Identification of a Potential Effector Pathway for the Trimeric Go Protein Associated with Secretory Granules

Tomosyn, a soluble R-SNARE protein identified as a binding partner of the Q-SNARE syntaxin 1A, is thought to be critical in setting the level of fusion-competent SNARE complexes for neurosecretion. To date, there has been no direct evaluation of the dynamics in which tomosyn transits through tomosyn-SNARE complexes or of the extent to which tomosyn-SNARE complexes are regulated by secretory demand. Here, we employed biochemical and optical approaches to characterize the dynamic properties of tomosyn-syntaxin 1A complexes in live adrenal chromaffin cells. We demonstrate that secretagogue stimulation results in the rapid translocation of tomosyn from the cytosol to plasma membrane regions and that this translocation is associated with an increase in the tomosyn-syntaxin 1A interaction, including increased cycling of tomosyn into tomosyn-SNARE complexes. The secretagogue-induced interaction was strongly reduced by pharmacological inhibition of the Rho-associated coiled-coil forming kinase, a result consistent with findings demonstrating secretagogue-induced activation of RhoA. Stimulation of chromaffin cells with lysophosphatidic acid, a nonsecretory stimulus that strongly activates RhoA, resulted in effects on tomosyn similar to that of application of the secretagogue. In PC-12 cells overexpressing tomosyn, secretagogue stimulation in the presence of lysophosphatidic acid resulted in reduced evoked secretory responses, an effect that was eliminated upon inhibition of Rho-associated coiled-coil forming kinase. Moreover, this effect required an intact interaction between tomosyn and syntaxin 1A. Thus, modulation of the tomosyn-syntaxin 1A interaction in response to secretagogue activation is an important mechanism allowing for dynamic regulation of the secretory response.Regulated neurotransmitter release requires the well orchestrated spatial and temporal actions of many presynaptic proteins (1). Although the primary molecular entities in the release pathway have been identified, the exact mechanics of synaptic vesicle fusion and its precise regulation are still not established. Central to the fusion process is the transient formation of SNARE 4 core complexes that include the target membrane SNARE proteins syntaxin 1A and SNAP25 and the vesicle SNARE protein synaptobrevin/VAMP (2-4). A SNARE core complex is a highly stable, four-␣-helix parallel bundle consisting of one SNARE motif from each of syntaxin 1A and synaptobrevin/VAMP, and two SNARE motifs from SNAP25 (5, 6). Although these proteins alone are sufficient to induce a slow fusion when reconstituted into liposomes (7), additional proteins are necessary to establish the properties that describe fast, Ca 2ϩ -dependent neurotransmitter release (8). For example, assembly of SNARE core complexes is subject to temporal and spatial regulation by a variety of protein families, including Rab-GTPases (9 -13), Sec/Munc18s (14 -16), exocyst tethering complexes (17-20), and Munc13s (21-24). In addition, recent evidence suggests that the temporal and spatial availability...

Section: Discussionmentioning

confidence: 97%

Receptor-mediated Regulation of Tomosyn-Syntaxin 1A Interactions in Bovine Adrenal Chromaffin Cells

Gladycheva

Lam

Liu

et al. 2007

“…Both phosphatidylinositol 3-kinase (PI3-kinase) and phosphatidylinositol 4-kinase (PI4-kinase) and their products are present in all eukaryotic organisms and tissues that have been investigated, including platelets (12,13). PI3-kinase and PI4-kinase have been shown to be regulated upstream by small GTP-binding proteins in platelets and other cells (14,15). Two different signal transduction pathways involving phosphoinositides have been identified.…”

mentioning

confidence: 99%

Phosphoinositides Are Required for Store-mediated Calcium Entry in Human Platelets

Rosado

Sage

2000

We have recently observed that small GTP-binding proteins are important for mediation of store-mediated Ca 2؉ entry in human platelets through the reorganization of the actin cytoskeleton. Because it has been shown in platelets and other cells that small GTP-binding proteins regulate the activity of phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase, whose products, phosphoinositides, play a key role in the reorganization of the actin cytoskeleton, we have investigated the role of these lipid kinases in store-mediated Ca 2؉ entry. Treatment of platelets with LY294002, an inhibitor of phosphatidylinositol 3-and phosphatidylinositol 4-kinases, resulted in a concentration-dependent inhibition of Ca 2؉ entry stimulated by thapsigargin or the physiological agonist, thrombin. In addition, wortmannin, another inhibitor of these kinases, which is structurally unrelated to LY294002, significantly reduced storemediated Ca 2؉ entry. The inhibitory effect of LY294002 was not mediated either by blockage of Ca 2؉ channels or by modification of membrane potential. LY294002 inhibited actin polymerization stimulated by thrombin or thapsigargin. These results indicate that both phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase are required for activation of store-mediated Ca 2؉ entry in human platelets and that the mechanism could involve the reorganization of the actin cytoskeleton.Store-mediated Ca 2ϩ entry (SMCE) 1 is a mechanism present in many cell types; however, the intracellular processes underlying SMCE remain unclear (1). Several hypotheses have considered both direct and indirect coupling mechanisms (2). Indirect coupling assumes the existence of a diffusible messenger generated by the intracellular Ca 2ϩ stores; in contrast, direct coupling models propose a physical interaction between the endoplasmic reticulum (ER) and the plasma membrane (PM; Ref. 2). Recently a new model for SMCE has been proposed in several different cell types. This involves a physical but reversible interaction between the ER and the PM that may require translocation of portions of the ER toward the PM and mechanical support provided by the actin cytoskeleton (3, 4).

“…Recent investigations lead to the idea that Rho family proteins are also involved in signaling pathways that control actin filament reorganization during exocytosis. In chromaffin cells, RhoA is specifically associated with the membrane of secretory chromaffin granules and is suggested to control the priming of exocytosis by modifying the cortical actin network (17,18). In mast cells, activation of GTP-binding proteins by GTP␥S 1 induces reorganization of actin filaments (19).…”

mentioning

confidence: 99%

Nadrin, a Novel Neuron-specific GTPase-activating Protein Involved in Regulated Exocytosis

Harada¹,

Furuta²,

Takeuchi³

et al. 2000

It has been proposed that the cortical actin filament networks act as a cortical barrier that must be reorganized to enable docking and fusion of the synaptic vesicles with the plasma membranes. We identified a novel neuron-associated developmentally regulated protein, designated as Nadrin. Expression of Nadrin is restricted to neurons and correlates well with the differentiation of neurons. Nadrin has a unique structure; it contains a GTPase-activating protein (GAP) domain for Rho family GTPases, a potential coiled-coil domain, and a succession of 29 glutamines. In vitro the GAP domain activates RhoA, Rac1, and Cdc42 GTPases. Expression of Nadrin in NIH3T3 cells markedly reduced the number of the actin stress fibers and the formation of the ruffled membranes, suggesting that Nadrin regulates actin filament reorganization. In PC12 cells, Nadrin colocalized with synaptotagmin in the neurite termini and also with cortical actin filaments in the subplasmalemmal regions. Expression of Nadrin or its mutant composed of the coiled-coil and GAP domain enhanced Ca 2؉-dependent exocytosis of PC12 cells, but a mutant lacking the GAP domain inhibited exocytosis. These results suggest that Nadrin plays a role in regulating Ca 2؉ -dependent exocytosis, most likely by catalyzing GTPase activity of Rho family proteins and by inducing the reorganization of the cortical actin filaments.