The mammalian anx7 gene codes for a Ca 2؉ -activated GTPase, which supports Ca 2؉ ͞GTP-dependent secretion events and Ca 2؉ channel activities in vitro and in vivo. To test whether anx7 might be involved in Ca 2؉ signaling in secreting pancreatic ␤ cells, we knocked out the anx7 gene in the mouse and tested the insulinsecretory properties of the ␤ cells. The nullizygous anx7 (؊͞؊) phenotype is lethal at embryonic day 10 because of cerebral hemorrhage. However, the heterozygous anx7 (؉͞؊) mouse, although expressing only low levels of ANX7 protein, is viable and fertile. The anx7 (؉͞؊) phenotype is associated with a substantial defect in insulin secretion, although the insulin content of the islets, is 8-to 10-fold higher in the mutants than in the normal littermate control. We infer from electrophysiological studies that both glucose-stimulated secretion and voltage-dependent Ca 2؉ channel functions are normal. However, electrooptical recordings indicate that the (؉͞؊) mutation has caused a change in the ability of inositol 1,4,5-trisphosphate (IP3)-generating agonists to release intracellular calcium. The principle molecular consequence of lower anx7 expression is a profound reduction in IP3 receptor expression and function in pancreatic islets. The profound increase in islets, ␤ cell number, and size may be a means of compensating for less efficient insulin secretion by individual defective pancreatic ␤ cells. This is a direct demonstration of a connection between glucoseactivated insulin secretion and Ca 2؉ signaling through IP3-sensitive Ca 2؉ stores.

The most common mutation in cystic fibrosis (CF) is ⌬F508, which is associated with failure of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) to traffic to the plasma membrane. By a still unknown mechanism, the loss of correctly trafficked ⌬F508-CFTR results in an excess of the epithelial sodium channel (ENaC) on the apical plasma membrane. ENaC trafficking is known to be regulated by a signaling pathway involving the glucocorticoid receptor, the serum-and glucocorticoid-regulated kinase SGK1, and the ubiquitin E3 ligase Nedd4-2. We show here that dexamethasone rescues functional expression of ⌬F508-CFTR. The half-life of ⌬F508-CFTR is also dramatically enhanced. Dexamethasone-activated ⌬F508-CFTR rescue is blocked either by the glucocorticoid receptor antagonist RU38486 or by the phosphatidylinositol 3-kinase inhibitor LY294002. Co-immunoprecipitation studies indicate that Nedd4-2 binds to both wild-type-and ⌬F508-CFTR. These complexes are inhibited by dexamethasone treatment, and CFTR ubiquitination is concomitantly decreased. We further show that knockdown of Nedd4-2 by small interfering RNA also corrects ⌬F508-CFTR trafficking. Conversely, knockdown of SGK1 by small interfering RNA completely blocks dexamethasone-activated ⌬F508-CFTR rescue. These data suggest that the SGK1/Nedd4-2 signaling pathway regulates both CFTR and ENaC trafficking in CF epithelial cells.

Exocytotic membrane fusion and secretion are promoted by the concerted action of GTP and Ca2 , although the precise site(s) of action in the process are not presently known. However, the calcium-dependent membrane fusion reaction driven by synexin (annexin VII) is an in vitro model for this process, which we have now found to be further activated by GTP. The mechanism of fusion activation depends on the unique ability of synexin to bind and hydrolyze GTP in a calcium-dependent manner, both in vitro and in vivo in streptolysin 0-permeabilized chromaffin cells. The required [Ca2+] for GTP binding by synexin is in the range of 50-200 ,AM, which is known to occur at exocytotic sites in chromaffin cells, neurons, and other cell types. Previous immunolocalization studies place synexin at exocytotic sites in chromaffin cells, and we conclude that synexin is an atypical G protein that may be responsible for both detecting and mediating the Ca2+/GTP signal for exocytotic membrane fusion.GTP and its nonhydrolyzable analogue guanosine 5'- [y-thio]triphosphate (GTP[,yS]) are known to promote Ca2+-dependent exocytotic secretion from many cell types by a mechanism thought to involve as yet unknown proteins in the GTPase superfamily (1-4). Specific members of this superfamily have been considered as mediators of these GTP effects on exocytosis, including heterotrimeric G proteins (5-9) and low molecular weight ras-like proteins such as Rab (10-15) and ARF (16). The current "fusion machine" hypothesis (17-19) envisions a core complex formed between plasma membrane syntaxin and SNAP-25 and the synaptic vesicle protein synaptobrevin/VAMP (20), with vesicular synaptotagmin putatively identified as a low-affinity calcium sensor that interacts with regulatory syntaxin 1 (21,22). However, none of the proteins presently identified in the hypothetical fusion machine have actually been shown to be activated by GTP or, indeed, even to fuse membranes (17,(23)(24)(25). Therefore, it has been suggested that other GTP-binding proteins, as yet unidentified, might control the activity of the fusion complex (19,25).The site of GTP action in exocytosis has been hypothesized to be closely associated with the site of calcium action in a common pathway (2,5,26,27). The most recent consensus on the nature of this calcium binding site is that it is involved in docking and fusion and that the affinity of the site for Ca2+ may be in the range of 50-200 ,tM (28-36