ß-arrestins Form a Protein Complex with Smo and Src Family Kinases to Drive Shh-Mediated Axon Guidance in Spinal Cord Commissural Neurons

Axon guidance is an essential process by which growing axons navigate toward their targets to establish proper wiring of the nervous system. Axons are guided by molecular cues that are secreted by cells into the extracellular environment or are expressed on the surface of cells along the target pathway. Such molecules mediate axon guidance by binding to receptors on the growth cone at the tip of the extending axon. This triggers an intracellular signaling cascade that leads to cytoskeletal remodeling and membrane trafficking, which promote growth cone extension or collapse, ultimately resulting in the attraction or repulsion of axon growth and driving the axon in the correct direction.

A well-studied example of axon guidance occurs at the ventral midline of the spinal cord. A subset of spinal cord neurons called commissural neurons extend their axons toward the floor plate by following long-range guidance cues (Lepelletier et al., 2017). The axons eventually cross the ventral midline and continue growing to reach appropriate postsynaptic targets. One molecule that guides these axons is Sonic hedgehog (Shh), which is secreted by floor plate cells and acts as an attractant to the midline. Shh, a morphogen that has numerous functions during embryonic development, acts through both canonical and noncanonical signaling pathways. Both pathways are initiated by Shh binding to the receptor Patched1 (Ptch1). This causes Ptch1 to dissociate from the G-protein–coupled receptor (GPCR) Smoothened (Smo), which it normally represses. In canonical Shh signaling, Smo then translocates to a signaling organelle in the cell, where it initiates a signaling cascade that leads to Gli-dependent transcription (Brennan et al., 2012). In noncanonical Shh signaling, Smo freed from Ptch1 repression activates Src family kinases (SFKs). Smo is required for SFK activation in Shh-mediated axon guidance (Yam et al., 2009). In growing axons, these SFKs phosphorylate proteins associated with cytoskeletal remodeling (Lepelletier et al., 2017; Sauvé et al., 2024), which allows axonal growth and turning.

Sauvé et al. hypothesized that ß-arrestins also have a role in Shh-mediated axon guidance. ß-arrestins are cytosolic adaptor proteins that are implicated in GPCR trafficking, including internalization and translocation of Smo. But ß-arrestins also serve as scaffolding proteins that bind multiple proteins to create functional signaling units. Importantly, ß-arrestins recruit SFKs to GPCRs in non-Shh signaling contexts (Chen et al., 2004; Jean-Charles et al., 2017). Therefore, Sauvé et al. investigated the role of ß-arrestins in Shh-mediated guidance of commissural axons.

The authors first asked whether ß-arrestin is expressed in commissural neurons during the developmental period in which Shh-mediated axon guidance occurs. Using in situ hybridization in E11.5 mouse commissural neurons, they determined that both ß-arrestin 1 and ß-arrestin 2 mRNA are expressed in commissural neurons marked by high DCC expression. This result, in combination with quantified mRNA transcript analysis, suggested that ß-arrestins are indeed expressed in commissural neurons during development.

To determine whether ß-arrestin is necessary for the interaction between Smo and SFKs, the authors coexpressed these proteins in Cos7 cells. When ß-arrestin 1 or ß-arrestin 2 was coexpressed with the SFK protein Fyn, which is required for Shh-mediated axon guidance, a pulldown assay for Fyn also pulled down ß-arrestins, indicating the proteins interact. In addition, a coimmunoprecipitation assay confirmed that ß-arrestin interacts with Smo and that this interaction is enhanced by Shh. Moreover, when Smo and Fyn were expressed, they coprecipitated only if ß-arrestin was also expressed. This indicates that Smo and Fyn do not bind directly to each other, but ß-arrestin allows them to be recruited into a stable protein complex. Finally, a coimmunoprecipitation assay determined that Smo and the SFK Src (another SFK required for Shh-mediated axon guidance) also interact in the lysate of commissural neurons, which have an endogenous expression of ß-arrestin.

