Faculty Bibliography

In addition to promoting cell survival, neurotrophins also can elicit apoptosis in restricted cell types. Recent results indicate that nerve growth factor (NGF) can induce Schwann cell death via engagement of the p75 neurotrophin receptor. Here we describe a novel interaction between the p75 receptor and receptor-interacting protein 2, RIP2 (RICK/CARDIAK), that accounts for the ability of neurotrophins to choose between a survival-versus-death pathway. RIP2, an adaptor protein with a serine threonine kinase and a caspase recruitment domain (CARD), is highly expressed in dissociated Schwann cells and displays an endogenous association with p75. RIP2 binds to the death domain of p75 via its CARD domain in an NGF-dependent manner. The introduction of RIP2 into Schwann cells deficient in RIP2 conferred NGF-dependent nuclear transcription factor-kappaB (NF-kappaB) activity and decreased the cell death induced by NGF. Conversely, the expression of a dominant-negative version of RIP2 protein resulted in a loss of NGF-induced NF-kappaB induction and increased NGF-mediated cell death. These results indicate that adaptor proteins like RIP2 can provide a bifunctional switch for cell survival or cell death decisions mediated by the p75 neurotrophin receptor

Dorsal root ganglion (DRG) neurons first express kainate receptor subunits, predominantly GluR5, during embryonic development. In the DRG and throughout the nervous system, substantial editing of GluR5 mRNA occurs with developmental maturation (Bernard et al., 1999). The accompanying change in Ca(2+) permeability of functional kainate receptors that is the predicted outcome of this developmental regulation of mRNA editing has not been investigated. Here we report that kainate receptors on DRG neurons from late embryonic and newborn rats are predominantly Ca(2+) permeable but then become fully Ca(2+) impermeable later in the first postnatal week. Using multiple markers for nociceptor subpopulations, we show that this switch in Ca(2+) permeability is not caused by the appearance of a new subpopulation of nociceptors with different receptor properties. Instead, the change in Ca(2+) permeability matches the time course of post-transcriptional RNA editing of GluR5 at the Q/R site within the pore of the channel, indicating that the change is probably caused by developmentally regulated RNA editing. We also report that, on the basis of the strong correlation of receptor expression with expression of the surface markers LA4, isolectin B4, and LD2, kainate receptors are present on C-fiber-type neurons projecting to lamina II of spinal cord dorsal horn. These results raise the possibility that kainate receptors in their Ca(2+)-permeable form serve a developmental role in synapse formation between this population of C-fibers and their targets in the spinal cord dorsal horn. Thereafter, the receptors may serve a new function that does not require Ca(2+) permeability

Tissue injury generates endogenous factors that heighten our sense of pain by increasing the response of sensory nerve endings to noxious stimuli. Bradykinin and nerve growth factor (NGF) are two such pro-algesic agents that activate G-protein-coupled (BK2) and tyrosine kinase (TrkA) receptors, respectively, to stimulate phospholipase C (PLC) signalling pathways in primary afferent neurons. How these actions produce sensitization to physical or chemical stimuli has not been elucidated at the molecular level. Here, we show that bradykinin- or NGF-mediated potentiation of thermal sensitivity in vivo requires expression of VR1, a heat-activated ion channel on sensory neurons. Diminution of plasma membrane phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) levels through antibody sequestration or PLC-mediated hydrolysis mimics the potentiating effects of bradykinin or NGF at the cellular level. Moreover, recruitment of PLC-gamma to TrkA is essential for NGF-mediated potentiation of channel activity, and biochemical studies suggest that VR1 associates with this complex. These studies delineate a biochemical mechanism through which bradykinin and NGF produce hypersensitivity and might explain how the activation of PLC signalling systems regulates other members of the TRP channel family

Neurotrophins rely on Trk tyrosine kinase and p75 receptors for signal transduction. Recently, other roles for these receptors have been identified. Many questions have been raised about the mechanism by which these receptors mediate diverse cellular functions. Studies indicate a great deal of neurotrophin signaling specificity may stem from ligand-receptor selectivity and intracellular protein recruitment

Neurotrophins regulate neuronal cell survival and synaptic plasticity through activation of Trk receptor tyrosine kinases. Binding of neurotrophins to Trk receptors results in receptor autophosphorylation and downstream phosphorylation cascades. Here, we describe an approach to use small molecule agonists to transactivate Trk neurotrophin receptors. Activation of TrkA receptors in PC12 cells and TrkB in hippocampal neurons was observed after treatment with adenosine, a neuromodulator that acts through G protein-coupled receptors. These effects were reproduced by using the adenosine agonist CGS 21680 and were counteracted with the antagonist ZM 241385, indicating that this transactivation event by adenosine involves adenosine 2A receptors. The increase in Trk activity could be inhibited by the use of the Src family-specific inhibitor, PP1, or K252a, an inhibitor of Trk receptors. In contrast to other G protein-coupled receptor transactivation events, adenosine used Trk receptor signaling with a longer time course. Moreover, adenosine activated phosphatidylinositol 3-kinase/Akt through a Trk-dependent mechanism that resulted in increased cell survival after nerve growth factor or brain-derived neurotrophic factor withdrawal. Therefore, adenosine acting through the A(2A) receptors exerts a trophic effect through the engagement of Trk receptors. These results provide an explanation for neuroprotective actions of adenosine through a unique signaling mechanism and raise the possibility that small molecules may be used to elicit neurotrophic effects for the treatment of neurodegenerative diseases

