Regulation of IgE receptor-mediated secretion from RBL-2H3 mast cells by GTP binding-proteins and calcium

Rat peritoneal mast cells, both intact and permeabilized, have been used widely as model secretory cells. GTP-binding proteins and calcium play a major role in controlling their secretory response. Here we have examined changes in the organization of actin filaments in intact mast cells after activation by compound 48/80, and in permeabilized cells after direct activation of GTP-binding proteins by GTP-gamma-S. In both cases, a centripetal redistribution of cellular F-actin was observed: the content of F-actin was reduced in the cortical region and increased in the cell interior. The overall F-actin content was increased. Using permeabilized cells, we show that AIF4-, an activator of heterotrimeric G proteins, induces the disassembly of F-actin at the cortex, while the appearance of actin filaments in the interior of the cell is dependent on two small GTPases, rho and rac. Rho was found to be responsible for de novo actin polymerization, presumably from a membrane-bound monomeric pool, while rac was required for an entrapment of the released cortical filaments. Thus, a heterotrimeric G-protein and the small GTPases, rho and rac, participate in affecting the changes in the actin cytoskeleton observed after activation of mast cells.

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The change of distribution of small GTP-binding proteins by GTPγS and its regulation by IgE receptor stimulation in α-toxin-permeabilized rat basophilic leukemia (RBL-2H3) cells

The Japanese Journal of Pharmacology ◽

10.1016/s0021-5198(19)48755-6 ◽

1992 ◽

Vol 58 ◽

pp. 85

Author(s):

Youichi Fukuda ◽

Tomoyuki Uekusa ◽

Atsushi Nakagawa ◽

Masaatsu K. Uchida ◽

Kazuhiko Oishi

Keyword(s):

Binding Proteins ◽

Ige Receptor ◽

Receptor Stimulation ◽

Gtp Binding Proteins ◽

Gtp Binding ◽

Basophilic Leukemia

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The small GTP-binding proteins, Rac and Rho, regulate cytoskeletal organization and exocytosis in mast cells by parallel pathways.

Molecular Biology of the Cell ◽

10.1091/mbc.7.9.1429 ◽

1996 ◽

Vol 7 (9) ◽

pp. 1429-1442 ◽

Cited By ~ 86

Author(s):

J C Norman ◽

L S Price ◽

A J Ridley ◽

A Koffer

Keyword(s):

Mast Cells ◽

Signaling Pathways ◽

Binding Proteins ◽

Small Gtpases ◽

Cortical Actin ◽

Actin Organization ◽

Gtp Binding Proteins ◽

Upstream Regulator ◽

Gtp Binding ◽

Constitutively Active Mutants

In mast cells, activation of GTP-binding proteins induces centripetal reorganization of actin filaments. This effect is due to disassembly, relocalization, and polymerization of F-actin and is dependent on two small GTPases, Rac and Rho. Activities of Rac and Rho are also essential for the secretory function of mast cells. In response to GTP-gamma-S and/or calcium, only a proportion of permeabilized mast cells is capable of secretory response. Here, we have compared actin organization of secreting and nonsecreting cell populations. We show that the cytoskeletal and secretory responses are strongly correlated, indicating a common upstream regulator of the two functions. The secreting cell population preferentially displays both relocalization and polymerization of actin. However, when actin relocalization or polymerization is inhibited by phalloidin or cytochalasin, respectively, secretion is unaffected. Moreover, the ability of the constitutively active mutants of Rac and Rho to enhance secretion is also unaffected in the presence of cytochalasin. Therefore, Rac and Rho control these two functions by divergent, parallel signaling pathways. Cortical actin disassembly occurs in both secreting and nonsecreting populations and does not, by itself, induce exocytosis. A model for the control of exocytosis is proposed that includes at least four GTP-binding proteins and suggests the presence of both shared and divergent signaling pathways from Rac and Rho.

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Small GTP-binding Protein RalA Associates with Weibel-Palade Bodies in Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614349 ◽

1999 ◽

Vol 82 (09) ◽

pp. 1177-1181 ◽

Cited By ~ 27

Author(s):

Hubert de Leeuw ◽

Pauline Wijers-Koster ◽

Jan van Mourik ◽

Jan Voorberg

Keyword(s):

Endothelial Cells ◽

Binding Proteins ◽

Binding Protein ◽

Control Release ◽

Small Gtpase ◽

Gtp Binding Protein ◽

Two Dimensional Gel Electrophoresis ◽

Gtp Binding Proteins ◽

Dense Granules ◽

Gtp Binding

SummaryIn endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules, so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.

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Rap1b Association with the Platelet Cytoskeleton Occurs in the Absence of Glycoproteins IIb/IIIa

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615073 ◽

1998 ◽

Vol 79 (04) ◽

pp. 832-836 ◽

Cited By ~ 3

Author(s):

Thomas Fischer ◽

Christina Duffy ◽

Gilbert White

Keyword(s):

Molecular Weight ◽

Divalent Cation ◽

Binding Proteins ◽

Binding Protein ◽

Platelet Membrane ◽

Low Molecular Weight ◽

Membrane Glycoproteins ◽

Gtp Binding Protein ◽

Gtp Binding Proteins ◽

Gtp Binding

SummaryPlatelet membrane glycoproteins (GP) IIb/IIIa and rap1b, a 21 kDa GTP binding protein, associate with the triton-insoluble, activation-dependent platelet cytoskeleton with similar rates and divalent cation requirement. To examine the possibility that GPIIb/IIIa was required for rap1b association with the cytoskeleton, experiments were performed to determine if the two proteins were linked under various conditions. Chromatography of lysates from resting platelets on Sephacryl S-300 showed that GPIIb/IIIa and rap1b were well separated and distinct proteins. Immunoprecipitation of GPIIb/IIIa from lysates of resting platelets did not produce rap1b or other low molecular weight GTP binding proteins and immunoprecipitation of rap1b from lysates of resting platelets did not produce GPIIb/IIIa. Finally, rap1b was associated with the activation-dependent cytoskeleton of platelets from a patient with Glanzmann’s thrombasthenia who lacks surface expressed glycoproteins IIb and IIIa. Based on these findings, we conclude that no association between GPIIb/IIIa and rap1b is found in resting platelets and that rap1b association with the activation-dependent cytoskeleton is at least partly independent of GPIIb/IIIa.

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