Smoothened Regulation: A Tale of Two Signals

Abstract

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the signal transducer of the developmentally and therapeutically relevant Hedgehog (Hh) pathway. Although recent structural analyses have advanced our understanding of Smo biology, several questions remain. Chief among them are the identity of its natural ligand, the regulatory processes controlling its activation, and the mechanisms by which it signals to downstream effectors. In this review, we discuss recent discoveries from multiple model systems that have set the stage for solving these mysteries. We focus on the roles of distinct Smo functional domains, post-translational modifications, and trafficking, and conclude by discussing their contributions to signal output.

Keywords: GPCR; Hedgehog; Smoothened; canonical signaling; noncanonical signaling.

Figure 1. Regulation of the Smoothened GPCR

A GPCR classes. Proteins of the GPCR superfamily, which possess 7 TM domains, are grouped into classes based upon their functionality and homology. The diagram indicates human GPCR classes, and denotes the number of proteins found in each class. The largest amount of structural data is available for Class A, the Rhodopsin family. Much of what is known about GPCR topology and activation has been deduced from Class A structural analyses. Smo is a member of the Class F Frizzled family. Classes D (mating pheromone receptors) and E (cAMP receptors) are not found in humans. B. The core Hh pathway. In the absence of Hh ligand, the Hh receptor Ptch localizes to the base of the primary cilium (PC) and inhibits Smo ciliary entry and signaling. Sufu inhibits Gli activator while Gli repressor keeps target genes silenced. Hh binding triggers Ptch internalization, which allows Smo to traffic to the tip of the PC where it signals for Sufu to release Gli activator. Gli activator then translocates to the nucleus to activate target gene expression. Many additional proteins contribute to regulation of these processes. Only the core pathway components are shown here. C. Smoothened functional domains. The cartoon illustrates the distinct Smo functional domains, ligand binding pockets, post-translational modifications and location of known activating mutations. CRD – cysteine rich domain; ATL – amino terminal linker; TM – transmembrane helix; ECL – extracellular loop; ICL – intracellular loop; C-term tail – carboxyl-terminal tail.

Figure 2. A Model for the Stages of Smo Activation

In the absence of ligand, inactive Smo (magenta) is ubiquitin modified (Ub, red), which signals for its internalization and membrane recycling or degradation. Stimulation with Hh ligand or a direct agonist such as a CRD-binding oxysterol (yellow) or the 7TM binding-compound SAG (orange) induces tail phosphorylation (P, white), leading to a conformation shift (light green) that promotes entry and accumulation in the primary cilium. Higher-order phosphorylation of the membrane-proximal GRK clusters leads to complete opening of the tail, which is permissive for Smo oligomerization and β-arrestin recruitment. This conformation (dark green) drives high-level signaling and correlates with increased accumulation of Smo in the tip of the primary cilium. We speculate that higher order signaling could be achieved by occupation of both ligand binding pockets by distinct ligands (left half of dimer) or by a larger peptide or fatty acid ligand bridging the two pockets (red ligand, right half of dimer). Recruitment of β-arrestin to the hyper-active conformation drives high-level signal propagation and eventual Smo desensitization. Extracellular N-linked glycans, which may contribute to extracellular conformation and/or ligand binding, are indicated in tan.

Figure 3. Models of Ptch-mediated Smo Regulation

Several models for Ptch-mediated Smo regulation have been hypothesized. In A, Ptch (yellow) pumps an antagonist (black), which binds to cell-surface localized Smo to block its accumulation and signaling (magenta, inactive Smo). In B, Ptch pumps a Smo agonist (green) out of the cell (B) or sequesters it in vesicles (B′) where it is unable to bind and activate Smo. In C, Ptch controls activity of a lipid regulator to block accumulation of a specific lipid species in membrane microdomains that are permissive for Smo signaling (purple haze, active Smo shown in green).