Function of kinase-deficient Trk receptor isoforms.TrkB and TrkC encode a number of isoforms, including those that lack the catalytic tyrosine kinase domain. Little is known about the function of these kinase deficient isoforms in Trk signaling. In vitro studies, and our own in vivo studies, have shown that truncated Trk receptors can inhibit the function of kinase-active receptor isoforms in a dominant-negative manner or by ligand sequestration. The physiological relevance of this activity is, however, still unclear. The high degree of sequence conservation of the intracellular domains of truncated receptors suggests the potential for specific interactions with cytoplasmic proteins and signaling capabilities. Indeed, it has been reported recently that BDNF induces the production of calcium waves in astroglia through the truncated TrkB T1 receptor. However, the molecular mechanism(s) linking the TrkB T1 receptor to calcium mobilization and its physiological role is still unknown. Interestingly, TrkB T1 is 50% overexpressed in the brain of the trisomy 16 (Ts16) mouse model of Down syndrome and Ts16 hippocampal neurons die prematurely in culture. Neurodegeneration is commonly associated with Down syndrome in humans and TrkB T1 is also overexpressed in Alzheimer's patients. To further investigate the role of TrkB T1 in neuronal survival, we generated a mouse lacking specifically the TrkBT1 kinase-deficient receptor isoform. This mutation caused no gross phenotype and could be used to correct the levels of TrkB T1 in Ts16 mice in vivo. Importantly, hippocampal neurons from TrkB T1 -/+; Ts16 mice escaped the premature cell death of Ts16 neurons in vitro (Dorsey et al. in preparation). This is a very exciting result because it contrasts with earlier hypotheses that neurodegeneration occurs due to insufficient supply of neurotrophic factors. Rather, our studies suggest that modulation of cell death and survival can occur at the level of the Trk receptor. We are now investigating the molecular mechanism underlying the detrimental effect of elevated TrkB T1 expression. Specifically, we are addressing both the effects of TrkBT1 on the activity of the full-length TrkB receptor and on the intracellular regulation of Ca++ levels. Truncated TrkC receptors, such as TrkC TK-, have never been implicated in intracellular signaling. To identify proteins that might bind the highly conserved intracellular domain of TrkC TK-, we conducted a yeast two-hybrid screen and identified an adaptor protein (GRASP/tamalin) that interacts specifically with TrkC TK- in a ligand dependent manner. Both tamalin and TrkC TK- are expressed in the brain with overlapping anatomical and subcellular distribution. We also found that NT-3 initiation of the TrkCTK-/Tamalin complex leads to activation of Rac1 GTPase through the ADP-ribosylation factor 6 (ARF6). NT-3 binding to TrkCTK-/Tamalin induces ARF6 translocation to the membrane, which in turn causes membrane ruffling and formation of cellular protrusions. Thus, we have shown that a truncated TrkC receptor lacking kinase activity can activate a specific intracellular signaling pathway that links NT-3 to key components of neuronal development and plasticity, such as regulation of the actin cytoskeleton and membrane trafficking. Moreover, we have established NT-3 as an unsuspected upstream activator of ARF6, a regulator of endosome membrane trafficking, endocytosis and actin remodeling at the cell surface; processes that are important for cell motility. (Esteban et al., submitted)To further investigate the function of tamalin in the nervous system, we have generated mice with a targeted deletion of tamalin. These mice are viable and are currently under investigation. The yeast two-hybrid screen also identified several other TrkC TK- binding candidates besides tamalin.
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