Misra U K, Pizzo S V
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Arch Biochem Biophys. 1999 Mar 1;363(1):68-80. doi: 10.1006/abbi.1998.1074.
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.
研究了用受体识别形式的α2M（α2M*）连接α2-巨球蛋白信号受体（α2MSR）对小鼠巨噬细胞、其质膜和细胞核中磷脂酶D（PLD）活性的影响。质膜和细胞核中的PLD活性随α2M或克隆表达的受体结合片段（RBF）配体浓度线性增加，直至约100 pM。与α2MSR具有高亲和力结合的RBF结合位点突变体K1370A也能增加细胞核PLD活性，与RBF和α2M相当。佛波酯二丁酸酯可使膜和细胞核PLD活性提高两到三倍，而蛋白激酶C下调则几乎消除PLD活性；用星形孢菌素、染料木黄酮、环孢素A、放线菌素D预处理；或螯合细胞内Ca2+。在通透的巨噬细胞、分离的质膜和细胞核中，GTP-γ-S通过百日咳毒素不敏感的G蛋白增加α2M刺激的PLD活性，且该作用在与GDP-β-S预孵育后被消除。用抗sARFII多克隆抗体孵育质膜，或添加用抗sARFII抗体免疫沉淀的胞质溶胶，在存在GTP-γ-S时可大大降低α2M刺激的PLD活性。在添加GTP-γ-S和重组ARF1之前，用GDP-β-S预孵育质膜可显著抑制α2M*对PLD活性的刺激。在同时存在GTP-γ-S和rARF1时，细胞核PLD活性受到最大刺激，而在存在rARF1、GTP-γ-S、RhoA和ATP时，质膜PLD活性受到最大刺激。相比之下，单独或与GTP-γ-S或ATP联合使用时，RhoA对细胞核PLD活性没有影响。
