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Critical role for Kalirin in nerve growth factor signaling through TrkA.

ABSTRACT: Kalirin is a multidomain guanine nucleotide exchange factor (GEF) that activates Rho proteins, inducing cytoskeletal rearrangement in neurons. Although much is known about the effects of Kalirin on Rho GTPases and neuronal morphology, little is known about the association of Kalirin with the receptor/signaling systems that affect neuronal morphology. Our experiments demonstrate that Kalirin binds to and colocalizes with the TrkA neurotrophin receptor in neurons. In PC12 cells, inhibition of Kalirin expression using antisense RNA decreased nerve growth factor (NGF)-induced TrkA autophosphorylation and process extension. Kalirin overexpression potentiated neurotrophin-stimulated TrkA autophosphorylation and neurite outgrowth in PC12 cells at a low concentration of NGF. Furthermore, elevated Kalirin expression resulted in catalytic activation of TrkA, as demonstrated by in vitro kinase assays and increased NGF-stimulated cellular activation of Rac, Mek, and CREB. Domain mapping demonstrated that the N-terminal Kalirin pleckstrin homology domain mediates the interaction with TrkA. The effects of Kalirin on TrkA provide a molecular basis for the requirement of Kalirin in process extension from PC12 cells and for previously observed effects on axonal extension and dendritic maintenance. The interaction of TrkA with the pleckstrin homology domain of Kalirin may be one example of a general mechanism whereby receptor/Rho GEF pairings play an important role in receptor tyrosine kinase activation and signal transduction.

SUBMITTER: Chakrabarti K

PROVIDER: S-EPMC1140581 | biostudies-literature | 2005 Jun

REPOSITORIES: biostudies-literature

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Critical role for Kalirin in nerve growth factor signaling through TrkA.

Chakrabarti Kausik K Lin Rong R Schiller Noraisha I NI Wang Yanping Y Koubi David D Fan Ying-Xin YX Rudkin Brian B BB Johnson Gibbes R GR Schiller Martin R MR

Molecular and cellular biology 20050601 12

Kalirin is a multidomain guanine nucleotide exchange factor (GEF) that activates Rho proteins, inducing cytoskeletal rearrangement in neurons. Although much is known about the effects of Kalirin on Rho GTPases and neuronal morphology, little is known about the association of Kalirin with the receptor/signaling systems that affect neuronal morphology. Our experiments demonstrate that Kalirin binds to and colocalizes with the TrkA neurotrophin receptor in neurons. In PC12 cells, inhibition of Kali ...[more]

PMID: 15923627

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Project description:Nerve growth factor (NGF) exerts multiple functions on target neurons throughout development. The recent discovery of a point mutation leading to a change from arginine to tryptophan at residue 100 in the mature NGF? sequence (NGFR100W) in patients with hereditary sensory and autonomic neuropathy type V (HSAN V) made it possible to distinguish the signaling mechanisms that lead to two functionally different outcomes of NGF: trophic versus nociceptive. We performed extensive biochemical, cellular, and live-imaging experiments to examine the binding and signaling properties of NGFR100W Our results show that, similar to the wild-type NGF (wtNGF), the naturally occurring NGFR100W mutant was capable of binding to and activating the TrkA receptor and its downstream signaling pathways to support neuronal survival and differentiation. However, NGFR100W failed to bind and stimulate the 75 kDa neurotrophic factor receptor (p75NTR)-mediated signaling cascades (i.e., the RhoA-Cofilin pathway). Intraplantar injection of NGFR100W into adult rats induced neither TrkA-mediated thermal nor mechanical acute hyperalgesia, but retained the ability to induce chronic hyperalgesia based on agonism for TrkA signaling. Together, our studies provide evidence that NGFR100W retains trophic support capability through TrkA and one aspect of its nociceptive signaling, but fails to engage p75NTR signaling pathways. Our findings suggest that wtNGF acts via TrkA to regulate the delayed priming of nociceptive responses. The integration of both TrkA and p75NTR signaling thus appears to regulate neuroplastic effects of NGF in peripheral nociception.SIGNIFICANCE STATEMENT In the present study, we characterized the naturally occurring nerve growth factor NGFR100W mutant that is associated with hereditary sensory and autonomic neuropathy type V. We have demonstrated for the first time that NGFR100W retains trophic support capability through TrkA, but fails to engage p75NTR signaling pathways. Furthermore, after intraplantar injection into adult rats, NGFR100W induced neither thermal nor mechanical acute hyperalgesia, but retained the ability to induce chronic hyperalgesia. We have also provided evidence that the integration of both TrkA- and p75NTR-mediated signaling appears to regulate neuroplastic effects of NGF in peripheral nociception. Our study with NGFR100W suggests that it is possible to uncouple trophic effect from nociceptive function, both induced by wild-type NGF.