Next, the authors addressed whether ß-arrestins are required for SFK activation in the context of Shh signaling. To do so, they knocked down ß-arrestins in commissural neuron cultures using siRNA. When neurons were exposed to Shh, levels of the active (phosphorylated) form of the Src were lower in ß-arrestin–deficient neurons than in controls.

After establishing ß-arrestin as required for Shh-mediated Src activation, Sauvé et al. (2024) asked whether ß-arrestin is necessary for commissural neuron attraction to Shh. Knocking down ß-arrestin in commissural neuron cultures prevented the normal axon turning that occurred when control neurons were exposed to an Shh gradient. Thus ß-arrestin is necessary for Shh-mediated axon attraction in commissural neuron culture.

Finally, the authors investigated which of the two main functions of ß-arrestin are required for Shh-mediated axon attraction by using two dominant-negative ß-arrestin constructs. One construct was able to bind SFKs but was unable to internalize Smo, whereas the second construct was able to internalize Smo but was impaired in its ability to bind Src and Fyn. Expressing either dominant-negative ß-arrestin construct in commissural axons led to impaired axon turning in vitro and expanded axon bundles in vivo. These results suggest that both the Smo internalization and the SFK-binding functions of ß-arrestins are required for Shh-mediated guidance of commissural axons.

In summary, Sauvé et al. demonstrated that ß-arrestin is necessary for Shh-mediated Smo internalization and SFK activation during Shh-mediated axon guidance in vivo. This expands our knowledge of the molecular pathway that allows Shh to guide axons. Shh binding to Ptch1 increases Smo binding to ß-arrestin, which results in Smo internalization and interaction with SFKs. Whether Smo internalization was necessary for SFK activation remains unclear, however. Regardless, activated SFKs phosphorylate downstream targets that cause axons to turn toward the Shh gradient.

Future work should flesh out some of the details of ß-arrestins’ role in Shh-mediated axon guidance, particularly the role of Smo internalization. SFK activation occurs downstream of Smo activity in noncanonical Shh signaling (Yam et al., 2009), whereas Smo internalization and accumulation at the primary cilium lead to Gli-dependent transcription in canonical Shh signaling. In other cases where ß-arrestin internalizes Smo, phosphorylation of Smo by GRK2 is also required for this process (Chen et al., 2004). However, there is little evidence that Smo internalization is required for SFK activation. This suggests that elements of both the noncanonical and canonical Shh pathways are required for axon guidance. On the other hand, Sauvé et al. speculate that Smo internalization may be necessary for quick membrane recycling and pathway resensitization. These possibilities could be tested by using the dominant-negative mutant that prevents Smo internalization to investigate the effect of perturbed Smo internalization on Shh-induced SFK phosphorylation. In addition, one could use knockouts or knockdowns to determine whether Gli-dependent transcription of other elements of the canonical Shh signaling pathway is required for guidance of commissural axons. Mass spectrometry, additionally, may elucidate other proteins involved in Smo internalization and the ß-arrestin–Smo-SFK complex.

Finally, addressing the spatiotemporal dynamics of this pathway in vitro or in vivo would help elucidate the developmental context in which ß-arrestins are most crucial. SFKs inhibit midline crossing by axons in Drosophila melanogaster (O’Donnell and Bashaw, 2013), so future work should investigate the role of ß-arrestin when growth cones reach the midline in vertebrate models. If the inhibitory role of SFKs in midline crossing is conserved in vertebrates, this may elucidate a possible reason why Smo internalization is required for Shh-mediated axon guidance; ß-arrestin–mediated internalization may direct Smo to degradation pathways as growth cones reach the midline. Using chick embryos before, during, and after midline crossing, dominant-negative SFK and Smo expression experiments like those in Sauvé et al. (2024) and RNA expression and sequencing analysis may help determine whether ß-arrestin binding to Smo and SFKs is restricted to a particular phase of commissural axon growth (e.g., before midline crossing).

Ultimately, Sauvé et al. set the stage for understanding ß-arrestin roles in axon guidance (2024). This role may occur in additional contexts both within and outside of spinal cord commissural neurons. Thus, there are several future avenues for exploring the function of ß-arrestin in both canonical and noncanonical Shh signaling.