Purification and identification of FOAD-II, a cytosolic protein that regulates secretion in streptolysin-O permeabilized mast cells, as a rac/rhoGDI complex.

Abstract

Mast cells permeabilized by treatment with streptolysin-O in the presence of Ca2+ and GTP-gamma-S can secrete almost 100% of their contained N-acetyl-beta-D-glucosaminidase. If these stimuli are provided to the permeabilized cells after a delay, the response is diminished and the ability of the cells to undergo secretion runs down progressively over a period of about 30 min. This is thought to be due to the loss of key proteins involved in the exocytotic mechanism. Using this effect as the basis of a biological assay, we have isolated a protein from bovine brain cytosol that retards the loss of responsiveness to stimulation by Ca2+ and GTP-gamma-S. Purification of this protein and peptide sequencing have enabled us to identify it as the small GTP-binding protein rac complexed to the guanine nucleotide exchange inhibitor rhoGDI. Both proteins are required to retard the loss of the secretory response, while purified rhoGDI applied alone accelerates the rundown.

Involvement of Rho and Rac small G proteins and Rho GDI in Ca2+‐dependent exocytosis from PC12 cells

“…Moreover, O'Sullivan et al (1996) have recently reported that Rac complexed with Rho GDI is involved in Ca 2þ -dependent exocytosis from permeabilized rat mast cells. In their experiments, when rat mast cells are permeabilized with streptolysin-O and immediately stimulated by GTPgS in the presence of Ca 2þ , Ca 2þ -dependent exocytosis occurs, but when stimulated after a delay, this reaction is markedly reduced.…”

Activation of exocytosis by cross-linking of the IgE receptor is dependent on ADP-ribosylation factor 1-regulated phospholipase D in RBL-2H3 mast cells: evidence that the mechanism of activation is via regulation of phosphatidylinositol 4,5-bisphosphate synthesis

“…The effects on exocytosis were independent of changes on the cytoskeleton [7]. Independent studies using a different approach also identified Rac as an effector of exocytosis from these cells [8]. In none of these reports has the downstream component regulated by these G-proteins been identified.…”

“…Subsequent work has revealed several candidate G-proteins that are involved in exocytosis. These include the Rho family members Rac and Rho [6][7][8], and the heterotrimeric G-protein G i$ [9]. The constitutively active mutant proteins V14RhoA and V12Rac1 enhanced Ca# + -regulated exocytosis from permeabilized mast cells by increasing the proportion of cells that were competent to respond to stimulation.…”

Calcium Signaling Regulates Translocation and Activation of Rac

“…Increased intracellular calcium [Ca 2ϩ ] i represents a ubiquitous second messenger system in cells, linking receptor activation to downstream signaling pathways. Previous studies (15)(16)(17)(18) have described relationships between intracellular calcium and the activation and function of Rho family GTPases in processes including muscle contraction and the exocytic response. Intracellular calcium is also required for thrombin-and collagen-induced Rac activation in platelets (19).…”