Schmidt M, Hüwe S M, Fasselt B, Homann D, Rümenapp U, Sandmann J, Jakobs K H
Institut für Pharmakologie, Universität GH Essen, Germany.
Eur J Biochem. 1994 Oct 15;225(2):667-75. doi: 10.1111/j.1432-1033.1994.00667.x.
In human embryonic kidney cells stably expressing the human m3 muscarinic acetylcholine receptor (mAChR) subtype, agonist (carbachol) activation stimulated phospholipase C, increased cytoplasmic calcium concentration, induced tyrosine phosphorylation of various cellular proteins and activated phospholipase D. Bypassing membrane receptors, phospholipase D was activated in these cells by direct activation of protein kinase C by phorbol esters, by direct activation of GTP-binding proteins by A1F4- and a stable GTP analogue (in permeabilized cells), by increasing cytoplasmic calcium concentration with the calcium ionophore A23187 and also apparently by tyrosine phosphorylation. In order to identify possible mechanisms by which the m3 mAChR couples to phospholipase D, various inhibitors of protein kinase C, tyrosine kinases and calcium-dependent events were studied. Prevention of an agonist-induced increase in cytoplasmic calcium concentration did not alter the mAChR-induced phospholipase D stimulation. The protein kinase C inhibitors, calphostin C and staurosporine, efficiently prevented phospholipase D activation by phorbol 12-myristate 13-acetate but only partially inhibited the activation induced by the mAChR agonist. Additionally, down-regulation of protein kinase C by prolonged exposure to phorbol 12-myristate 13-acetate abrogated phospholipase D activation by this effector but had only minor or no effects on the response to the mAChR agonist and direct activators of GTP-binding proteins. In contrast, the tyrosine kinase inhibitor genistein abolished the carbachol-induced and A1F4(-)-induced phospholipase D activation but had no effect on enzyme activation by phorbol 12-myristate 13-acetate. The data indicate that phospholipase D in m3 mAChR-expressing human embryonic kidney cells can be activated by various different mechanisms, i.e. receptor agonists, GTP-binding proteins, protein kinase C-dependent and calcium-dependent events and tyrosine phosphorylation. The coupling of m3 mAChR to phospholipase D appears to be largely independent of concomitant phospholipase C activation with subsequent increase in cytoplasmic calcium concentration and protein kinase C activity. The data instead suggest the involvement of an essential protein tyrosine phosphorylation mechanism in phopsholipase D activation by the m3 mAChR and heterotrimeric GTP-binding proteins.
在稳定表达人m3型毒蕈碱型乙酰胆碱受体（mAChR）亚型的人胚肾细胞中，激动剂（卡巴胆碱）激活可刺激磷脂酶C，增加细胞质钙浓度，诱导多种细胞蛋白的酪氨酸磷酸化并激活磷脂酶D。绕过膜受体，在这些细胞中，佛波酯通过直接激活蛋白激酶C、A1F4 - 和一种稳定的GTP类似物（在通透细胞中）通过直接激活GTP结合蛋白、钙离子载体A23187通过增加细胞质钙浓度以及显然通过酪氨酸磷酸化来激活磷脂酶D。为了确定m3 mAChR与磷脂酶D偶联的可能机制，研究了蛋白激酶C、酪氨酸激酶和钙依赖性事件的各种抑制剂。防止激动剂诱导的细胞质钙浓度增加并未改变mAChR诱导的磷脂酶D刺激。蛋白激酶C抑制剂钙泊三醇C和星形孢菌素有效地阻止了佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯对磷脂酶D的激活，但仅部分抑制了mAChR激动剂诱导的激活。此外，通过长时间暴露于佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯使蛋白激酶C下调消除了该效应物对磷脂酶D的激活，但对mAChR激动剂和GTP结合蛋白直接激活剂的反应只有轻微影响或无影响。相反，酪氨酸激酶抑制剂染料木黄酮消除了卡巴胆碱诱导的和A1F4（ - ）诱导的磷脂酶D激活，但对佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯对酶的激活没有影响。数据表明，在表达m3 mAChR的人胚肾细胞中，磷脂酶D可通过多种不同机制激活，即受体激动剂、GTP结合蛋白、蛋白激酶C依赖性和钙依赖性事件以及酪氨酸磷酸化。m3 mAChR与磷脂酶D的偶联似乎在很大程度上独立于伴随的磷脂酶C激活以及随后细胞质钙浓度和蛋白激酶C活性的增加。相反，数据表明一种必需的蛋白酪氨酸磷酸化机制参与了m3 mAChR和异三聚体GTP结合蛋白对磷脂酶D的激活。
