, 1999). Given the broad expression patterns of TrkC and PTPσ during development, some loss-of-function defects may be because of loss of critical trans interaction of TrkC and PTPσ outside synapses. Our study raises a number of questions for future research. We show here

that the bidirectional synaptic organizing function of TrkC-PTPσ occurs independently of kinase and phosphatase activity, but whether this interaction triggers or otherwise regulates catalytic activity is unknown. This is the first trans interaction of which we are aware between a tyrosine kinase and a tyrosine phosphatase, and it may represent a mechanism for regulating the balance of tyrosine phosphorylation. It will be important to determine whether binding of PTPσ activates TrkC Romidepsin kinase and/or whether binding of TrkC activates PTPσ phosphatase. It will be particularly interesting to determine how the TrkC-PTPσ adhesion complex regulates glutamatergic synaptic signaling pathways and how NT-3 modulates this process. TrkC kinase activation initiates multiple pathways including Ras-Erk1/2, Src, and PI3K-Akt (Huang and Reichardt, 2003), pathways shown to alter

AMPA and NMDA-mediated transmission (Sheng and Kim, 2002). N-cadherin is a major substrate of PTPσ (Siu et al., 2007), raising the potential for TrkC-PTPσ modulation VX-809 datasheet of other synaptic adhesion complexes. The distinct binding sites could allow for simultaneous binding of PTPσ and NT-3 to TrkC, via LRR-Ig1 and Ig2, respectively. NT-3 may modulate the synaptogenic activity of TrkC, for example, by inducing TrkC dimerization and internalization. PTPσ also binds via its first Ig domain to chondroitin and heparan sulfate proteoglycans to inhibit axon regeneration (Aricescu et al., 2002 and Shen et al., 2009). Whether TrkC and proteoglycans compete for binding to PTPσ and consequences for axon regeneration and synaptogenic activity remain to be determined. The TrkC-PTPσ interaction may function in diverse processes including cell proliferation

and differentiation, axon guidance and regeneration, and excitatory synaptic assembly and signaling. Cultures of hippocampal neurons, neuron-fibroblast cocultures, and immunocytochemistry were performed essentially as described (Linhoff et al., 2009). Transfections into very COS-7 cells and hippocampal neurons were done by using FuGENE 6 (Roche) and the ProFection Mammalian Transfection System (Promega), respectively. The cDNAs for full-length rat TrkCTK- (BC078844), TrkCTK+ (NM_019248), TrkCKI25 (AAB26724.1), TrkA (NM_021589), TrkBTK- (NM_001163168), TrkBTK+ (NM_012731), and p75NTR (NM_012610) were cloned by RT-PCR from a P11 rat brain cDNA library (Linhoff et al., 2009) and subcloned into pcDNA3 vectors. Deletion and swap mutants of TrkC were made based on domain structures described by Barbacid (1994). Additional details are provided in Supplemental Experimental Procedures.