Trafficking, Acidification, and Growth Factor Signaling

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Science Signaling  10 Aug 2010:
Vol. 3, Issue 134, pp. pe26
DOI: 10.1126/scisignal.3134pe26


Wnt and Notch signaling pathways play key roles in development and disease. Despite great progress, the mechanism of signal transduction of their receptor-ligand complexes still holds surprises. For example, in both pathways, endocytosis is required for downstream signaling, but the mechanism by which endocytosis permits signaling is still unknown. New evidence indicates that endocytosis is required for the receptor-ligand complex to reach an acidified vesicular compartment. In turn, enzymes responsible for acidification are essential for Notch and Wnt signaling and also directly interact with the receptors. These findings raise new questions concerning the mechanism by which low pH promotes signal transduction and may open new possibilities for therapeutic intervention through the targeting of acidifying enzymes.

Cell-cell signaling through the Wnt and Notch pathways is involved in various developmental processes, and their dysregulation is associated with human disease, most notably cancer (1, 2). In response to binding of the ligands Delta or Serrate (also known as Jagged), the single-pass transmembrane Notch receptor undergoes a series of proteolytic cleavages. The first metalloprotease-mediated cleavage (S2) releases the extracellular portion to produce a membrane-anchored form that is subsequently cleaved by γ-secretase (S3), which releases the Notch intracellular domain (NICD). The NICD enters the nucleus, where it cooperates with DNA-binding proteins of the CSL [CBF1/ RBP-Jκ, Su(H), Lag-1] family and regulates the transcription of target genes (3).

In contrast to Notch signaling, Wnt signaling involves various receptors and engages several pathways, including the β-catenin and the planar cell polarity (PCP) pathways (4, 5). The best understood is the Wnt/β-catenin pathway, which is mediated by high-affinity Wnt receptors of the seven-pass transmembrane Frizzled (Fz) family, and a coreceptor, the low-density lipoprotein (LDL)–receptor–related proteins (LRP) 5 and 6. Upon ligand binding, Wnt, Fz, and LRP6 form a ternary signaling complex, which rapidly clusters on polymers of the scaffold proteins Dishevelled (Dvl) and Axin to form multiprotein complexes termed signalosomes (6). Signalosome formation promotes phosphorylation of LRP6, which ultimately leads to the inactivation of GSK-3, which relieves proteasomal degradation of β-catenin, allowing it to accumulate, enter the nucleus, and engage in transcriptional activation (7) (Fig. 1). Like the Wnt/β-catenin pathway, the Wnt/PCP pathway similarly involves Fz receptors and the adaptor protein Dvl, but requires a distinct set of proteins of downstream molecules that lead to the asymmetric localization of Fz-Dvl complexes during PCP (8). Although the requirement for Dvl appears to be universal among the different pathways, its protein domains are differentially used for the activation of downstream pathways.

Fig. 1

Acidification and trafficking in Wnt and Notch signaling pathways. The activation of both pathways relies on endocytosis of the receptor complexes and trafficking to a subcellular compartment that is acidified by V-ATPase. Cofactors, such as ATP6AP2 and Rbcn-3, are required for proper localization and may act as adaptors to bring receptor complexes in close proximity to V-ATPases.

Increasing evidence suggests that endocytosis is involved in activation of the Notch receptor and Fz during signal transduction. In Notch signaling, for example, a role for endocytosis was first suggested by the observation that transient removal of the vesicular traffic regulator dynamin in developing flies phenocopies the loss of Notch signaling (9). Since then, it has been shown that mutations that block endocytic transport from the cell surface to the endosome inhibit Notch signal transduction, whereas mutations that block endosomal sorting to lysosomes lead to excess signaling (1013).

Similarly, mounting evidence indicates that Wnt signaling requires endocytosis. In Drosophila, expression of dominant-negative forms of dynamin or Rab5 (a small guanosine triphosphatase involved in endosomal trafficking) in wing imaginal discs inhibits early endosomal fusion and reduces Wnt pathway activity (14). Subsequent analysis in mammalian cultured cells indicated that endocytosis is likely to be directly involved, because, when inhibited, it affects β-catenin accumulation within 1 hour of Wnt treatment (15, 16)

The precise mechanism by which endocytosis promotes both Notch and Wnt signaling has been unclear. Evidence indicates that the role of endocytosis is to transport the ligand-receptor complex to an acidic compartment, where the low pH is required for signaling (17). A screen carried out in Drosophila identified a role for Rabconnectin 3 (Rbcn-3) in endocytic trafficking and Notch signaling. Rbcn-3 shares homology with the yeast protein Rav1, which, as part of the RAVE complex, promotes the assembly of the vacuolar adenosine triphosphatase (V-ATPase) (18, 19). This multisubunit, adenosine triphosphate (ATP)–dependent proton pump is found at the plasma membrane, as well as in intracellular membranes, such as secretory compartments and cargo vesicles. It regulates the pH of intracellular compartments, the extracellular space, and the cytoplasm. V-ATPase regulates receptor-mediated endocytosis, vesicular traffic, protein processing, and the coupled transport of small molecules and ions. Moreover, V-ATPase is implicated in various bone and kidney diseases, as well as in tumor cell invasion (20, 21).

