Mechanism for Src activation by the CCK2 receptor: Pathophysiological functions of this receptor in pancreas
Mechanism for Src activation by the CCK2 receptor: Pathophysiological functions of this receptor in pancreas
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DOI：
10.3748/wjg.v12.i28.4498
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发表时间：
2006-07-28
影响因子：
4.3
通讯作者：
Seva, Catherine
中科院分区：
文献类型：
--
作者：
Ferrand, Audrey;Vatinel, Sebastien;Seva, Catherine
目的：在体内研究CCK2受体（CCK2R）是否调节已知在细胞增殖和癌症发展中起关键作用的蛋白质，并在体外分析导致Src激活的分子机制；特别是在CCK2R序列中识别与这种激活有关的结构域。方法：采用免疫荧光和western-blot技术研究Src和ERK在体内的表达和活化。我们使用来自野生型或elas2 - cck2小鼠的胰腺组织，这些胰腺组织在胰腺腺泡中表达CCK2R，显示胰腺生长增加并发生癌前病变。以表达内源性CCK2R的胰腺肿瘤细胞系AR4-2J或瞬时转染野生型或突变型CCK2R的COS-7细胞为体外模型，研究Src激活的机制。Src激活通过体外激酶检测，ERK激活通过抗磷酸化ERK抗体的western blot检测，Src参与胃泌素诱导的细胞增殖通过MTT试验。结果：我们在体内发现，在elasi - cck2小鼠胰腺中靶向表达CCK2R，导致Src和ERK通路的激活。Src在胰腺肿瘤细胞的ERK通路上游被CCK2R激活，并参与了该受体介导的增殖作用。体外实验结果表明，CCK2R激活Src/ERK通路需要NPXXY基序，该基序位于CCK2R序列第7（th）跨膜结构域末端，并提示Gq可能在该机制中发挥作用。结论：CCK2R对Src/ERK通路的调控可能是促进细胞增殖、癌前病变形成和胰腺肿瘤发展的早期步骤。(C) 2006 WJG出版社。版权所有。
AIM: To investigate in vivo, whether CCK2 receptors (CCK2R) regulate proteins known to play a crucial role in cell proliferation and cancer development and analyse in vitro the molecular mechanisms that lead to Src activation; in particular, to identify the domains within the CCK2R sequence that are implicated in this activation.METHODS: The expression and activation of Src and ERK were studied in vivo using immunofluorescence and western-blot techniques. We used pancreatic tissues derived from wild type or Elas-CCK2 mice that expressed the CCK2R in pancreatic acini, displayed an increased pancreatic growth and developed preneoplastic lesions. The pancreatic tumor cell line AR4-2J expressing the endogenous CCK2R or COS-7 cells transiently transfected with wild type or mutant CCK2R were used as in vitro models to study the mechanism of Src activation. Src activation was measured by in vitro kinase assays, ERK activation by western blot using anti-phospho-ERK antibodies and the involvement of Src in gastrin-induced cell proliferation by MTT test.RESULTS: We showed in vivo that the targeted CCK2R expression in the pancreas of Elas-CCK2 mice, led to the activation of Src and the ERK pathway. Src was activated upstream of the ERK pathway by the CCK2R in pancreatic tumoral cells and contributed to the proliferative effects mediated by this receptor. In vitro results demonstrated that activation of the Src/ERK pathway by the CCK2R required the NPXXY motif, located within the CCK2R sequence at the end of the 7(th) transmembrane domain, and suggested the putative role of Gq in this mechanism.CONCLUSION: Deregulation of the Src/ERK pathway by the CCK2R might represent an early step that contributes to cell proliferation, formation of preneoplastic lesions and pancreatic tumor development. (C) 2006 The WJG Press. All rights reserved.