assemblies should be broadly useful for unraveling the complexities of signaling in key developmental transitions and for developing future therapeutic applications.


Asunto(s)
Diferenciación Celular , Factores de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos , Transducción de Señal , Animales , Humanos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Ratones , Ligandos , Calcio/metabolismo , Sistema de Señalización de MAP Quinasas
2.
Proc Natl Acad Sci U S A ; 120(7): e2219128120, 2023 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-36745784

RESUMEN

While important insights were gained about how FGF21 and other endocrine fibroblast growth factors (FGFs) bind to Klotho proteins, the exact mechanism of Klotho/FGF receptor assembly that drives receptor dimerization and activation has not been elucidated. The prevailing dogma is that Klotho proteins substitute for the loss of heparan sulfate proteoglycan (HSPG) binding to endocrine FGFs by high-affinity binding of endocrine FGF molecules to Klotho receptors. To explore a potential role of HSPG in FGF21 signaling, we have analyzed the dynamic properties of FGF21-induced FGF21-ßKlotho-FGFR1c complexes on the surface of living wild-type (WT) or HSPG-deficient Chinese hamster ovary (CHO) cells by employing quantitative single-molecule fluorescence imaging analyses. Moreover, detailed analyses of FGF21 and FGF1 stimulation of cellular signaling pathways activated in WT or in HSPG-deficient CHO cells are also analyzed and compared. These experiments demonstrate that heparin is required for the formation of FGF21-ßKlotho-FGFR1c complexes on the cell membrane and that binding of heparin or HSPG to FGFR1c is essential for optimal FGF21 stimulation of FGFR1c activation, mitogen-activated protein kinase responses, and intracellular Ca2+ release. It is also shown that FGF1 binding stimulates assembly of ßKlotho and FGFR1c on cell membranes, resulting in endocytosis and degradation of ßKlotho. We conclude that heparin or HSPG is essential for FGF21 signaling and for regulation of ßKlotho cellular stability by acting as a coligand of FGFR1c.


Asunto(s)
Proteoglicanos de Heparán Sulfato , Proteínas Klotho , Cricetinae , Animales , Células CHO , Cricetulus , Heparina , Factor 1 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/fisiología
3.
Proc Natl Acad Sci U S A ; 119(48): e2208947119, 2022 11 29.
Artículo en Inglés | MEDLINE | ID: mdl-36417441

RESUMEN

The phosphoinositide-3 kinase (PI-3K)/AKT cell survival pathway is an important pathway activated by EGFR signaling. Here we show, that in addition to previously described critical components of this pathway, i.e., the docking protein Gab1, the PI-3K/AKT pathway in epithelial cells is regulated by the exocyst complex, which is a vesicle tether that is essential for exocytosis. Using live-cell imaging, we demonstrate that PI(3,4,5)P3 levels fluctuate at the membrane on a minutes time scale and that these fluctuations are associated with local PI(3,4,5)P3 increases at sites where recycling vesicles undergo exocytic fusion. Supporting a role for exocytosis in PI(3,4,5)P3 generation, acute promotion of exocytosis by optogenetically driving exocyst-mediated vesicle tethering up-regulates PI(3,4,5)P3 production and AKT activation. Conversely, acute inhibition of exocytosis using Endosidin2, a small-molecule inhibitor of the exocyst subunit Exo70 (also designated EXOC7), or inhibition of exocyst function by siRNA-mediated knockdown of the exocyst subunit Sec15 (EXOC6), impairs PI(3,4,5)P3 production and AKT activation induced by EGF stimulation of epithelial cells. Moreover, prolonged inhibition of EGF signaling by EGFR tyrosine kinase inhibitors results in spontaneous reactivation of AKT without a concomitant relief of EGFR inhibition. However, this reactivation can be negated by acutely inhibiting the exocyst. These experiments demonstrate that exocyst-mediated exocytosis-by regulating PI(3,4,5)P3 levels at the plasma membrane-subserves activation of the PI-3K/AKT pathway by EGFR in epithelial cells.


