Inhibition of Calcium Release-activated Calcium Current by Rac/Cdc42-inactivating Clostridial Cytotoxins in RBL Cells

Abstract

Using large clostridial cytotoxins as tools, the role of Rho GTPases in activation of RBL 2H3 hm1 cells was studied. Clostridium difficile toxin B, which glucosylates Rho, Rac, and Cdc42 and Clostridium sordellii lethal toxin, which glucosylates Rac and Cdc42 but not Rho, inhibited the release of hexosaminidase from RBL cells mediated by the high affinity antigen receptor (Fc⑀RI entry is suggested to depend on the depletion of intracellular Ca 2ϩ stores and is termed capacitative Ca 2ϩ entry (for review, see Refs. 7 and 8). The inward Ca 2ϩ current in RBL cells that seems to contribute to this entry is designated I CRAC for calcium release-activated calcium current. However, the regulatory mechanisms leading to activation of I CRAC is still unclear. Small GTPases have been suggested to participate in receptormediated Ca 2ϩ influx in RBL cells and mast cells (9). The small GTPases of the Rho family including Rho, Rac, and Cdc42 play important roles in regulation of the actin cytoskeleton (10). RhoA participates in growth factor-mediated formation of stress fibers and cell adhesions (11), Rac regulates membranes ruffling and lamellipodia (12) Recently, various bacterial toxins have been established as tools to study the function of small GTPases (23). Clostridium botulinum C3 transferase and related C3-like exoenzymes, including the C3 chimeric toxin C2IN-C3, selectively ADP-ribosylate RhoA, RhoB, and RhoC at Asn-41, thereby inhibiting their biological functions (24 -27). The family of large clostridial cytotoxins inactivate small GTPases by glucosylation (28). Whereas Clostridium difficile toxins A and B monoglucosylate Rho GTPases including Rho, Rac, and Cdc42 at Thr-37 or respectively (29), the lethal toxin from Clostridium sordellii modifies Rac, possibly Cdc42, but not Rho (30, 31). In addition, Ras subfamily proteins (e.g. Ras, Ral, and Rap) are glucosylated by the lethal toxin.Here we studied the effects of toxins on degranulation, Ca 2ϩ mobilization, and I CRAC in RBL 2H3 hm1 cells. We report that toxin B and lethal toxin but not the Rho-modifying chimeric toxin C2IN-C3 inhibit secretion and increase of [Ca 2ϩ ] i by the Fc⑀RI receptor in RBL cells. Moreover, the toxins inhibit thapsigargin-induced Ca 2ϩ mobilization and the activation of I CRAC by depletion of intracellular Ca 2ϩ stores, indicating that Rac/ Cdc42 but not Rho participates in regulation of capacitative Ca 2ϩ entry.* This work was supported by grants from the Deutsche Forschungsgemeinschaft (to K. A. and A. C.) and a grant from the BMBF (clinical research group "Pathomechanismen der allergischen Entzü ndung" (to K. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.§ To whom correspondence should be addressed: Inst. fü r Pharmakologie und Toxikologie, Albert-Ludwigs-Universitä t Freiburg, Hermann-Herder-Str. 5, 1 The abbreviations used are: Fc⑀RI, high affini...

The Uptake and Degradation of Matrix-bound Lipoproteins by Macrophages Require an Intact Actin Cytoskeleton, Rho Family GTPases, and Myosin ATPase Activity

“…LDL receptor knockout mice and C57BL6 wild-type mice were from Jackson Research Laboratories (Bar Harbor, ME). Clostridium difficile toxin B, C. sordellii lethal toxin, C2IN-C3 exoenzyme fusion protein and C2IIa, derived from C. botulinum C2 toxin and C. limosum C3-like exoenzyme, were prepared as previously described (26,27). ML-7 (1-(5-isoquinoline sulfonyl)-2-methyl piperazine) was purchased from Alexis Biochemicals (San Diego, CA).…”

“…To further examine the role of Rho family GTPases, macrophages were treated with C. sordellii lethal toxin, which monoglucosylates and inhibits Rac and possibly Cdc42, but not Rho; Ras proteins, including Ras, Ral, and Rap, are also glucosylated by this compound (26). The degradation of retained and aggregated LDL by macrophages treated with this compound was inhibited markedly, although in this case there was also partial inhibition of the degradation of non-retained LDL (Fig.…”

Ca2+-Dependent Regulation of Rho GTPases Triggers Turning of Nerve Growth Cones

“…However, Rho GTPase activities have also been shown to regulate the [Ca 2ϩ ] i dynamics (Wilk-Blaszczak et al, 1997;Costello et al, 1999;Djouder et al, 2000;Singleton and Bourguignon, 2002). In view of the essential role of both Ca 2ϩ and Rho GTPases in growth cone guidance, it is important to determine their causal relationship in transducing guidance signal.…”

Modulation of Ca2+ Signaling by Na+/Ca2+ Exchangers in Mast Cells

“…The Ca 2ϩ signals were initiated in RBL cells either by the blockade of sarco(endo)plasmic reticulum Ca 2ϩ -ATPases pumps in the internal Ca 2ϩ stores with thapsigargin or by activation of the stably expressed muscarinic receptors with carbachol (13,19). Fig.…”

“…Ionic currents were recorded in the whole-cell mode of the patch-clamp technique (19) using an EPC 9-2 amplifier (Heka Electronics). Recording pipettes with resistances of 2.5-3 M⍀ were pulled from borosilicate glass capillaries (Kimax-51; Kimble Products).…”