The TrkA NGF receptor extracellular region contains three leucine repeats flanked by cysteine clusters and two immunoglobulin-like domains that are required for specific ligand binding. Deletion of the immunoglobulin-like domains abolishes NGF binding and causes ligand independent activation of the receptor. Here we report a specific mutation that increases the binding affinity of the TrkA receptor for NGF. A change of proline 203 to alanine (P203A) in the linker region between the leucine repeats and the first Ig-like domain increased NGF binding by decreasing the ligand rate of dissociation. This mutated receptor was appropriately expressed on the cell surface and promoted ligand-independent neurite outgrowth in PC12nnr5 cells. The mutant receptor was capable of spontaneous dimerization and was constitutively phosphorylated in the absence of ligand. Moreover, expression of TrkA-P203A receptor in fibroblasts induced DNA synthesis and transformation and generated tumours in nude mice. These data suggest that domains outside of the immunoglobulin-like structure contribute to ligand binding and constitutive activation of Trk receptors

NGF initiates the majority of its neurotrophic effects by promoting the activation of the tyrosine kinase receptor TrkA. Here we describe a novel interaction between TrkA and GIPC, a PDZ domain protein. GIPC binds to the juxtamembrane region of TrkA through its PDZ domain. The PDZ domain of GIPC also interacts with GAIP, an RGS (regulators of G protein signaling) protein. GIPC and GAIP are components of a G protein-coupled signaling complex thought to be involved in vesicular trafficking. In transfected HEK 293T cells GIPC, GAIP, and TrkA form a coprecipitable protein complex. Both TrkA and GAIP bind to the PDZ domain of GIPC, but their binding sites within the PDZ domain are different. The association of endogenous GIPC with the TrkA receptor was confirmed by coimmunoprecipitation in PC12 (615) cells stably expressing TrkA. By immunofluorescence GIPC colocalizes with phosphorylated TrkA receptors in retrograde transport vesicles located in the neurites and cell bodies of differentiated PC12 (615) cells. These results suggest that GIPC, like other PDZ domain proteins, serves to cluster transmembrane receptors with signaling molecules. When GIPC is overexpressed in PC12 (615) cells, NGF-induced phosphorylation of mitogen-activated protein (MAP) kinase (Erk1/2) decreases; however, there is no effect on phosphorylation of Akt, phospholipase C-gamma1, or Shc. The association of TrkA receptors with GIPC and GAIP plus the inhibition of MAP kinase by GIPC suggests that GIPC may provide a link between TrkA and G protein signaling pathways

Nerve growth factor (NGF) initiates its trophic effects by long-range signaling through binding, internalization, and transport of a ligand-receptor complex from the axon terminal to the cell body. However, the mechanism by which retrograde transport of NGF takes place has not been elucidated. Here we describe an interaction between the Trk receptor tyrosine kinase and a 14 kDa light chain of cytoplasmic dynein. After transfection in human embryonic kidney 293 cells, this 14 kDa dynein light chain was found to bind to TrkA, TrkB, and TrkC receptors. Mapping experiments indicated that the 14 kDa dynein light chain binds to the distal region of the TrkA juxtamembrane domain. Coimmunoprecipitation experiments in vivo indicate that Trk receptors are in a complex with the 14 kDa light chain and 74 kDa intermediate chain of dynein. Confirming the physiological relevance of this association, a marked accumulation of Trk with the 14 kDa and the 74 kDa dynein components was observed after ligation of the sciatic nerve. The association of Trk receptors with components of cytoplasmic dynein suggests that transport of neurotrophins during vesicular trafficking may occur through a direct interaction of the Trk receptor with the dynein motor machinery

The Akt family of serine/threonine-directed kinases promotes cellular survival in part by phosphorylating and inhibiting death-inducing proteins. Here we describe a novel functional interaction between Akt and apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase. Akt decreased ASK1 kinase activity stimulated by both oxidative stress and overexpression in 293 cells by phosphorylating a consensus Akt site at serine 83 of ASK1. Activation of the phosphoinositide 3-kinase (PI3-K)/Akt pathway also inhibited the serum deprivation-induced activity of endogenous ASK1 in L929 cells. An association between Akt and ASK1 was detected in cells by coimmunoprecipitation. Phosphorylation by Akt inhibited ASK1-mediated c-Jun N-terminal kinase and activating transcription factor 2 activities in intact cells. Finally, activation of the PI3-K/Akt pathway reduced apoptosis induced by ASK1 in a manner dependent on phosphorylation of serine 83 of ASK1. These results provide the first direct link between Akt and the family of stress-activated kinases

Appropriate development of nervous system connectivity involves a variety of processes, including neuronal life-and-death decisions, differentiation, axon guidance and migration, and synaptogenesis. Although these activities likely require specialized signaling events, few substrates unique to these neurotrophic functions have been identified. Here we describe the cloning of ankyrin repeat-rich membrane spanning (ARMS), which encodes a novel downstream target of neurotrophin and ephrin receptor tyrosine kinases, Trk and Eph, respectively. The amino acid sequence of ARMS is highly conserved from nematode to human, suggesting an evolutionarily conserved role for this protein. The ARMS protein consists of 1715 amino acids containing four putative transmembrane domains, multiple ankyrin repeats, a sterile alpha motif domain, and a potential PDZ-binding motif. In the rat, ARMS is specifically expressed in the developing nervous system and in highly plastic areas of the adult brain, regions enriched in Trks and Eph receptors. ARMS can physically associate with TrkA and p75 neurotrophin receptors. Moreover, endogenous ARMS protein is tyrosine phosphorylated after neurotrophin treatment of pheochromocytoma 12 cells and primary hippocampal neurons or ephrin B treatment of NG108-15 cells, demonstrating that ARMS is a downstream target for both neurotrophin and ephrin receptors