Asunto(s)
Factor de Crecimiento Epidérmico , Exocitosis , Fosfatidilinositol 3-Quinasa , Humanos , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB , Proteínas Proto-Oncogénicas c-akt , Vesículas Extracelulares
4.
Proc Natl Acad Sci U S A ; 117(50): 31800-31807, 2020 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-33257569

RESUMEN

The three members of the endocrine-fibroblast growth factor (FGF) family, FGF19, 21, and 23 are circulating hormones that regulate critical metabolic processes. FGF23 stimulates the assembly of a signaling complex composed of α-Klotho (KLA) and FGF receptor (FGFR) resulting in kinase activation, regulation of phosphate homeostasis, and vitamin D levels. Here we report that the C-terminal tail of FGF23, a region responsible for KLA binding, contains two tandem repeats, repeat 1 (R1) and repeat 2 (R2) that function as two distinct ligands for KLA. FGF23 variants with a single KLA binding site, FGF23-R1, FGF23-R2, or FGF23-wild type (WT) with both R1 and R2, bind to KLA with similar binding affinity and stimulate FGFR1 activation and MAPK response. R2 is flanked by two cysteines that form a disulfide bridge in FGF23-WT; disulfide bridge formation in FGF23-WT is dispensable for KLA binding and for cell signaling via FGFRs. We show that FGF23-WT stimulates dimerization and activation of a chimeric receptor molecule composed of the extracellular domain of KLA fused to the cytoplasmic domain of FGFR and employ total internal reflection fluorescence microscopy to visualize individual KLA molecules on the cell surface. These experiments demonstrate that FGF23-WT can act as a bivalent ligand of KLA in the cell membrane. Finally, an engineered Fc-R2 protein acts as an FGF23 antagonist offering new pharmacological intervention for treating diseases caused by excessive FGF23 abundance or activity.


Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Glucuronidasa/metabolismo , Multimerización de Proteína/fisiología , Sitios de Unión , Calcinosis/tratamiento farmacológico , Calcinosis/genética , Membrana Celular/metabolismo , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/uso terapéutico , Células HEK293 , Humanos , Hiperostosis Cortical Congénita/tratamiento farmacológico , Hiperostosis Cortical Congénita/genética , Hiperfosfatemia/tratamiento farmacológico , Hiperfosfatemia/genética , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Proteínas Klotho , Mutación , Osteomalacia/tratamiento farmacológico , Osteomalacia/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Dominios Proteicos , Multimerización de Proteína/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/uso terapéutico , Raquitismo Hipofosfatémico/tratamiento farmacológico , Raquitismo Hipofosfatémico/genética
5.
bioRxiv ; 2023 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-36993355

RESUMEN

Growth factors and cytokines signal by binding to the extracellular domains of their receptors and drive association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affects signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo designed fibroblast growth-factor receptor (FGFR) binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and MAPK pathway activation. The high specificity of the designed agonists reveal distinct roles for two FGFR splice variants in driving endothelial and mesenchymal cell fates during early vascular development. The ability to incorporate receptor binding domains and repeat extensions in a modular fashion makes our designed scaffolds broadly useful for probing and manipulating cellular signaling pathways.

6.
Cell Rep Methods ; 2(4): 100199, 2022 04 25.
Artículo en Inglés | MEDLINE | ID: mdl-35497490

RESUMEN

A complete understanding of synaptic-vesicle recycling requires the use of multiple microscopy methods to obtain complementary information. However, many currently available probes are limited to a specific microscopy modality, which necessitates the use of multiple probes and labeling paradigms. Given the complexity of vesicle populations and recycling pathways, having new single-vesicle probes that could be used for multiple microscopy techniques would complement existing sets of tools for studying vesicle function. Here, we present a probe based on the membrane-binding C2 domain of cytosolic phospholipase A2 (cPLA2) that fulfills this need. By conjugating the C2 domain with different detectable tags, we demonstrate that a single, modular probe can allow synaptic vesicles to be imaged at multiple levels of spatial and temporal resolution. Moreover, as a general endocytic marker, the C2 domain may also be used to study membrane recycling in many cell types.