Project description:Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurovirulent coronavirus and causes neurological dysfunction in the central nervous system (CNS), but the neuropathological mechanism of PHEV remains poorly understood. We report that Unc51-like kinase 1 (Ulk1/Unc51.1) is a pivotal regulator of PHEV-induced neurological disorders and functions to selectively control the initiation of nerve growth factor (NGF)/TrkA endosome trafficking. We first identified the function of Ulk1 by histopathologic evaluation in a PHEV-infected mouse model in which neuronal loss was accompanied by the suppression of Ulk1 expression. Morphogenesis assessments in the primary cortical neurons revealed that overexpression or mutations of Ulk1 modulated neurite outgrowth, collateral sprouting, and endosomal transport. Likewise, Ulk1 expression was decreased following PHEV infection, suggesting that there was a correlation between the neurodegeneration and functional Ulk1 deficiency. We then showed that Ulk1 forms a multiprotein complex with TrkA and the early endosome marker Rab5 and that Ulk1 defects lead to either blocking of NGF/TrkA endocytosis or premature degradation of pTrkA via constitutive activation of the Rab5 GTPase. Further investigation determined that the ectopic expression of Rab5 mutants induces aberrant endosomal accumulation of activated pTrkA, proving that targeting of Ulk1-TrkA-NGF signaling to the retrograde transport route in the neurodegenerative process that underlies PHEV infection is dependent on Rab5 GTPase activity. Therefore, we described a long-distance signaling mechanism of PHEV-driven deficits in neurons and suggested that such Ulk1 repression may result in limited NGF/TrkA retrograde signaling within activated Rab5 endosomes, explaining the progressive failure of neurite outgrowth and survival.IMPORTANCE Porcine hemagglutinating encephalomyelitis virus (PHEV) is a neurotropic coronavirus and targets neurons in the nervous system for proliferation, frequently leaving behind grievous neurodegeneration. Structural plasticity disorders occur in the axons, dendrites, and dendritic spines of PHEV-infected neurons, and dysfunction of this neural process may contribute to neurologic pathologies, but the mechanisms remain undetermined. Further understanding of the neurological manifestations underlying PHEV infection in the CNS may provide insights into both neurodevelopmental and neurodegenerative diseases that may be conducive to targeted approaches for treatment. The significance of our research is in identifying an Ulk1-related neurodegenerative mechanism, focusing on the regulatory functions of Ulk1 in the transport of long-distance trophic signaling endosomes, thereby explaining the progressive failure of neurite outgrowth and survival associated with PHEV aggression. This is the first report to define a mechanistic link between alterations in signaling from endocytic pathways and the neuropathogenesis of PHEV-induced CNS disease.

Project description:Distal sensory polyneuropathy (DSP) with associated neuropathic pain is the most common neurological disorder affecting patients with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). Viral protein R (Vpr) is a neurotoxic protein encoded by HIV-1 and secreted by infected macrophages. Vpr reduces neuronal viability, increases cytosolic calcium and membrane excitability of cultured dorsal root ganglion (DRG) sensory neurons, and is associated with mechanical allodynia in vivo. A clinical trial with HIV/AIDS patients demonstrated that nerve growth factor (NGF) reduced the severity of DSP-associated neuropathic pain, a problem linked to damage to small diameter, potentially NGF-responsive fibers. Herein, the actions of NGF were investigated in our Vpr model of DSP and we demonstrated that NGF significantly protected sensory neurons from the effects of Vpr. Footpads of immunodeficient Vpr transgenic (vpr/RAG1(-/-)) mice displayed allodynia (p<0.05), diminished epidermalinnervation (p<0.01) and reduced NGF mRNA expression (p<0.001) compared to immunodeficient (wildtype/RAG1(-/-)) littermate control mice. Compartmented cultures confirmed recombinant Vpr exposure to the DRG neuronal perikarya decreased distal neurite extension (p<0.01), whereas NGF exposure at these distal axons protected the DRG neurons from the Vpr-induced effect on their cell bodies. NGF prevented Vpr-induced attenuation of the phosphorylated glycogen synthase-3 axon extension pathway and tropomyosin-related kinase A (TrkA) receptor expression in DRG neurons (p<0.05) and it directly counteracted the cytosolic calcium burst caused by Vpr exposure to DRG neurons (p<0.01). TrkA receptor agonist indicated that NGFacted through the TrkA receptor to block the Vpr-mediated decrease in axon outgrowth in neonatal and adult rat and fetal human DRG neurons (p<0.05). Similarly, inhibiting the lower affinity NGF receptor, p75, blocked Vpr's effect on DRG neurons. Overall, NGF/TrkA signaling or p75 receptor inhibition protects somatic sensory neurons exposed to Vpr, thus laying the groundwork for potential therapeutic options for HIV/AIDS patients suffering from DSP.

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Critical role for Kalirin in nerve growth factor signaling through TrkA.

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Critical role for Kalirin in nerve growth factor signaling through TrkA.

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