INDUCTION OF EXOCYTOSIS FROM PERMEABILIZED MAST-CELLS BY THE GUANOSINE TRIPHOSPHATASES RAC AND CDC42

We applied recombinant forms of the Rho-related small guanosine tripho sphatases (GTPases) Rac2 and Cdc42/G25K to permeabilized mast cells to test their ability to regulate exocytotic secretion. Mast cells perme abilized with streptolysin-O leak soluble (cytosol) proteins over a pe riod of 5 min and become refractory to stimulation by Ca2+ and guanosi ne triphosphate (GTP)gamma S over about 20-30 min. This loss of sensit ivity is likely to be due to loss of key regulatory proteins that are normally tethered at intracellular locations. Exogenous proteins that retard this loss of sensitivity to stimulation may be similar, if not identical, to those secretory regulators that are lost. Recombinant Ra c and Cdc42/G25K, preactivated by binding GTP gamma S, retard the loss of sensitivity (rundown) and, more importantly, enable secretion to b e stimulated by Ca2+ alone. Investigation of the concentration depende nce of each of these two GTPases applied individually to the permeabil ized cells, and of Cdc42/G25K applied in the presence of an optimal co ncentration of Rac2, has provided evidence for a shared effector pathw ay and also a second effector pathway activated by Cdc42/G25K alone. D ominant negative mutant (N17) forms of Rac2 and Cdc42/G25K inhibit sec retion induced by Ca2+ and GTP gamma S. Our data suggest that Rac2 and Cdc42 should be considered as candidates for G(E), GTPases that media te exocytosis in cells of hematopoietic origin.