RHO-GUANINE-NUCLEOTIDE DISSOCIATION INHIBITOR PROTEIN (RHOGDI) INHIBITS EXOCYTOSIS IN MAST-CELLS

Introducing non-hydrolysable analogues of GTP into the cytosolic compa rtment of mast cells results in exocytotic secretion through the activ ation of GTP binding proteins. The identity and mechanism of action of these proteins are not established, We have investigated the effects of Rho GDP dissociation inhibitor (RhoGDI) on exocytosis induced by gu anosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) in rat mast cells, int roducing the protein into cells by means of a patch pipette and record ing the progress of exocytosis by monitoring cell capacitance. To allo w time for the protein to enter the cells and find its correct locatio n, stimulation was provided 5-10 min after patch rupture by photolysin g caged GTP-gamma-S included in the pipette solution, When bovine RhoG DI was introduced into mast cells, exocytosis was inhibited at concent rations of 200-400 nM for native protein and 800 nM to 8 mu M for the recombinant form. Protein denatured by heat or N-ethylmaleimide treatm ent did not inhibit. In permeabilized cells, recombinant RhoGDI increa sed the rate at which cells lose their ability to respond to GTP-gamma -S, These data demonstrate that one or more small GTP binding proteins of the Rho family has a central role in the exocytotic mechanism in m ast cells.