Asunto(s)
Imagen Multimodal , Vesículas Sinápticas , Vesículas Sinápticas/química
7.
Nat Commun ; 12(1): 5434, 2021 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-34521845

RESUMEN

Vesicle tethers are thought to underpin the efficiency of intracellular fusion by bridging vesicles to their target membranes. However, the interplay between tethering and fusion has remained enigmatic. Here, through optogenetic control of either a natural tether-the exocyst complex-or an artificial tether, we report that tethering regulates the mode of fusion. We find that vesicles mainly undergo kiss-and-run instead of full fusion in the absence of functional exocyst. Full fusion is rescued by optogenetically restoring exocyst function, in a manner likely dependent on the stoichiometry of tether engagement with the plasma membrane. In contrast, a passive artificial tether produces mostly kissing events, suggesting that kiss-and-run is the default mode of vesicle fusion. Optogenetic control of tethering further shows that fusion mode has physiological relevance since only full fusion could trigger lamellipodial expansion. These findings demonstrate that active coupling between tethering and fusion is critical for robust membrane merger.


Asunto(s)
Criptocromos/genética , Exosomas/metabolismo , Receptores de Transferrina/genética , Vesículas Secretoras/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Unión al GTP rab/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Criptocromos/metabolismo , Exosomas/ultraestructura , Expresión Génica , Genes Reporteros , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Fusión de Membrana/genética , Microscopía Fluorescente , Optogenética/métodos , Receptores de Transferrina/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Vesículas Secretoras/ultraestructura , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteína Fluorescente Roja
8.
Nat Neurosci ; 13(5): 577-83, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20383135

RESUMEN

Understanding the fundamental role of soluble NSF attachment protein receptor (SNARE) complexes in membrane fusion requires knowledge of the spatiotemporal dynamics of their assembly. We visualized complexin (cplx), a cytosolic protein that binds assembled SNARE complexes, during single exocytic events in live cells. We found that cplx appeared briefly during full fusion. However, a truncated version of cplx containing only the SNARE-complex binding region persisted at fusion sites for seconds and caused fusion to be transient. Resealing pores with the mutant cplx only partially released transmitter and lipid probes, indicating that the pores are narrow and not purely lipidic in structure. Depletion of cplx similarly caused secretory cargo to be retained. These data suggest that cplx is recruited at a late step in exocytosis and modulates fusion pores composed of SNARE complexes.


Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Exocitosis/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Dopamina/metabolismo , Exocitosis/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Humanos , Microscopía Confocal/métodos , Proteínas del Tejido Nervioso/genética , Neuropéptido Y/genética , Neuropéptido Y/metabolismo , Células PC12 , Fotoblanqueo , Unión Proteica , Compuestos de Piridinio/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Interferencia de ARN/fisiología , Ratas , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Transfección/métodos
9.
Science ; 306(5698): 1042-6, 2004 Nov 05.
Artículo en Inglés | MEDLINE | ID: mdl-15528447

RESUMEN

Syntaxin, synaptosome-associated protein of 25 kD (SNAP25), and vesicle-associated membrane protein/synaptobrevin are collectively called SNAP receptor (SNARE) proteins, and they catalyze neuronal exocytosis by forming a "core complex." The steps in core complex formation are unknown. Here, we monitored SNARE complex formation in vivo with the use of a fluorescent version of SNAP25. In PC12 cells, we found evidence for a syntaxin-SNAP25 complex that formed with high affinity, required only the amino-terminal SNARE motif of SNAP25, tolerated a mutation that blocks formation of other syntaxin-SNAP25 complexes, and assembled reversibly when Ca2+ entered cells during depolarization. The complex may represent a precursor to the core complex formed during a Ca2+-dependent priming step of exocytosis.


Asunto(s)
Proteínas de Transporte Vesicular/fisiología , Médula Suprarrenal/citología , Animales , Proteínas Bacterianas , Línea Celular , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Células PC12 , Proteínas Qa-SNARE , Ratas , Proteínas Recombinantes de Fusión , Proteínas SNARE , Proteína 25 Asociada a Sinaptosomas
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA