RESUMEN
The small GTP-binding protein Ral has been implicated in regulated exocytosis via its interaction with the mammalian exocyst complex. We have previously demonstrated that Ral is involved in exocytosis of Weibel-Palade bodies (WPBs). Little is known about intracellular signaling pathways that promote activation of Ral in response to ligand binding of G protein-coupled receptors. Here we show that RNAi-mediated knockdown of RalGDS, an exchange factor for Ral, results in inhibition of thrombin- and epinephrine-induced exocytosis of WPBs, while overexpression of RalGDS promotes exocytosis of WPBs. A RalGDS variant lacking its exchange domain behaves in a dominant negative manner by blocking release of WPBs. We also provide evidence that RalGDS binds calmodulin (CaM) via an amino-terminal CaM-binding domain. RalGDS association to CaM is required for Ral activation because a cell-permeable peptide comprising this RalGDS CaM-binding domain inhibits Ral activation and WPB exocytosis. Together our findings suggest that RalGDS plays a vital role in the regulation of Ral-dependent WPB exocytosis after stimulation with Ca(2+)- or cAMP-raising agonists.
Asunto(s)
Exocitosis/fisiología , Cuerpos de Weibel-Palade/fisiología , Factor de Intercambio de Guanina Nucleótido ral/fisiología , Secuencia de Aminoácidos , Sitios de Unión/genética , Calmodulina/metabolismo , Línea Celular , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Células Endoteliales/ultraestructura , Epinefrina/farmacología , Exocitosis/efectos de los fármacos , Variación Genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Trombina/farmacología , Transfección , Proteínas de Unión al GTP ral/metabolismo , Factor de Intercambio de Guanina Nucleótido ral/antagonistas & inhibidores , Factor de Intercambio de Guanina Nucleótido ral/química , Factor de Intercambio de Guanina Nucleótido ral/genéticaRESUMEN
OBJECTIVE: Perinuclear clustering is observed for several different organelles and illustrates dynamic regulation of the secretory pathway and organelle distribution. Previously, we observed that a subset of Weibel-Palade bodies (WPBs), endothelial cell-specific storage organelles, undergo centralization when endothelial cells are stimulated with cAMP-raising agonists of von Willebrand factor (vWF) secretion. In this study, we investigated this phenomenon of WPB clustering in more detail. METHODS AND RESULTS: Our results demonstrate that the clustered WPBs are localized at the microtubule organizing center and that cluster formation depends on an intact microtubule network. Disruption of the microtubules by nocodazole completely abolished clustering, whereas treatment with the actin depolymerizing compound cytochalasin B had no effect on WPB clustering. Interfering with the dynein-dynactin interaction by overexpression of the p50 dynamitin subunit or the CC1 domain of the p150glued subunit of the dynactin complex completely inhibited perinuclear clustering of WPBs, suggesting that dynein activity mediates this process. Furthermore, we found that inhibition of dephosphorylation resulted in an increase in clustering, whereas inhibition of protein kinase A (PKA) markedly reduced WPB clustering. CONCLUSIONS: These results suggest that perinuclear clustering of WPBs involves PKA-dependent regulation of the dynein-dynactin complex. Endothelial cell stimulation with epinephrine results in retrograde movement of a subset of WPBs to the microtubule organizing center. This minus-end directed transport requires an intact microtubular network and is mediated by the motor protein dynein. Together, our results suggest that epinephrine-induced clustering of WPBs involves PKA-dependent regulation of the dynein-dynactin complex.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dineínas/metabolismo , Endotelio Vascular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Cuerpos de Weibel-Palade/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Bucladesina/farmacología , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Complejo Dinactina , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Epinefrina/farmacología , Humanos , Centro Organizador de los Microtúbulos/metabolismo , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Fosforilación , Venas Umbilicales/citología , Vasoconstrictores/farmacologíaRESUMEN
OBJECTIVE: von Willebrand factor (vWF) is synthesized by endothelial cells and stored in specialized vesicles called Weibel-Palade bodies (WPBs). Recently, we have shown that the small GTP-binding protein Ral is involved in thrombin-induced exocytosis of WPBs. In addition to Ca2+-elevating secretagogues such as histamine and thrombin, release of WPB is also observed after administration of cAMP-raising substances such as epinephrine and vasopressin. In the present study, we investigated whether Ral is also involved in cAMP-mediated vWF release. METHODS AND RESULTS: Activation of Ral was observed 15 to 20 minutes after stimulation of endothelial cells with epinephrine, forskolin, or dibutyryl-cAMP. A cell-permeable peptide comprising the carboxy-terminal part of the Ral protein reduced both thrombin-induced and epinephrine-induced vWF secretion supporting a crucial role for Ral in this process. Furthermore, inhibition of protein kinase A by H-89 resulted in a marked reduction of vWF release and greatly diminished levels of GTP-Ral on stimulation with epinephrine. Activation of Ral was independent of the activation of Epac, a cAMP-regulated exchange factor for the small GTPases Rap1 and Rap2. CONCLUSIONS: These results suggest that protein kinase A-dependent activation of Ral regulates cAMP-mediated exocytosis of WPB in endothelial cells.