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 α2-macroglobulin signaling receptor (α2MSR) with receptor-recognized forms of α2M (α2M*) 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 α2M* or a cloned and expressed receptor binding fragment (RBF). The RBF binding site mutant K1370A, which binds with high affinity to α2MSR, also increased nuclear PLD activity comparable to RBF and α2M*. 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-γ-S increased α2M*-stimulated PLD activity via a pertussis toxin-insensitive G protein and this effect was abolished on preincubation with GDP-β-S. Incubation of plasma membranes with polyclonal antibody against sARFII, or the addition of cytosol which was immunoprecipitated with antibody against sARFII, greatly reduced α2M*-stimulated PLD activity in the presence of GTP-γ-S. Preincubation of plasma membranes with GDP-β-S prior to the addition of GTP-γ-S and recombinant ARF1 significantly inhibited α2M*-stimulation of PLD activity. Nuclear PLD activity was maximally stimulated in the presence of both GTP-γ-S and rARF1, whereas plasma membrane PLD activity was maximally stimulated in the presence of rARF1, GTP-γ-S, RhoA, and ATP. In contrast, nuclear PLD activity was not affected by RhoA either alone or in combination with GTP-γ-S or ATP.