胃平滑肌中通过 PI3 激酶 γ 和整合素连接激酶的毒蕈碱 M2 受体介导的肌动蛋白聚合。

Muscarinic m2 receptor-mediated actin polymerization via PI3 kinase γ and integrin-linked kinase in gastric smooth muscle.

机构信息

Department of Physiology and Biophysics, VCU Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, Virginia.

出版信息

Neurogastroenterol Motil. 2019 Feb;31(2):e13495. doi: 10.1111/nmo.13495. Epub 2018 Nov 4.

DOI:10.1111/nmo.13495

PMID:30393912

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6347515/

Abstract

BACKGROUND

Actin polymerization plays an important role in smooth muscle contraction. Integrin-linked kinase (ILK) was shown to mediate actin polymerization in airway smooth muscle. The role of ILK in actin polymerization in response to m2 receptor activation was not in gastric smooth muscle.

METHODS

Phosphorylation of paxillin, neuronal Wiskott-Aldrich syndrome protein (N-WASp), and association of paxillin with GEF proteins (Cool2/αPix [Cool2/PAK-interacting exchange factor alpha], Cool1/βPix [Cool1/PAK-interacting exchange factor beta], and DOCK 180 [Dedicator of cytokinesis]) and N-WASp with Arp2/3 complex were measured by western blot. Activation of Cdc42 was determined using an antibody for activated Cdc42. Actin polymerization was measured as an increase in F-actin/G-actin ratio.

RESULTS

Phosphorylation of paxillin, an association of paxillin with GEF proteins, Cdc42 activity, and actin polymerization were increased in response to m2 receptor activation in gastric smooth muscle cells. The increases in paxillin phosphorylation, Cdc42 activity, and actin polymerization were inhibited by a PI3Kγ inhibitor (AS-605240), ILK siRNA, and ILK dominant negative mutant (ILK [R211]). Increase in actin polymerization was also inhibited by Cdc42 dominant negative mutant (Cdc42 [T17N]). Increases in the association of paxillin with GEF proteins, phosphorylation of N-WASp and its association with Arp2/3 complex were inhibited by ILK (R211).

CONCLUSION

In gastric smooth muscle cells, activation of PI3Kγ by muscarinic m2 receptors causes ILK-dependent phosphorylation of paxillin, an association of paxillin with Cdc42 GEF proteins and activation of Cdc42, which, in turn, causes phosphorylation of N-WASp and its association with Arp2/3 complex leading to actin polymerization.

摘要

背景

肌动蛋白聚合在平滑肌收缩中起着重要作用。整合素连接激酶（ILK）被证明可介导气道平滑肌中的肌动蛋白聚合。ILK 在 m2 受体激活引起的肌动蛋白聚合中的作用在胃平滑肌中并不存在。

方法

通过 Western blot 测定 paxillin、神经元 Wiskott-Aldrich 综合征蛋白（N-WASp）的磷酸化以及 paxillin 与 GEF 蛋白（Cool2/αPix [Cool2/PAK 相互作用交换因子 alpha]、Cool1/βPix [Cool1/PAK 相互作用交换因子 beta]和 DOCK 180 [细胞分裂的 dedicator of cytokinesis]）和 N-WASp 与 Arp2/3 复合物的关联。使用针对激活的 Cdc42 的抗体测定 Cdc42 的激活。通过 F-肌动蛋白/G-肌动蛋白比值的增加来测量肌动蛋白聚合。

结果

胃平滑肌细胞中 m2 受体激活后，paxillin 磷酸化、paxillin 与 GEF 蛋白的关联、Cdc42 活性和肌动蛋白聚合增加。PI3Kγ 抑制剂（AS-605240）、ILK siRNA 和 ILK 显性负突变体（ILK [R211]）抑制 paxillin 磷酸化、Cdc42 活性和肌动蛋白聚合的增加。Cdc42 显性负突变体（Cdc42 [T17N]）也抑制肌动蛋白聚合的增加。ILK [R211] 抑制 paxillin 与 GEF 蛋白的关联、N-WASp 的磷酸化及其与 Arp2/3 复合物的关联的增加。