Upregulation of macrophage plasma membrane and nuclear phospholipase D activity on ligation of the alpha(2)-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 studie d with respect to phospholipase D (PLD) activity in murine macrophages, the ir plasma membranes, and nuclei. PLD activity in plasma membranes and nucle i 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)M SR, also increased nuclear PLD activity comparable to RBF and alpha(2)M*. P horbol dibutyrate caused a two- to threefold stimulation of membrane and nu clear PLD activity, whereas PLD activity was nearly abolished by downregula tion of protein kinase C; prior treatment with staurosporin, genestein, cyc losporin A, actinomycin D; or chelation of intracellular Ca2+. In permeabil ized macrophages, isolated plasma membranes, and nuclei, GTP-gamma-S increa sed alpha(2)M*-stimulated PLD activity via a pertussis toxin-insensitive G protein and this effect was abolished on preincubation with GDP-beta-S. Inc ubation of plasma membranes with polyclonal antibody against sARFII, or the addition of cytosol which was immunoprecipitated with antibody against sAR FII, 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 stimu lated in the presence of both GTP-gamma-S and rARF1, whereas plasma membran e 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 Rh oA either alone or in combination with GTP-gamma-S or ATP. (C) 1999 Academi c Press.