NTRAP interacts with receptor tyrosine kinases through a novel mechanism. Most signaling proteins interact with receptor tyrosine kinases through their SH2 domain (Src homology domain 2) or PTB domain (phosphotyrosine-binding domain), such as Shc and PLC-γ, which is dependent on receptor activation and phosphorylation of key tyrosine residues (Schlessinger, 2000
). However, a small number of signaling proteins interact with receptor tyrosine kinases independent of activation status of the receptors. For example, ARMS (an ankyrin-rich membrane-spanning protein) is required for sustained neurotrophin signaling and interacts with the transmembrane domain of Trk receptors (Arevalo et al., 2004
). Our results indicate that NTRAP does not belong to either one of the two classes. First, NTRAP interacts with inactive Trk receptors in both yeast and mammalian cells. Second, neurotrophin activation greatly enhances the interaction of NTRAP with Trk receptors. Although we have not identified the Trk sequence that interacts with NTRAP, the interaction site must include the tyrosine kinase domain and/or the short carboxy terminus of a Trk receptor because deletion of these two regions completely abolishes the interaction of TrkC with NTRAP.
How does neurotrophin activation enhance the interaction of NTRAP and Trk receptors? C2
zinc finger proteins comprise a large family of regulatory proteins, many of which are transcription factors, bind to specific DNA sequences, and participate in a variety of cellular activities (Iuchi, 2001
). Some C2
zinc fingers bind to RNAs or mediate self-association or selective association with other proteins (Iuchi, 2001
; Edelstein and Collins, 2005
). NTRAP contains five tandem C2
zinc fingers, and the third C2
zinc finger motif is required for the interaction with Trk receptor. Because a majority of C2
zinc fingers bind to nucleic acids, phosphorylation of tyrosine residues may bestow some properties of nucleic acids on Trk receptors. TrkC contains five key tyrosine residues at positions 485, 674, 678, 679, and 789, which serve as the docking sites for signaling proteins Shc, Frs2, SH2-B, and PLC-γ when they are phosphorylated (Reichardt, 2006
). However, we found that substitution of these tyrosine residues with phenylalanine residues did not weaken the interaction of the two proteins (Supplemental Figure S4), indicating that phosphotyrosine residues are not required for binding of NTRAP to Trk receptors. Therefore, it is more likely that neurotrophin activation alters the conformation of the Trk intracellular domain, which enhances the association of the two proteins. These observations also suggest that the active site and the three autophosphorylation sites (tyrosine residues at 674, 678, and 679 in the rat TrkC) in the tyrosine kinase domain have distinct roles in neurotrophic signaling. Although the third C2
zinc finger is critical for the interaction of NTRAP and Trk receptors, other C2
zinc fingers of NTRAP enhance the interaction. These C2
zinc fingers may affect the interaction by either interacting weakly with Trk receptors or mediating dimerization of NTRAP.
Our results from both PC12 cells and DRG neurons indicate that NTRAP plays a crucial role in retrograde neurotrophic signaling. Inhibiting NTRAP function with expression of its N-terminal fragment diminished NGF-dependent differentiation without affecting survival in PC12 cells. Because signaling cascades activated by TrkA receptor on cell surface are sufficient to mediate NGF-induced survival, but NGF-induced differentiation requires internalization of the Trk receptor in PC12 cells (Zhang et al., 2000
), Our observation suggests that NTRAP may be involved in the internalization and/or subsequent endocytic sorting of activated Trk receptors. The observation that expression of NTRAP shRNA or ZF-HA did not affect internalization of Trk receptors in PC12 cells and DRG neurons indicates that NTRAP is likely required for intracellular sorting of internalized Trk receptors. Our several observations support this argument. First, a significant fraction of NTRAP is localized to early endosomes and, after neurotrophin stimulation, to late endosomes. Second, membrane fractionation analysis showed that endocytic TrkA trafficking was normal in the first 10 min of NGF treatment, which should include the internalization step, and aberrant in the second 10 min of NGF treatment in cells expressing ZF-HA. Finally, expression of ZF-HA impaired NGF-induced persistent ERK activation in PC12 cells, which is dependent on TrkA signaling in Rap1-associated endosomes (York et al., 1998
; Wu et al., 2001
). In light of substantial evidence indicating that retrograde neurotrophic signaling is mediated by endosomes carrying internalized Trk receptors (Cosker et al., 2008
; Wu et al.
