Le Page Sophie L, Bi Yan, Williams John A
Department. of Molecular & Integrative Physiology, University of Michigan, 7744 Medical Science II, Ann Arbor, MI 48109-0622, USA.
Am J Physiol Cell Physiol. 2003 Nov;285(5):C1197-206. doi: 10.1152/ajpcell.00083.2003. Epub 2003 Jul 9.
Cholecystokinin (CCK) is a major regulator of pancreatic acinar cells and was shown previously to be capable of inducing cytoskeletal changes in these cells. In the present study, using NIH3T3 cells stably transfected with CCK-A receptors as a model cell, we demonstrate that CCK can induce actin stress fibers through a G13- and RhoA-dependent mechanism. CCK induced stress fibers within minutes similar to those induced by lysophosphatidic acid (LPA), the active component of serum. The effects of CCK were mimicked by active RhoV14 and blocked by dominant-negative RhoN19, Clostridium botulinum C3 transferase, and the Rho-kinase inhibitor Y-27632. CCK rapidly induced active Rho in cells as shown with a pull-down assay using the Rho binding domain of rhotekin and by a serum response element (SRE)-luciferase reporter assay. To evaluate the G protein mediating the action of CCK, cells were transfected with active alpha-subunits; Galpha13 and Galpha12 but not Galphaq induced stress fibers and in some cases cell rounding. A p115 Rho guanine nucleotide exchange factor (GEF) regulator of G protein signaling (RGS) domain known to interact with G12/13 inhibited active alpha12/13-and CCK-induced stress fibers, whereas RGS2 and RGS4, which are known to inhibit Gq, had no effect. Cotransfection with plasmids coding for the G protein alpha-subunit carboxy-terminal peptide from alpha13 and, to a lesser extent alpha12, also inhibited the effect of CCK, whereas the peptide from alphaq did not. These results show that in NIH3T3 cells bearing CCK-A receptors, CCK activates Rho primarily through G13, leading to rearrangement of the actin cytoskeleton.
胆囊收缩素（CCK）是胰腺腺泡细胞的主要调节因子，先前已证明其能够诱导这些细胞的细胞骨架变化。在本研究中，我们以稳定转染CCK-A受体的NIH3T3细胞作为模型细胞，证明CCK可通过G13和RhoA依赖性机制诱导肌动蛋白应激纤维。CCK在数分钟内即可诱导应激纤维的形成，类似于血清中的活性成分溶血磷脂酸（LPA）所诱导的应激纤维。活性RhoV14可模拟CCK的作用，而显性负性RhoN19、肉毒杆菌C3转移酶和Rho激酶抑制剂Y-27632则可阻断CCK的作用。如使用rhotekin的Rho结合结构域进行的下拉试验以及血清反应元件（SRE）-荧光素酶报告基因试验所示，CCK可迅速诱导细胞内活性Rho的产生。为了评估介导CCK作用的G蛋白，我们用活性α亚基转染细胞；Gα13和Gα12而非Gαq可诱导应激纤维形成，在某些情况下还会导致细胞变圆。一种已知与G12/13相互作用的p115 Rho鸟嘌呤核苷酸交换因子（GEF）调节G蛋白信号（RGS）结构域可抑制活性α12/13和CCK诱导的应激纤维，而已知抑制Gq的RGS2和RGS4则无此作用。与编码来自α13以及程度较轻的来自α12的G蛋白α亚基羧基末端肽的质粒共转染，也可抑制CCK的作用，而来自αq的肽则无此作用。这些结果表明，在表达CCK-A受体的NIH3T3细胞中，CCK主要通过G13激活Rho，从而导致肌动蛋白细胞骨架的重排。
