Mechanism for Src activation by the CCK2 receptor: Patho-physiological functions of this receptor in pancreas.

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 immuno-fluorescence 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 7th 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.