In this report, we have identified APPL1 as a novel TrkA-associated protein in neurons. APPL1, which is known to play roles in endosomal signaling and Akt regulation, was also found to be associated with GIPC1, another TrkA-interacting protein previously shown to cluster with TrkA in intracellular vesicles. The association of APPL1 and TrkA could occur by at least two mechanisms, one requiring GIPC1 and the second through the direct binding of the APPL1 PTB domain to TrkA. The binding of APPL1 to GIPC1 was mediated by the C-terminal region of APPL1 and the PDZ domain of GIPC1. APPL1, GIPC1, and activated TrkA cofractionated in the same highly purified endosomal membrane preparation from PC12 cells. Both APPL1 and GIPC1 were required for TrkA signal transduction, as reduction of the levels of either of these proteins suppressed NGF-stimulated ERK1/2 and Akt activation and neurite outgrowth in PC12 cells.
What is the function of APPL1 in NGF-mediated signal transduction? We found that the suppression of APPL1 levels inhibited NGF-mediated Akt and Erk activation, which are required for NGF-mediated survival and neuritogenesis, respectively (19
). APPL1 has been reported to be a direct binding partner for Akt2 and postulated to potentiate Akt activation by tethering this kinase to PI 3-kinase (27
). Consistent with this hypothesis, APPL1 enhanced Akt-mediated suppression of androgen receptor transactivation in a PI 3-kinase-dependent manner and was required for Akt phosphorylation on serine residue 473 in response to insulin-like growth factor in PC-3 cells (38
). A similar role for APPL1 is suggested by our observation that reducing APPL1 levels decreased NGF-mediated Akt phosphorylation. The reduction of Akt activity by suppression of APPL1 levels, however, did not suppress NGF-induced PC12 cell survival, likely because the residual levels of Akt were sufficient to mediate survival. In support of this conclusion, sympathetic neurons are known to require 10-fold less NGF for survival than for axonal growth (3
Our experiments also revealed a requirement for APPL1 in NGF-induced activation of MEK and ERK1/2 but not TrkA activation or ShcA phosphorylation. In PC12 cells and sympathetic neurons, activation of the MAPK pathway is required for NGF-mediated neuritogenesis (19
). Consistent with this role of the MAPK pathway downstream of NGF, we demonstrated that the suppression of APPL1 levels also attenuated NGF-induced neuritogenesis. Interestingly, APPL1 was previously shown to weakly associate with Grb2 (27
), an adaptor protein that regulates TrkA-induced MAPK activity, providing a possible means by which APPL1 regulates the activity of this pathway.
The observation that APPL1 associates with TrkA and cofractionates with TrkA in a subpopulation of endosomes is consistent with a model whereby APPL1 may function in endosomal signaling downstream of NGF. APPL proteins have previously been implicated in an EGF-induced endosomal signaling pathway that links trafficking events at the plasma membrane to chromatin remodeling in the nucleus (26
). In EGF-treated HeLa cells, APPL was required for EGF-induced cell proliferation through uptake of EGF into APPL- and Rab5-containing endosomes at membranes and transport and release of APPL to the nuclear compartment. While we have thus far not observed an NGF-mediated translocation of APPL1, the association between these proteins may serve to couple NGF-TrkA endosomal signaling complexes to other trafficking events governed by the Rab proteins. This hypothesis is consistent with the emerging theme that Rab GTPases are significant players in TrkA trafficking and signaling. Indeed, Rab7 was recently identified to associate with TrkA to regulate the signaling endosome residence time (29
) and Rab5 has been found with GIPC1, APPL1, and pTrkA in endosomal fractions of NGF-treated PC12 cells (M. Grimes, personal communication).
We found that APPL1 associated with GIPC1, a TrkA-binding protein that has previously been shown to be required for activation of NGF-induced MAPK signaling (24
). We show that GIPC1 is also required for efficient NGF-induced neurite outgrowth, as well as activation of MEK, MAPK, and Akt. The precise mechanism through which GIPC1 mediates these functions remains to be elucidated. However, the observed effects of reducing GIPC1 levels are similar to those of decreasing APPL1 levels. Furthermore, we have identified a direct PDZ domain-mediated association between GIPC1 and APPL1 and have detected APPL1, GIPC1, and phosphorylated TrkA in the same endosomal fractions. Based on these observations, we propose that GIPC1 may function as a bridging protein to connect APPL1 with TrkA. Despite containing only a single protein-protein interaction domain, GIPC1 has been hypothesized to function as a scaffolding molecule to organize multiprotein complexes. Interestingly, mutation of the carboxylate binding loop of the GIPC1 PDZ domain does not abolish the interaction between TrkA and GIPC1, raising the possibility that the binding pocket of the GIPC1 PDZ domain remains open to recruit other proteins (24
). Therefore, it is conceivable that GIPC1 can simultaneously engage both TrkA and APPL1 since the binding of the internal sequence in the juxtamembrane region of TrkA occurs at a site within the GIPC1 PDZ domain that is distinct from the carboxylate binding pocket that interacts with the C-terminal sequence of APPL1. Another mechanism by which GIPC1 can organize higher-order complexes containing both APPL1 and TrkA is through the ability of GIPC1 to multimerize, a process involving its N-terminal domain (5
). This versatility in protein interactions allows GIPC1 to bridge distinct pathways by simultaneously binding different proteins. For example, GIPC1 can recruit GAIP, a regulator of heterotrimeric G proteins, to TrkA (24
) and to the D2
R dopamine receptor (17
). We suggest that GIPC1 may play an important role in TrkA function by bridging the association between APPL1 and TrkA.
The APPL1 PTB domain bound TrkA directly without the involvement of GIPC1. The availability of both GIPC1-dependent and GIPC1-independent routes for APPL1 association with TrkA suggests that the composition of the internalized TrkA signaling complex may be dynamic. One possibility is that the GIPC-dependent association may be important in early events after TrkA activation while the GIPC1-independent interaction comes into play during later events. Several lines of evidence suggest GIPC1 is involved in endocytosis and early trafficking events after receptor internalization. In nonneuronal cells, GIPC1 localizes to membrane-associated vesicles and tubular structures and to clathrin-coated pits (23
). We have shown the presence of GIPC1, APPL1, and activated TrkA in endosomal fractions 10 min after NGF treatment. This observation is in agreement with fractionation studies with PC12 cells and cortical neurons showing similar profiles of enrichment within fractions of intracellular membranes for GIPC1 and Trk (39
). During the course of TrkA endocytosis and trafficking, however, GIPC1 or APPL1 may not remain associated with TrkA. The dissociation of GIPC1 from vesicles after fusion with early endosomes in epithelial cells (1
) and nonexhaustive colabeling of GIPC1 with D2
R and D3
R dopamine receptors (16
) suggest that GIPC1 is released during later sorting events. At these later time points, GIPC1-independent binding via the APPL1 PTB domain may be the mechanism through which TrkA and APPL1 continue to associate in neuronal cells.
Based upon our observations, we propose a model whereby APPL1 and GIPC1 are constitutively bound to TrkA. Upon activation of TrkA by NGF, APPL1 potentiates Akt activation at the plasma membrane. Both GIPC1 and APPL1 are cointernalized with activated TrkA, residing in a population of endosomes that target TrkA to signaling proteins, such as MAPK, or to the nuclear membrane. Future experiments assessing the effect of disrupting APPL1, GIPC, and TrkA interactions in primary neurons in compartmentalized cultures will allow us to further elucidate the role of these proteins in the transmission of NGF retrograde signals.