Signaling from axon guidance receptors

Abstract

Determining how axon guidance receptors transmit signals to allow precise pathfinding decisions is fundamental to our understanding of nervous system development and may suggest new strategies to promote axon regeneration after injury or disease. Signaling mechanisms that act downstream of four prominent families of axon guidance cues--netrins, semaphorins, ephrins, and slits--have been extensively studied in both invertebrate and vertebrate model systems. Although details of these signaling mechanisms are still fragmentary and there appears to be considerable diversity in how different guidance receptors regulate the motility of the axonal growth cone, a number of common themes have emerged. Here, we review recent insights into how specific receptors for each of these guidance cues engage downstream regulators of the growth cone cytoskeleton to control axon guidance.

Figure 1.

Regulation of guidance receptor activation and signaling by endocytosis. (A) In response to ephrin binding to Ephs, the Rho family GEF Vav2 is recruited to the activated Eph receptor. Vav family GEFs are required for EphA endocytosis and ephrinA1-induced growth cone (GC) collapse, suggesting a role for Vavs in trafficking Ephs into signaling endosomes. (B) EphrinB binding to EphBs in neuronal GCs leads to collapse triggered by Eph forward signaling. Bi-directional endocytosis is necessary to remove the Ephrin-Eph protein complex from the cell surface, thereby allowing efficient cell detachment. (C) Endocytosis is a mechanism by which GCs adapt to guidance cues. Xenopus retinal GCs undergo collapse in response to Sema3A binding to its receptors Plxn-A and Nrpn. Exposure to a low dose of Sema3A desensitized the GCs, because Sema3A receptors were rapidly endocytosed. (D) Neural cell adhesion molecules (CAMs) such as L1 and TAG-1 modulate the response of certain axons to Sema3A. L1 binding to Nrpn (shown) and TAG-1 binding to L1 (not shown) facilitate internalization of Sema3A receptors, thereby enhancing the sensitivity of the GCs to Sema3A. (E) Endocytosis switches chemorepulsion to chemoattraction. Chemorepulsion by netrin is mediated by a complex of DCC and Unc5 receptors, whereas chemoattraction of netrin is mediated by DCC alone. Activation of protein kinase Cα (PKCα) specifically promotes internalization of the repellent Unc5 receptor, thereby converting netrin-mediated repulsion to attraction.

Figure 2.

Regulation of receptor activation and signaling by proteolytic processing. (A) Following ligand-receptor complex formation, ADAM10 cleaves the ephrinA5 ligand. This regulated proteolytic event both leads to release from the initial cell–cell adhesion, allowing for growth cone retraction, and is necessary for the transduction of the EphA3 forward signal. (B) Processive cleavage of ephrinB leads to the release of the ephrinB intracellular domain (ICD), which may activate SRC-family kinases to contribute to reverse signaling. On the other hand, cleavage of the EphB2 receptor, in this case by matrix metalloproteases (not pictured), is required for receptor activation in vitro. (C) Regulated proteolysis of DCC occurs by ADAM10-mediated creation of a carboxy-terminal fragment (CTF), followed by γ-secretase mediated intramembrane cleavage releasing DCC ICD. This ICD is competent to translocate to the nucleus when fused with Gal4. The cleavage event by ADAM10 leads to attenuation of neuritogenesis in vitro. (D) Kuzbanian appears to act positively in the Slit-Robo signaling pathway. Based on genetic observations and the abnormal presence of Robo protein on the commissural portions of axons in kuz mutants, we speculate that Kuz may cleave Robo to regulate receptor activity.

Figure 3.

Second messengers and guidance receptor signaling. (A) In vitro application of chemoattractants like Netrin and BDNF (not pictured) leads to rapid membrane depolarization and triggers an asymmetric elevation of intracellular calcium on the side of the growth cone facing the source of the chemoattractant. Activation of DCC (or TRK receptors for BDNF) leads to the release of calcium from intracellular stores through IP3 receptors, which in turn activates plasma membrane TRP channels and voltage‐dependent calcium channels (VDCC). The resulting gradient of intracellular calcium directs growth cone attraction. (B) In vitro application of chemorepellants like Semaphorin and Slit (not pictured) leads to rapid membrane hyperpolarization and a local elevation of intracellular calcium. Activation of PlexA receptors leads to the production of cGMP (likely through nitric oxide synthase), which in turn activates cyclic nucleotide gated (CNG) channels in the plasma membrane. In this case, the gradient of calcium and cGMP across the growth cone leads to growth cone repulsion.

Figure 4.

Eph forward signaling via GEFs and GAPs. (A) In the absence of ephrin stimulation, ephexin1 (ex) is bound to Eph receptors and activates RhoA, Rac1, and Cdc42, leading to a balance of GTPase activation that promotes actin polymerization and axonal growth. α2-chimaerin (α2) and Vav proteins (V) do not bind unclustered Ephs. (B) Upon ephrin-induced clustering and autophosphorylation of Ephs, ephexin1 is tyrosine phosphorylated (P), which shifts its exchange activity toward RhoA. α2-chimaerin is recruited to the Eph cluster and becomes tyrosine phosphorylated. This modification activates its intrinsic GAP activity, causing inactivation of Rac1. RhoA activation and Rac1 inactivation promote actin depolymerization and axon retraction. The specific role of Vav-mediated Rac1 activation is currently unclear. It may be linked to Vavs role in Eph endocytosis and may help to polymerize actin near the plasma membrane, where the endocytic vesicle forms.