Activation of PLC-delta1 by Gi/o-coupled receptor agonists.
Activation of PLC-delta1 by Gi/o-coupled receptor agonists.
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发表时间：
2004
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通讯作者：
K. Murthy;Huiping Zhou;Jie'an Huang;S. Pentyala
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作者：
K. Murthy;Huiping Zhou;Jie'an Huang;S. Pentyala
本文研究了G蛋白偶联受体激动剂激活兔胃平滑肌磷脂酶(PLC)-Delta的机制。Ca(2+)刺激通透性肌肉细胞PLC-Delta1活性增加8倍。用三种G(I/O)偶联受体激动剂(生长抑素、增量阿片激动剂[D-Pen(2)、D-Pen(5)]脑啡肽和A(1)激动剂环戊基腺苷)处理分散或培养的肌细胞后，磷脂酰肌醇(PI)的水解率延迟增加(8-10倍)，而显性负性PLC-Delta1(E341R/D343R；65-76%)或成分活性RhoA(G14V)的过度表达强烈抑制了这一作用。这种反应与钙离子的容积性内流相一致，在无细胞外钙离子的情况下观察不到这种反应，但硝苯地平(16-30%)能部分抑制这种反应，而钙离子通道阻断剂SKF-96365则能强烈地抑制这种反应。用G(Q/13)偶联受体激动剂CCK-8处理细胞，只引起短暂的PLC-β1介导的PI水解。与G(I/O)偶联受体激动剂不同，CCK-8激活RhoA并刺激RhoA：PLC-Delta1结合。用C3胞外酶抑制RhoA的活性或过表达显性负的RhoA(T19N)或Galpha(13)微型基因揭示了PI水解率的延迟增加，而PLC-Delta1(E341R/D343R)的共表达或SKF-96365强烈地抑制了这一延迟的增加。Thapsigarin诱导的非激动剂电容性Ca(2+)内流可刺激PI的水解率(8倍)，硝苯地平(约25%)部分抑制PI水解率，SKF-96365(约75%)和表达PLC-Delta1的细胞(E341R/D343R)强烈抑制PI水解率。非激动剂依赖的Ca(2+)释放或通过电压门控的Ca(2+)通道内流只能刺激2~3倍的PI适度水解，这种作用可被PLC-Delta1抗体或硝苯地平所阻断。我们得出结论，PLC-Delta1被G(I/O)偶联受体激动剂激活，而不激活RhoA。这种激活主要是由钙离子通过商店操作的钙离子通道内流所介导的。
The mechanism of phospholipase (PLC)-delta activation by G protein-coupled receptor agonists was examined in rabbit gastric smooth muscle. Ca(2+) stimulated an eightfold increase in PLC-delta1 activity in permeabilized muscle cells. Treatment of dispersed or cultured muscle cells with three G(i/o)-coupled receptor agonists (somatostatin, delta-opioid agonist [D-Pen(2),D-Pen(5)]enkephalin, and A(1) agonist cyclopentyl adenosine) caused delayed increase in phosphoinositide (PI) hydrolysis (8- to 10-fold) that was strongly inhibited by overexpression of dominant-negative PLC-delta1(E341R/D343R; 65-76%) or constitutively active RhoA(G14V). The response coincided with capacitative Ca(2+) influx and was not observed in the absence of extracellular Ca(2+), but was partly inhibited by nifedipine (16-30%) and strongly inhibited by SKF-96365, a blocker of store-operated Ca(2+) channels. Treatment of the cells with a G(q/13)-coupled receptor agonist, CCK-8, caused only transient, PLC-beta1-mediated PI hydrolysis. Unlike G(i/o)-coupled receptor agonists, CCK-8 activated RhoA and stimulated RhoA:PLC-delta1 association. Inhibition of RhoA activity with C3 exoenzyme or by overexpression of dominant-negative RhoA(T19N) or Galpha(13) minigene unmasked a delayed increase in PI hydrolysis that was strongly inhibited by coexpression of PLC-delta1(E341R/D343R) or by SKF-96365. Agonist-independent capacitative Ca(2+) influx induced by thapsigargin stimulated PI hydrolysis (8-fold), which was partly inhibited by nifedipine ( approximately 25%) and strongly inhibited by SKF-96365 ( approximately 75%) and in cells expressing PLC-delta1(E341R/D343R). Agonist-independent Ca(2+) release or Ca(2+) influx via voltage-gated Ca(2+) channels stimulated only moderate PI hydrolysis (2- to 3-fold), which was abolished by PLC-delta1 antibody or nifedipine. We conclude that PLC-delta1 is activated by G(i/o)-coupled receptor agonists that do not activate RhoA. The activation is preferentially mediated by Ca(2+) influx via store-operated Ca(2+) channels.