, 2008), these observations further suggest that NTRAP may play a critical role in retrograde neurotrophic signaling. Indeed, NTRAP is localized to signaling endosomes in DRG neurons, as indicated by colocalization of NTRAP and NGF in small-sized DRG neurons. Importantly, we show that NTRAP knockdown inhibits the ability of neurotrophins applied to the side compartment to promote neuronal survival and to activate CREB without affecting the action of neurotrophins applied to cell bodies in compartmentalized cultures of DRG neurons. Taken together, these results support the notion that NTRAP regulates retrograde neurotrophic signaling by controlling endocytic trafficking of internalized Trk receptors.
In the endocytic pathway, GTPases Rab5 and Rab7 act in a sequential manner by controlling targeting and fusion of clathrin-coated vesicles to early endosomes and regulating progression from early to late endosomes, respectively (Pfeffer, 2003
; Rink et al., 2005
; Deinhardt et al., 2006
). Internalized molecules in early and late endosomes can be recycled back to the plasma membrane. They can also be stored in endosomes for a long time or transported to lysosomes in which they are degraded (Sorkin and Von Zastrow, 2002
). Several studies indicate that Trk receptors at axonal terminals are internalized upon neurotrophin activation via clathrin-mediated (Grimes et al., 1996
; Grimes et al., 1997
; Howe et al., 2001
) or pincher-dependent endocytosis (Valdez et al., 2005
). Characterization of endosomes localized in axons suggests that the retrograde neurotrophic signal is carried in either early endosomes (Delcroix et al., 2003
) or late endosomes (Deinhardt et al., 2006
). Activated TrkA receptors in either early or late endosomes are able to induce sustained activation of ERK1/2 (Delcroix et al., 2003
; Hisata et al., 2007
). However, it is unknown whether signaling endosomes are a specialized population of early or late endosomes and, if so, which protein controls their generation. Biochemical fractionation analyses revealed that in PC12 cells with disrupted NTRAP function the internalized TrkA receptor was stuck in fractions enriched in late endosomes and was not associated with activated ERK1/2. These results suggest that signaling endosomes are indeed specialized ones and require NTRAP for their formation or maturation. This argument is further supported by our observation that inhibition of NTRAP reduced the sustained activation of ERK1/2, Rap1 activation, and differentiation in PC12 cells after NGF treatment. Future live imaging of Trk trafficking along different membrane compartments is necessary to reveal the precise role of NTRAP in the formation of signaling endosomes.
NTRAP likely regulates endocytic trafficking of Trk receptors by linking Trk receptors to other factors important for sorting of endosomes. NTRAP has a RING finger motif at its N-terminus, which is commonly found in E3 ubiquitin ligases (Jackson et al., 2000
). Although we failed to detect any effect of NTRAP on ubiquitination or sumoylation of Trk receptors (data not shown), NTRAP may mediate posttranslational modifications of other associated proteins. It can recruit proteins to Trk-containing endosomes with its two proline-rich regions and modify these proteins. Posttranslational modifications can change the property of the endosomes and specify the endosomes as retrograde cargoes. As an example, the RNA-binding protein La is anterogradely transported into axons by kinesin motors, and sumoylation in axons triggers retrograde transport of La because sumoylated La exclusively binds to dynein (van Niekerk et al., 2007
). Rab5 is regulated through the binding of guanine nucleotides and is active when bound to GTP. Its activation is required to complete vesicle targeting and fusion processes and is positively regulated by several guanine exchange factors, which stimulate the release of GDP, allowing GTP to enter the guanine-binding site (Carney et al., 2006
). It is intriguing to speculate that NTRAP may control the endocytic pathway by posttranslationally modifying the regulators of Rab5. Further investigation of the ubiquitination activity and interacting partners of NTRAP should provide new insights into the molecular mechanism of retrograde neurotrophic signaling.