Abstract
Receptor activation of phospholipase D has been implicated in signal transduction in a variety of cells. Reconstitution of cell-free guanosine 5'-O-(3-thiotriphosphate)(GTP gamma S)-dependent phospholipase D activity from human neutrophils requires protein factors in both the plasma membrane and the cytosol. We previously proposed that one of the factors is a Ras-family small molecular weight GTPase of the Rho subtype (Bowman, E. P., Uhlinger, D. J., and Lambeth, J. D. (1993) J. Biol. Chem. 268, 21509-21512). Herein, we have used RhoGDI (GDP dissociation inhibitor), an inhibitory Rho-binding protein, to selectively extract Rho-type GTPases from the plasma membrane, and have used immunoprecipitation as well as chromatographic methods to remove cytosolic Rho. Depletion of RhoA from either the plasma membrane or the cytosol resulted in a partial loss in GTP gamma S dependent activity, while removal of RhoA from both fractions resulted in a nearly complete loss in activity. Activity was nearly completely restored by adding purified recombinant RhoA, which showed an EC50 of 52 nM, while Rac1 showed little activity. Cytosol fractionated using DEAE-cellulose chromatography separated ADP-ribosylation factor and Rho from the major activating fraction. Gel exclusion chromatography of this fraction revealed an activating factor of 50 kDa apparent molecular mass. Using RhoA-depleted membranes, reconstitution of phospholipase D activity required both RhoA and the 50-kDa factor. Thus, RhoA along with a non-Rho, non-ADP-ribosylation factor 50-kDa cytosolic factor are both required to reconstitute GTP gamma S-dependent phospholipase D activity by neutrophil plasma membranes.
Highlights
Responses such as the respiratory burst of granulocytes [4, 5]
We recently showed that the major cytosolic activating factor is approximately 50 kDa by gel exclusion chromatography [9] and separates from fractions containing ADP-ribosylation factor (ARF)
Depletion of Rho from Plasma Membranes—In preliminary studies, we used antibodies to RhoA, RhoB, Rac (1 and 2), Rac2, and CDC42 to determine which of these Rho family small GTPases were present in neutrophil lysates and plasma membranes
Summary
Responses such as the respiratory burst of granulocytes [4, 5]. While the occurrence of receptor-activated PLD has been widely documented, its molecular mechanism of activation remains poorly understood. The identification of the GTP-dependent activating factor has been confounded by recent studies implicating ADP-ribosylation factor (ARF) as the guanine nucleotide-dependent factor and as a cytosolic rather than a membrane factor [10, 11] These studies utilized permeabilized HL-60 cells or HL-60 plasma membranes (or their extracts) along with cytosol from bovine brain. The remainder of RhoA and the majority of the other small GTPases are cytosolic, largely associated with the inhibitor protein RhoGDI The latter is complexed with Rho proteins in such a way as to mask their isoprenyl group, maintaining solubility and preventing membrane interactions [15, 16]. We have used this Rho-depleted system in reconstitution studies to investigate the factors required for PLD activation We find that both RhoA and a (non-Rho, non-ARF) 50-kDa cytosolic factor are required to reconstitute GTP␥S-stimulated phospholipase D activity in neutrophil plasma membranes
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