NEUTROPHIL PHOSPHOLIPASE-D IS ACTIVATED BY A MEMBRANE-ASSOCIATED RHO FAMILY SMALL MOLECULAR-WEIGHT GTP-BINDING PROTEIN

Phospholipase D in human neutrophil lysates is activated by GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), implying the participation of a GTP-binding protein. Reconstitution of GTPgammaS-dependent activity r equires protein factors in both the plasma membrane and the cytosol (O lson, S. C., Bowman, E. P., and Lambeth, J. D. (1991) J. Biol. Chem. 2 66,17236-17242). The location of the GTP-binding protein was investiga ted by preincubating either cytosol or plasma membrane with GTPgammaS, followed by removal of all but tightly bound nucleotide and reconstit uting activity with the complementing untreated subcellular fraction. This approach indicated that the GTP-binding protein was membrane-asso ciated. A low magnesium requirement for GTPgammaS prebinding, as well as a failure of aluminum fluoride to activate, suggested a Ras-like sm all M(r) GTP-binding protein. smg GDP dissociation stimulator, which s timulates the exchange of GDP for GTP on a variety of small GTP-bindin g proteins, stimulated GTP-dependent phospholipase D activity. Rho GDP dissociation inhibitor, a regulatory protein that binds specifically to and inhibits the functions of Rho family small GTP-binding proteins , inhibited GTPgammaS-dependent activity. Thus, neutrophil phospholipa se D is regulated by a membrane-associated small molecular weight GTP- binding protein, likely to be a member of the Rho family.