Nerve growth factor (NGF) is a neurotrophic factor secreted by cells that are the targets of innervation of sympathetic and some sensory neurons. However, the mechanism by which the NGF signal is propagated from the axon terminal to the cell body, which can be more than 1 meter away, to influence biochemical events critical for growth and survival of neurons has remained unclear. An NGF-mediated signal transmitted from the terminals and distal axons of cultured rat sympathetic neurons to their nuclei regulated phosphorylation of the transcription factor CREB (cyclic adenosine monophosphate response element-binding protein). Internalization of NGF and its receptor tyrosine kinase TrkA, and their transport to the cell body, were required for transmission of this signal. The tyrosine kinase activity of TrkA was required to maintain it in an autophosphorylated state upon its arrival in the cell body and for propagation of the signal to CREB within neuronal nuclei. Thus, an NGF-TrkA complex is a messenger that delivers the NGF signal from axon terminals to cell bodies of sympathetic neurons.

SUMMARY Growing axons encounter multiple guidance cues, but it is unclear how separate signals are resolved and integrated into coherent instructions for growth cone navigation. We report that glycosylphosphatidylinositol (GPI)-anchored ephrin-As function as “reverse” signaling receptors for motor axons when contacted by transmembrane EphAs present in the dorsal limb. Ephrin-A receptors are thought to depend on transmembrane co-receptors for transmitting signals intracellularly. We show that the receptor tyrosine kinase Ret is required for motor axon attraction mediated by ephrin-A reverse signaling. Ret also mediates GPI-anchored GFRα1 signaling in response to GDNF, a diffusible chemoattractant in the limb - indicating that Ret is a multifunctional co-receptor for guidance molecules. Axons respond synergistically to co-activation by GDNF and EphA ligands and these cooperative interactions are gated by GFRα1 levels. Our studies uncover a hierarchical GPI-receptor signaling network that is constructed from combinatorial components and integrated through Ret using ligand coincidence detection.

The receptor tyrosine kinase (RTK) Ret is activated by the formation of a complex consisting of ligands such as glial cell line-derived neurotrophic factor (GDNF) and glycerophosphatidylinositol-anchored coreceptors termed GFR␣s. During activation, Ret translocates into lipid rafts, which is critical for functional responses to GDNF. We found that Ret was rapidly ubiquitinated and degraded in sympathetic neurons when activated with GDNF, but, unlike other RTKs that are trafficked to lysosomes for degradation, Ret was degraded predominantly by the proteasome. After GDNF stimulation, the majority of ubiquitinated Ret was located outside of lipid rafts and Ret was lost predominantly from nonraft membrane domains. Consistent with the predominance of Ret degradation outside of rafts, disruption of lipid rafts in neurons did not alter either the GDNF-dependent ubiquitination or degradation of Ret. GDNF-mediated survival of sympathetic neurons was inhibited by lipid raft depletion, and this inhibitory effect of raft disruption on GDNF-mediated survival was reversed if Ret degradation was blocked via proteasome inhibition. Therefore, lipid rafts sequester Ret away from the degradation machinery located in nonraft membrane domains, such as Cbl family E3 ligases, thereby sustaining Ret signaling.

Podocytes are morphologically complex cells, the junctions of which form critical elements of the final filtration barrier. Disruption of their foot processes and slit diaphragms occur early in the development of many glomerular diseases. Here, we biochemically purified fractions enriched with slit diaphragm proteins and performed a proteomic analysis to identify new components of this important structure. Several known slit diaphragm proteins were found, such as podocin and nephrin, confirming the validity of the purification scheme. However, proteins on the apical membrane such as podocalyxin were neither enriched nor identified in our analysis. The chloride intracellular channel protein 5 (CLIC5), predominantly expressed in podocytes, was enriched in these fractions and localized in the foot process apical and basal membranes. CLIC5 colocalized and associated with the ezrin/radixin/moesin complex and with podocalyxin in podocytes in vivo. It is important to note that CLIC5−/− mice were found to have significantly decreased foot process length, widespread foot process abnormalities, and developed proteinuria. The ezrin/radixin/moesin complex and podocalyxin were significantly decreased in podocytes from CLIC5−/− mice. Thus, our study identifies CLIC5 as a new component that is enriched in and necessary for foot process integrity and podocyte function in vivo.

The p75NTR neurotrophin receptor has been implicated in multiple biological and pathological processes. While significant advances have recently been made in understanding the physiologic role of p75NTR, many details and aspects remain to be determined. This is in part because the two existing knockout mouse models (exons 3 or 4 deleted, respectively), both display features that defy definitive conclusions. Here we describe the generation of mice that carry a conditional p75NTR (p75NTR-FX) allele made by flanking exons 4–6, which encode the transmembrane and all cytoplasmic domains, by loxP sites. To validate this novel conditional allele, both neural crest-specific p75NTR/Wnt1-Cre mutants and conventional p75NTR null mutants were generated. Both mutants displayed abnormal hind limb reflexes, implying that loss of p75NTR in neural crest-derived cells causes a peripheral neuropathy similar to that seen in conventional p75NTR mutants. This novel conditional p75NTR allele will offer new opportunities to investigate the role of p75NTR in specific tissues and cells.

