Evidence supporting the functionality of Smoothened (SMO), an essential transducer in most pathways engaged by Hedgehog (Hh), as a Gi-coupled receptor contrasts with the lack of an apparently consistent requirement for Gi in Hh signal transduction. In the present study, we sought to evaluate the role of SMO-Gi coupling in fibroblast migration induced by Sonic Hedgehog (Shh). Our results demonstrate an absolute requirement for Gi in Shh-induced fibroblast migration. We found that Shh acutely stimulates the small Rho GTPases Rac1 and RhoA via SMO through a Gi protein- and PI3K-dependent mechanism, and that these are required for cell migration. These responses were independent of transcription by Gli and of the C-terminal domain of SMO, as we show using a combination of molecular and genetic tools. Our findings provide a mechanistic model for fibroblast migration in response to Shh and underscore the role of SMO-Gi coupling in non-canonical Hh signaling.

The vertebrate Hedgehog (Hh) pathway has essential functions during development and tissue homeostasis during normal physiology while its dysregulation is a common theme in cancer. The Hh ligands (Sonic, Indian, and Desert Hh), bind to the receptors Patched1 and 2, resulting in inhibition of their repressive effect on Smoothened (Smo). Smo is a 7-transmembrane protein, which was only recently proved to function as a GPCR with specificity toward Gi. In addition to Gi activation, Smo signals via its C-tail to inhibit Suppressor of Fused (Sufu), resulting in stabilization and activation of the Gli family of transcription factors, which execute a transcriptional response to the so-called “canonical Hh signaling”. In this presentation we illustrate two outcomes of Hh signaling that are independent of Gli transcriptional activity and, thus, are defined as “non-canonical”. One outcome is dependent on Smo coupling to Gi proteins and exerts changes in the actin cytoskeleton via stimulation of RhoA and Rac1. These cytoskeletal changes promote migration in fibroblasts and tubulogenesis in endothelial cells. The other non-canonical Hh pathway is independent of Smo, and consists of inhibition of Patched1-induced cell death.

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