Not only does Rbcn-3 link Notch signaling to V-ATPase, but various mutants for subunits of the V-ATPase also induce impaired acidification and phenocopy Notch mutants (17, 22). V-ATPase is required cell autonomously in signal-receiving cells, and hence, low pH is required for Notch receptor activation (23). How does V-ATPase regulate Notch signaling? The simplest possibility is that V-ATPase is required indirectly, because it is essential for endocytosis, where it regulates transport of vesicles from early to late endosomes (24). However, in cells expressing V-ATPase mutants, Notch reaches and accumulates in late endosomal compartments, although at a reduced rate (17, 22). Furthermore, defects in genes encoding proteins that act at the late endosome, such as 3-phosphate 5-kinase Fab1, lead to Notch accumulation in the late endosome, comparable to that observed in cells expressing V-ATPase mutants. However, under these conditions, Notch signaling is not perturbed (11). Alternatively, it has been suggested that V-ATPase–dependent acidification is required for S3 cleavage of Notch. In this model, both endocytosis and V-ATPase are required for activated Notch to reach a low pH compartment and to activate γ-secretase, which has optimal catalytic activity at an acidic pH (25). However, γ-secretase–mediated cleavage of Notch1 is not affected by alkalizing drug treatments (26), which raises the possibility that another process in Notch activation requires low pH. This may involve more indirect steps, such as maturation or trafficking of γ-secretase.

V-ATPase–mediated acidification is also involved in Wnt signaling. Genome-wide screens for regulators of Wnt signaling identified ATP6AP2, a V-ATPase–associated, single-pass transmembrane protein that is also known as the prorenin receptor (PRR) (2729). Binding of prorenin and renin to ATP6AP2 can induce intracellular signaling, but the physiological relevance of this binding is unclear, because ATP6AP2 has a function in early vertebrate embryos before prorenin can be detected (30). Despite the established role of prorenin and renin in kidney physiology, a hypomorphic mutant of ATP6AP2 is associated with a mental retardation syndrome (XLMR) and epilepsy (31), rather than renal disease.

ATP6AP2 is required for Wnt/β-catenin signaling and binds to the Wnt receptor proteins Fz8 and LRP6, as well as to V-ATPase. Blocking V-ATPase activity by means of knockdown of one of its subunits or by pharmacological inhibitors blocks Wnt signaling and induces neural patterning defects in Xenopus embryos that are characteristic of defects in Wnt signaling. ATP6AP2 may act as an adaptor between V-ATPase and Wnt receptors, which enables acidification in the vicinity of the activated receptor complex, which appears to be necessary for phosphorylation of LRP6 and subsequent signaling (27).

ATP6AP2 (also known as PRR) is conserved in Drosophila, whereas a prorenin homolog does not appear to be present in Drosophila (30). Depletion of the Drosophila homolog to PRR (dPRR) leads to defects in Wnt signaling and planar cell polarity, and dPRR biochemically interacts with both Fz1 and Fz2, consistent with a role in canonical and noncanonical signaling (28, 29). dPRR seems necessary for the correct localization of Fz receptors, but not of other PCP signaling components such as Fmi. Moreover, the Na+-H+ exchanger Nhe2 is required for Fz–planar cell polarity signaling in Drosophila (32), which suggests that electrochemical regulation may have multiple roles in Wnt receptor signaling.

How does V-ATPase function in Wnt signaling? Within a few minutes of activation, the Wnt receptor reaches an acidic vesicular compartment, which is likely endosomal. Pharmacological inhibition of V-ATPase blocks LRP6 phosphorylation, a process that normally requires receptor clustering in endocytic signalosomes, which suggests that low pH is required for signalosome formation (27). This could be a direct effect, if low pH promotes a conformational change in the receptor which triggers clustering, or it may be indirect, if signalosome formation occurs only in endosomes, and this, in turn, requires V-ATPase for vesicular traffic.

However, many questions remain—in particular, with regard to the effect of V-ATPase function on the trafficking of receptor complexes. For example, although the ESCRT complex is required for the attenuation of Notch signaling, a similar requirement for Wnt signaling has not yet been described. The ESCRT complex is important for sorting proteins into multivesicular bodies and lysosomes, which suggests that down-regulation of Wnt signaling might depend on a different mechanism. Differences for V-ATPase function in Wnt/β-catenin and PCP signaling most likely also exist. During PCP, Fz accumulates at the plasma membrane, a localization that is lost in cells lacking dPRR. This could, for example, suggest that Fz receptors in PCP are recycled back to the membrane after reaching an acidified endosomal compartment, rather than remaining in endosomes or being targeted for degradation.

During development and tumorigenesis, Wnt and Notch signaling often occur in a coordinated manner (33, 34). The identification of V-ATPase function in both Wnt and Notch signaling might be a link between these pathways and also may open new avenues for therapeutic interventions. Small-molecule inhibitors against V-ATPases might be particularly suitable for tumors that are dependent on synergistic activation of Notch and Wnt signaling (34).


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