Upregulation of macrophage plasma membrane and nuclear phospholipase D activity on ligation of the alpha2-macroglobulin signaling receptor: involvement of heterotrimeric and monomeric G proteins.
The effect of ligating the alpha2-macroglobulin signaling receptor (alpha2MSR) with receptor-recognized forms of alpha2M (alpha2M*) 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 alpha2M* or a cloned and expressed receptor binding fragment (RBF). The RBF binding site mutant K1370A, which binds with high affinity to alpha2MSR, also increased nuclear PLD activity comparable to RBF and alpha2M*. 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 alpha2M*-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 alpha2M*-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 alpha2M*-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.
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Related Subject Headings
- rhoA GTP-Binding Protein
- alpha-Macroglobulins
- Up-Regulation
- Signal Transduction
- Receptors, Immunologic
- Protein Binding
- Phospholipase D
- Mice, Inbred C57BL
- Mice
- Macrophages, Peritoneal
Citation
Published In
DOI
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- rhoA GTP-Binding Protein
- alpha-Macroglobulins
- Up-Regulation
- Signal Transduction
- Receptors, Immunologic
- Protein Binding
- Phospholipase D
- Mice, Inbred C57BL
- Mice
- Macrophages, Peritoneal