Differential signalling by muscarinic receptors in smooth muscle:: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway
Differential signalling by muscarinic receptors in smooth muscle:: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway
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DOI：
10.1042/bj20021274
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发表时间：
2003-08-15
影响因子：
4.1
通讯作者：
Makhlouf, GM
中科院分区：
文献类型：
--
作者：
Murthy, KS;Zhou, HP;Makhlouf, GM
平滑肌中通过m3和m2受体的信号传导涉及每个受体激活两个G蛋白依赖性通路。m2受体通过G β γ（13）偶联，激活磷脂酶C-β 3、磷酸肌醇3-激酶和Cdc 42/Rac 1（其中Cdc代表细胞分裂周期）和p21激活激酶1（PAK 1），导致肌球蛋白轻链激酶（MLCK）磷酸化和失活。每一步都被甲氧曲明和百日咳毒素抑制。PAK 1活性在同时表达Cdc 42-DN（其中DN代表显性阴性）和Rac 1-DN的细胞中被消除。MLCK磷酸化被PAK 1抗体以及表达Cdc 42-DN和Rac 1-DN的细胞抑制。m3受体通过Ga（q/11）与磷脂酶C-β 1的活化偶联，并通过RhoA与Rho相关激酶（Rho激酶）、磷脂酶D和蛋白激酶C（PKC）的活化偶联。Rho激酶和磷脂酶D活性被C3外切酶和表达RhoA-DN的细胞抑制。PKC活性被双吲哚马来酰亚胺抑制，在表达RhoA-DN的细胞中，PKC活性也被Y27632部分抑制（44 +/- 5%）。PKC诱导的PKC激活的1型磷酸酶17 kDa抑制蛋白（CPI-17）在Thr（38）的磷酸化被双吲哚马来酰亚胺消除，并被Y27632部分抑制（28 +/-3%）。Rho激酶诱导的肌球蛋白磷酸酶靶向亚基（MYPT 1）磷酸化，并被Y27632消除。双吲哚马来酰亚胺Y27632和C3外切酶以及表达RhoA-DN的细胞中，肌球蛋白II的20 kDa调节轻链（MLC 20）的持续磷酸化和收缩被废除。结果表明，Rho激酶依赖的MYPT 1磷酸化和PKC依赖的磷酸化以及CPI-17与MLC磷酸酶催化亚基（MLCP）结合的增强协同作用以抑制MLCP活性，导致MLCZO磷酸化和收缩的持续刺激。由于Y27632抑制Rho激酶和PKC活性，因此不能用于确定MYPT 1对MLCP活性抑制的贡献。MLCK的M2依赖性磷酸化和失活排除了其参与持续的MLCZO磷酸化和收缩。
Signalling via m3 and m2 receptors in smooth muscles involved activation of two G-protein-dependent pathways by each receptor. m2 receptors were coupled via Gbetagamma(13) with activation of phospholipase C-beta3, phosphoinositide 3-kinase and Cdc42/Rac1 (where Cdc stands for cell division cycle) and p21-activated kinase 1 (PAK1), resulting in phosphorylation and inactivation of myosin light chain kinase (MLCK). Each step was inhibited by methoctramine and pertussis toxin. PAK1 activity was abolished in cells expressing both Cdc42-DN (where DN stands for dominant negative) and Rac1-DN. MLCK phosphorylation was inhibited by PAK1 antibody, and in cells expressing Cdc42-DN and Rac1-DN. m3 receptors were coupled via Galpha(q/11) with activation of phospholipase C-beta1 and via RhoA with activation of Rho-associated kinase (Rho kinase), phospholipase D and protein kinase C (PKC). Rho kinase and phospholipase D activities were inhibited by C3 exoenzyme and in cells expressing RhoA-DN. PKC activity was inhibited by bisindolylmaleimide, and in cells expressing RhoA-DN; PKC activity was also inhibited partly by Y27632 (44 +/- 5%). PKC-induced phosphorylation of PKC-activated 17 kDa inhibitor protein of type 1 phosphatase (CPI-17) at Thr(38) was abolished by bisindolylmaleimide and inhibited partly by Y27632 (28 +/- 3 %). Rho-kinase-induced phosphorylation of myosin phosphatase targeting subunit (MYPT1) and was abolished by Y27632. Sustained phosphorylation of 20 kDa regulatory light chain of myosin II (MLC20) and contraction were abolished by bisindolylmaleimide Y27632 and C3 exoenzyme and in cells expressing RhoA-DN. The results suggest that Rho-kinase-dependent phosphorylation of MYPT1 and PKC-dependent phosphorylation and enhancement of CPI-17 binding to the catalytic subunit of MLC phosphatase (MLCP) act co-operatively to inhibit MLCP activity, leading to sustained stimulation of MLCZO phosphorylation and contraction. Because Y27632 inhibited both Rho kinase and PKC activities, it could not be used to ascertain the contribution of MYPT1 to inhibition of MLCP activity. m2-dependent phosphorylation and inactivation of MLCK precluded its involvement in sustained MLCZO phosphorylation and contraction.