RHOA AND A CYTOSOLIC 50-KDA FACTOR RECONSTITUTE GTP-GAMMA-S-DEPENDENTPHOSPHOLIPASE-D ACTIVITY IN HUMAN NEUTROPHIL SUBCELLULAR-FRACTIONS

Receptor activation of phospholipase D has been implicated in signal t ransduction in a variety of cells, Reconstitution of cell free guanosi ne 5'-O-(3-thiotriphosphate)(GTP gamma S)-dependent phospholipase D ac tivity from human neutrophils requires protein factors in both the pla sma membrane and the cytosol, We previously proposed that one of the f actors is a Ras-family small molecular weight GTPase of the Rho subtyp e (Bowman, E.P., Uhlinger, D.J., and Lambeth, J.D. (1993) J, Biol, Che m. 268, 21509-21512), Herein, we have used RhoGDI (GDP dissociation in hibitor), an inhibitory Rho-binding protein, to selectively extract Rh o-type GTPases from the plasma membrane, and have used immunoprecipita tion as well as chromatographic methods to remove cytosolic Rho, Deple tion of RhoA from either the plasma membrane or the cytosol re suited in a partial loss in GTP gamma S dependent activity, while removal of RhoA from both fractions resulted in a nearly complete loss in activit y, Activity was nearly completely restored by adding purified recombin ant RhoA, which showed an EC(50) of 52 nM, while Rad showed little act ivity, Cytosol fractionated using DEAE- cellulose chromatography separ ated ADP-ribosylation factor and Rho from the major activating fractio n, Gel exclusion chromatography of this fraction revealed an activatin g factor of 50 KDa apparent molecular-mass, Using RhoA-depleted membra nes, reconstitution of phospholipase D activity required both RhoA and the 50-kDa factor, Thus, RhoA along with a non-Rho, non-ADP-ribosylat ion factor 50-kDa cytosolic factor are both required to reconstitute G TP gamma S dependent phospholipase D activity by neutrophil plasma mem branes.