The Signaling Adapter, FRS2, Facilitates Neuronal Branching in Primary Cortical Neurons via Both Grb2- and Shp2-Dependent Mechanisms

The neurotrophins are a family of closely related growth factors that regulate proliferation and differentiation in the developing and mature nervous systems. Neurotrophins stimulate a family of receptor tyrosine kinases (Trk receptors) and utilize an intracellular docking protein termed fibroblast growth factor (FGF) receptor substrate 2 (FRS2) as a major downstream adapter to activate Ras, phosphatidylinositide 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK) signaling cascades. The goals of this study were twofold: first, to investigate the complexity of neurotrophin-induced FRS2 interactions in primary cortical neurons and to determine which pathway(s) are important in regulating neuronal growth and, second, to determine whether the related signaling adapter, FRS3, stimulates neuron growth comparable to FRS2. We find that neurotrophin treatment of primary cortical neurons stimulates the tyrosine phosphorylation of FRS2 and the subsequent recruitment of Shp2, Grb2, and Gab2. With FRS2 mutants deficient in Grb2 or Shp2 binding, we demonstrate that FRS2 binds Gab1 and Gab2 through Grb2, providing an alternative route to activate PI3 kinase and Shp2. Using recombinant adenoviruses expressing FRS2, we demonstrate that FRS2 overexpression promotes neurite outgrowth and branching in cortical neurons relative to controls. In contrast, overexpression of FRS3 does not stimulate neuronal growth. Moreover, we find that while loss of Shp2, but not Grb2, reduces brain-derived neurotrophic factor (BDNF)-induced MAPK activation, the loss of either pathway impairs neuronal growth. Collectively, these experiments demonstrate that FRS2 functions as an adapter of a multiprotein complex that is activated by the Trk receptors and that the activation of both Grb2- and Shp2-dependent pathways facilitates cortical neuronal growth.