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Glial cell line-derived neurotrophic factor (GDNF) signals through a unique receptor system that includes Ret receptor tyrosine kinase and a glycosyl-phosphatidylinositol-linked cell surface protein. In the present study, we have identified several proteins in neuroblastoma cells that are phosphorylated on tyrosine in response to GDNF. The phosphorylated proteins include focal adhesion kinase (FAK), paxillin and Crk-associated substrate, p130Cas, all of which are known to be associated with focal adhesions. Of these, paxillin and p130Cas interacted with Crk proteins in GDNF-treated neuroblastoma cells. GDNF also induced reorganization of the actin cytoskelton. Tyrosine phosphorylation of FAK, paxillin and p130Cas was inhibited by cytochalasin D or two specific inhibitors of phosphatidylinositol-3′ kinase (PI-3′ kinase), wortmannin and LY294002, indicating that their tyrosine phosphorylation depends on the formation of actin stress fiber and activation of PI-3′ kinase. In addition, phosphorylation of FAK but not of paxillin and p130Cas was markedly impaired by the Clostridium botulinum C3 exoenzyme that specifically ADP-ribosylates and inactivates Rho. These results suggested the presence of Rho-dependent and -independent signaling pathways downstream of PI-3′ kinase that mediate tyrosine phosphorylation of FAK, paxillin and p130Cas through Ret kinase. [ABSTRACT FROM AUTHOR]

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) define a new family of neurotrophic factors that play crucial roles in survival and differentiation of various neurons. Recent studies demonstrated that GDNF and NTN use a multicomponent receptor system in which glycosyl-phosphatidylinositol (GPI)-linked cell surface proteins and Ret receptor tyrosine kinase function as the ligand-binding and signalling components, respectively. In the present study, we investigated the role of Ca2+ ions for biochemical and biological activities of Ret because Ret has a unique structure of the extracellular domain with the cadherin-like motif. The results demonstrated that Ca2+ ions might be required for the complex formation of Ret and GDNF or NTN that induces Ret oligomerization and autophosphorylation. Full morphological differentiation of neuroblastoma cells by these neurotrophic factors was also Ca2+-dependent. These findings thus suggested that, in addition to GPI-linked cell surface proteins, Ca2+ ions are components of the signal transducing complex formed by Ret and GDNF protein family. [ABSTRACT FROM AUTHOR]