Upregulation of macrophage plasma membrane and nuclear phospholipase D activity on ligation of the α2-macroglobulin signaling receptor:: Involvement of heterotrimeric and monomeric G proteins

The effect of ligating the alpha(2)-macroglobulin signaling receptor (alpha(2)MSR) with receptor-recognized forms of alpha(2)M (alpha(2)M*) was studied with respect to phospholipase D (PLD) activity in murine macrophages, their plasma membranes, and nuclei. PLD activity in plasma membranes and nuclei increased linearly up to a ligand concentration of about 100 pM of either alpha(2)M* or a cloned and expressed receptor binding fragment (RBF), The RBF binding site mutant K1370A, which binds with high affinity to alpha(2)MSR, also increased nuclear PLD activity comparable to RBF and alpha(2)M*. Phorbol dibutyrate caused a two- to threefold stimulation of membrane and nuclear PLD activity, whereas PLD activity was nearly abolished by downregulation of protein kinase C; prior treatment with staurosporin, genestein, cyclosporin A, actinomycin D; or chelation of intracellular Ca2+. In permeabilized macrophages, isolated plasma membranes, and nuclei, GTP-gamma-S increased alpha(2)M*-stimulated PLD activity via a pertussis toxin-insensitive G protein and this effect was abolished on preincubation with GDP-beta-S. Incubation of plasma membranes with polyclonal antibody against sARFII, or the addition of cytosol which was immunoprecipitated with antibody against sARFII, greatly reduced alpha(2)M*-stimulated PLD activity in the presence of GTP-gamma-S. Preincubation of plasma membranes with GDP-beta-S prior to the addition of GTP-gamma-S and recombinant ARF1 significantly inhibited alpha(2)M*-stimulation of PLD activity. Nuclear PLD activity was maximally stimulated in the presence of both GTP-gamma-S and rARF1, whereas plasma membrane PLD activity was maximally stimulated in the presence of rARF1, GTP-gamma-S, RhoA, and ATP, In contrast, nuclear PLD activity was not affected by RhoA either alone or in combination with GTP-gamma-S or ATP. (C) 1999 Academic Press.