SignificanceWhile knowledge of signaling mechanisms orchestrating the development and diversification of peripheral somatosensory neurons is extensive, our understanding of the mechanisms controlling chemosensory neuron specification remains rudimentary. Lingually projecting sensory neurons of the geniculate ganglion are receptive to the five taste qualities, as well as temperature and tactile stimuli, but the mechanisms responsible for the diversification of the unique subpopulations that respond to one, or several, of these stimuli remain unknown. Here we demonstrate that the GDNF-Ret signaling pathway exerts a unique, dual function in peripheral taste system development and postnatal function. Ret acts embryonically to regulate the expression of the chemosensory master regulator Phox2b, thus inducing chemosensory differentiation, while postnatally acting to specify a molecularly unique subpopulation of lingual mechanoreceptors.

The immunosuppressant rapamycin prevents cell cycle progression in several mammalian cell lines and the yeast Saccharomyces cerevisiae. In mammalian cells, rapamycin binds to the small FK506-binding protein, FKBP12, allowing the drug-receptor complex to interact with the 289-kDa RAFT1/FRAP proteins. These proteins, along with their yeast homologs, TOR1/DRR1 and TOR2/ DRR2, contain a C-terminal domain with amino acid homology to several phosphatidylinositol (PI) 4-and 3-kinases. However, no direct demonstration of kinase activity for this family of proteins has been reported. We now show that RAFT1, immunoprecipitated from rat brain and MG63 and HEK293 cells, contains PI 4-kinase activity and that rapamycin-FKBP12 has no effect on this activity. Thus, it is likely that, in vivo, rapamycin does not directly inhibit the PI 4-kinase activity and affects the RAFT1/FRAP protein through another mechanism.The ability of rapamycin, a potent immunosuppressant drug, to arrest a variety of mammalian cells and the yeast Saccharomyces cerevisiae in the G 1 stage of the cell cycle (1-4) has made it a valuable tool for studying the intermediate signaling events that convey mitogenic stimuli to the cell nucleus.Rapamycin binds with low nanomolar affinity to the FK506-binding protein (FKBP12), a small soluble protein that is also the intracellular receptor for FK506, a structurally similar immunosuppressant (5-7). Analogous to the mechanism of action of FK506, it is the drug-receptor complex that mediates the effects of the rapamycin, which include inhibition of the 70-kDa S6 kinase (8, 9) and of several cyclin-dependent kinases (2-4). The immunosuppressant effects of FK506 derive from the binding of the FK506-FKBP12 complex to the calcium-activated phosphatase, calcineurin, and the resulting inhibition of its activity (10). Although rapamycin binds to FKBP12, the complex does not interact with calcineurin. Instead, rapamycin-FKBP12 binds to a recently purified and molecularly cloned protein designated rapamycin and FKBP12 target-1 (RAFT1) in rats (11) and FKBP-rapamycin-associated protein (FRAP) in humans (12). Several other groups have subsequently purified and/or cloned the same protein (13-15).RAFT1 is a 2549-amino acid protein with a predicted molecular mass of 289 kDa and is thought to be a mammalian homolog of the products of two yeast genes, TOR1/DRR1 and TOR2/DRR2, which when mutated lead to dominant rapamycin resistance in yeast (1, 16, 17). The C-terminal 600-amino acid domain of RAFT1 and the yeast TOR proteins has homology to several PI 1 kinases, including the p110 subunit of mammalian PI 3-kinase (18); the yeast PI 3-kinase VPS34 (19); two yeast PI 4-kinases, STT4 (20) and PIK1 (21, 22); and the recently cloned mammalian PI 4-kinase, PI4K␣ (23). This homology to known lipid kinases has lead to the suggestion that RAFT1 may also be a PI kinase and that rapamycin exerts its effects by inhibiting this activity (11). However, no direct demonstration of kinase activity for RAFT1/FRAP or the yeast TOR proteins has been r...

Glomerulosclerosis correlates with a reduction in podocyte number that occurs through mechanisms that include apoptosis. Whether glial cell line-derived neurotrophic factor (GDNF), a growth factor that is critical for neural and renal development, is a survival factor for injured podocytes was investigated. Ret, the GDNF receptor tyrosine kinase, was upregulated in podocytes in the passive Heymann nephritis and puromycin aminonucleoside (PA) nephrosis rat models of podocyte injury. In addition, Ret mRNA and protein were upregulated in mouse podocytes in vitro after injury that was induced by sublytic C5b-9 and PA. GDNF, which also was induced during podocyte injury, inhibited significantly the apoptosis of podocytes that was induced by ultraviolet C irradiation. Knockdown of Ret expression by small interference RNA in podocytes exacerbated apoptosis that was induced by both ultraviolet C and PA. Ret knockdown, upon injury, decreased AKT phosphorylation, suggesting that the phosphoinositol-3 kinase/AKT pathway mediated the survival effect of GDNF on podocytes. Consistent with this hypothesis, the selective phosphoinositol-3 kinase inhibitor LY294002 blocked the survival-promoting effects of GDNF. In conclusion, GDNF is a novel podocyte survival factor. Furthermore, Ret is highly upregulated during podocyte injury in vitro and in vivo, suggesting that Ret activation is a critical adaptive response for podocyte remodeling and repair.