Preininger A M, Henage L G, Oldham W M, Yoon E J, Hamm H E, Brown H A
Department of Pharmacology: 442 RRB, Vanderbilt University School of Medicine, 23rd Ave. South and Pierce, Nashville, TN 37232-6600, USA.
Mol Pharmacol. 2006 Jul;70(1):311-8. doi: 10.1124/mol.105.021451. Epub 2006 Apr 25.
Phospholipase D-mediated hydrolysis of phosphatidylcholine is stimulated by protein kinase C and the monomeric G proteins Arf, RhoA, Cdc42, and Rac1, resulting in complex regulation of this enzyme. Using purified proteins, we have identified a novel inhibitor of phospholipase D activity, Gbetagamma subunits of heterotrimeric G proteins. G protein-coupled receptor activation alters affinity between Galpha and Gbetagamma subunits, allowing subsequent interaction with distinct effectors. Gbeta1gamma1 inhibited phospholipase D1 and phospholipase D2 activity, and both Gbeta1gamma1 and Gbeta1gamma2 inhibited stimulated phospholipase D1 activity in a dosedependent manner in reconstitution assays. Reconstitution assays suggest this interaction occurs through the amino terminus of phospholipase D, because Gbeta1gamma1 is unable to inhibit an amino-terminally truncated phospholipase D construct, PLD1.d311, which like full-length phospholipase D isoforms, requires phosphatidylinositol-4,5-bisphosphate for activity. Furthermore, a truncated protein consisting of the amino-terminal region of phospholipase D containing the phox/pleckstrin homology domains was found to interact with Gbeta1gamma1, unlike the PLD1.d311 recombinant protein, which lacks this domain. In vivo, expressed recombinant Gbeta1gamma2 was also found to inhibit phospholipase D activity under basal and stimulated conditions in MDA-MB-231 cells, which natively express both phospholipase D1 and phospholipase D2. These data demonstrate that Gbetagamma directly regulates phospholipase D activity in vitro and suggest a novel mechanism to negatively regulate phospholipase D signaling in vivo.
磷脂酶D介导的磷脂酰胆碱水解受到蛋白激酶C以及单体G蛋白Arf、RhoA、Cdc42和Rac1的刺激，从而导致对该酶的复杂调控。我们使用纯化的蛋白质鉴定出了一种磷脂酶D活性的新型抑制剂——异源三聚体G蛋白的Gβγ亚基。G蛋白偶联受体激活会改变Gα和Gβγ亚基之间的亲和力，从而允许随后与不同的效应器相互作用。Gβ1γ1抑制磷脂酶D1和磷脂酶D2的活性，并且在重组实验中，Gβ1γ1和Gβ1γ2均以剂量依赖性方式抑制刺激后的磷脂酶D1活性。重组实验表明这种相互作用是通过磷脂酶D的氨基末端发生的，因为Gβ1γ1无法抑制氨基末端截短的磷脂酶D构建体PLD1.d311，与全长磷脂酶D同工型一样，该构建体的活性需要磷脂酰肌醇-4,5-二磷酸。此外，发现由含有phox/普列克底物蛋白同源结构域的磷脂酶D氨基末端区域组成的截短蛋白与Gβ1γ1相互作用，这与缺乏该结构域的PLD1.d311重组蛋白不同。在体内，还发现表达的重组Gβ1γ2在MDA-MB-231细胞的基础和刺激条件下均抑制磷脂酶D活性，该细胞天然表达磷脂酶D1和磷脂酶D2。这些数据表明Gβγ在体外直接调节磷脂酶D活性，并提示了一种在体内对磷脂酶D信号进行负调控的新机制。
