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.
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
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D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D002467:Cell Nucleus / Q000201:enzymology* / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008262:Macrophage Activation / Q000187:drug effects; D017737:Macrophages, Peritoneal / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D010739:Phospholipase D / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011971:Receptors, Immunologic / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D015854:Up-Regulation; D000511:alpha-Macroglobulins / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein
D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011971:Receptors, Immunologic; D000511:alpha-Macroglobulins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D020742:rhoA GTP-Binding Protein; D002118:Calcium