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]