Lin Mei - US grants

DESCRIPTION (provided by applicant): Netrins are a family of well-characterized guidance cues that are conserved across different species and are homologous to laminin beta2. They promote axon outgrowth and guide neuronal growth cones in the developing nervous system. The intracellular signaling mechanism of netrin is unclear. In preliminary studies, we found that DCC (deleted in colorectal cancer), a netrin receptor, interacts directly with the focal adhesion kinase (FAK), a tyrosine kinase implicated in integrin signaling. Netrin-1 stimulation results in increased DCC-FAK interaction and FAK tyrosine phosphorylation at multiple residues including tyrosines 397 and 861. Furthermore, netrin-1 induces DCC/neogenin tyrosine phosphorylation in a manner dependent on FAK and an Src family kinase. The tyrosine phosphorylated DCC, on the other hand, binds to Fyn, an Src family kinase enriched in the brain. Functionally, treatment of rat cortical explants with PP2, an inhibitor of Src kinases, prevents netrin-1 from inducing neurite outgrowth. Expression of dominant negative FAK mutant blocks netrin-1 induced attractive turning in Xenopus spinal neurons. Based on these preliminary results, we hypothesize that FAK is important in mediating and/or regulating netrin functions. To test this hypothesis, we will: (1) determine the role of DCC tyrosine phosphorylation in netrin-induced axon outgrowth and turning, (2) determine the role of FAK in netrin-induced axon outgrowth and turning, and (3) investigate the effects of laminin on netrin-induced axon outgrowth and turning. The proposed research will provide not only key pieces of the signaling pathway by which netrin-DCC regulate axon outgrowth and growth cone guidance, but also insight into how signaling networks between netrin-DCC and extracellular matrix-integrins are organized. Such knowledge is essential for understating the mechanisms involved in axon path-finding in neural development and birth defects with malformation of the brain and spinal cord.

[unreadable] DESCRIPTION (provided by applicant): AChR is a ligand-gated ion channel that is essential for neurotransmission at the NMJ. AChR gene transcription is controlled by multiple mechanisms. It is inhibited by electrical activity in all nuclei in the muscle, but maintains in synaptic nuclei probably due to factors released from motoneurons. One such factor is NRG, which stimulates ErbB protein tyrosine kinases and subsequently activates Erk to mediate transcription of AChR subunit genes. An important question yet to be addressed is how NRG signaling is regulated so that AChR is maintained at optimal level. In preliminary studies, we demonstrate that expression of SHP2 inhibits NRG-induced AChR. epsilon subunit promoter activity. The inhibitory effect of SHP2 is dependent on the N-terminal SH2 (N-SH2) domain and executed at the level above Ras. We find that SHP2 binds to ErbB proteins in response to NRG and at the same time interacts with the mu2 subunit ofAP2, a complex implicated in receptor endocytosis. Moreover, NRG signaling is enhanced in SHP2 mutant cells where ErbB proteins are hyper-phosphorylated and NRG-induced ErbB endocytosis is impaired. Based on these results, we propose a working hypothesis that upon NRG stimulation, ErbB proteins become tyrosine-phosphorylated. Tyrosine phosphorylated ErbB proteins recruit SHP2, which switch off NRG signaling by 1) dephosphorylating ErbB residues that bind to downstream signaling proteins and 2) regulating ErbB endocytosis and thus down-regulate AChR synthesis. To test this hypothesis, we will characterize the temporal regulation of NRG signaling by SHP2; identify and characterize the phosphotyrosines of ErbB2 and ErbB4 for binding to SHP2; study the role of the ErbB-SHP2-mu2 complex in NRG-induced endocytosis of ErbB proteins; and investigate the role of SHP2 in AChR expression using in vivo mouse models. Results of proposed research will provide a better understanding of how the NMJ is formed and maintained. Such information is prerequisite to developing methods for diagnosis and therapeutic interventions of neurological disorders at the NMJ. In addition to providing insight into mechanisms by which neuregulin mediates expression of neuronal AChR, the primary target of nicotine, the proposed studies will identify novel targets that may be altered during nicotine abuse. [unreadable] [unreadable]