The control of activities of individual Rho GTPases is crucial to neurotrophin-induced neurite outgrowth (3
). Our results suggest that Grit, a novel TrkA-interacting protein, regulates neurite outgrowth through modulating Rho GTPases. In PC12 cells, NGF oppositely regulated the activities of Rac1 and RhoA, activating Rac1 and suppressing RhoA (58
). Sos1 has been implicated in NGF-induced Rac activation, and the Sos1/E3b1/eps8 complex, endowed with Rac-specific GEF activity, was proposed to be located somewhat distantly from the activated receptors (21
). In contrast, RhoA was shown to interact constitutively with p75NTR
, a low-affinity neurotrophin receptor (59
). While p75NTR
-mediated down-regulation of RhoA upon neurotrophin stimulation was previously reported (59
), we assume a possible involvement of TrkA/Grit in the NGF-induced RhoA suppression to explain the result of our neurite extension assay (Fig. ), because RhoA was one of preferred targets of Grit GAP activity (Fig. ). The association between Trk and p75NTR
was demonstrated previously, and neurotrophin was suggested to reinforce the Trk-p75NTR
). Based on this finding, it is possible that TrkA/Grit could be prompted to associate with p75/RhoA complex upon NGF stimulation; Grit could thereby down-regulate RhoA in close proximity to the two neurotrophin receptors and positively contribute to NGF-induced neurite outgrowth.
The remarkable difference between the positive effect of the full-length Grit protein and the negative one of the Grit GAP domain on NGF-induced neurite outgrowth supports the idea that RhoGAP activity of the Grit GAP domain should be properly regulated spatiotemporally by other domains including the TrkA-binding region. As in the case of p190, overexpression of Rho-specific GAP could promote neurite outgrowth (9
). However, Grit had a GAP activity toward Rho/Rac/Cdc42; thus, it is not surprising that overexpression of the Grit GAP domain throughout the cell (free from the spatiotemporal control by other domains of Grit) did not accelerate neurite extension, because down-regulation of Rho could increase neurite production, whereas down-regulation of Rac/Cdc42 could decrease neurite outgrowth (26
). During the 2.5-day incubation in the neurite extension assay, a large excess of Grit GAP domain markedly reduced cell adhesion; the number of Grit GAP domain-expressing cells (mean ± standard deviation) that remained on the substrate was 47.5% ± 6.0% relative to that of controls (100%), whereas the RhoGAP R58A expression did not affect cell adhesiveness (104.0% ± 26.3%). Because the neurite growth rate is changed depending on the strength of cell adhesion (29
), the decrease in neurite outgrowth in the Grit GAP domain-expressing cells could be largely attributed to the decreased adhesiveness.
Another pool of Grit was associated with phosphotyrosine adapter molecules, N-Shc and CrkL/Crk, and thereby could be relocated to a close vicinity of activated receptor tyrosine kinases. Membrane relocation has been described for other Rho regulators, e.g., Tiam1 (10
), KIAA0380 (53
), and p190 RhoGAP (8
). However, to our knowledge, the present study provides the first demonstration of a relocation mechanism through a direct adapter binding of a RhoGAP. The relocated Grit would down-regulate Rho/Rac/Cdc42 depending on which GTPase was positioned close to the activated receptors. The N-Shc-Grit interaction and the CrkL/Crk-Grit interaction were independent, and thus Grit relocation was separately mediated by N-Shc and CrkL/Crk. In the case of neurotrophin stimulation, both N-Shc and Crk/CrkL can recruit their partners to the Trk-containing complex (34
). Thus, we suppose that Grit is recruited to this complex and may turn off RhoA also relocated to this complex through the induced Trk-p75 interaction, thereby contributing to neurotrophin-induced neurite outgrowth.
GAP activity itself can be modulated, and the regulation of RhoGAP activity has been classified into two major mechanisms, i.e., RhoGAP phosphorylation and protein-protein interaction. Tyrosine phosphorylation of p190 RhoGAP by Src or focal adhesion kinase has been proposed to regulate its GAP activity (2
). A similar regulation was reported for other GEF/GAPs (18
). However, in the present study, neither an increase nor a decrease in Grit GAP activity was detected following its tyrosine phosphorylation, and thus the functional significance of Grit phosphorylation remains to be clarified. The regulation of GAP activity via protein-protein interaction was recently demonstrated for CdGAP (23
), another member of the CdGAP/Grit family. The CdGAP sequence containing five SH3 binding motifs was responsible for down-regulation of GAP activity by the SH3-containing protein intersectin, and showed a partial homology to the corresponding Grit sequence having three SH3 binding motifs. We have obtained several candidate partners containing SH3 domains by the two-hybrid screening using this region of Grit as a bait (T. Nakamura and N. Mori, unpublished data). Modulation of Grit GAP activity by these candidate binders will be a subject of future research.
Accumulating evidence indicates the critical roles of Rho GTPases in neuronal morphogenesis, including axon growth and guidance (19
), dendrite elaboration (31
), migration, polarity, and plasticity (reviewed in reference 30
). A variety of effects of different Rho GTPases in different neuronal compartments indicate that functions and regulations of Rho GTPases may be more complex in neurons than in fibroblasts (30
). Further, the expression of constitutive-active and dominant-negative Rho GTPase mutants often produced similar phenotypes (1
), indicating that Rho GTPase signaling pathway has a cyclic mode of action, particularly prominent in neuronal cells, e.g., a rapid cycling between extension and retraction of filopodia during axonal growth. These views make it increasingly important to elucidate the elaborate mechanism of spatiotemporal control of Rho GTPase activity in individual neuronal compartments. Recently identified Rho GTPase regulators (GEF/GAPs) associated with specific receptors (48
), including Grit, should advance our understanding of these signaling pathways linking extracellular cues, receptors, and molecular switches (GTPases).